001 - Simplexa COVID-19 Flu AB Direct PreMarket Notification 30MAR22 Final

Simplexa™ COVID-19 & Flu A/B Direct REF MOL4250
Simplexa™ COVID-19 & Flu A/B Positive Control Pack REF MOL4260
Revision Date: March 30, 2022
Page 1 of 250
Table of Contents (Sections)
1. Medical Device User Fee Cover Sheet (FORM FDA 3601) .................................. 7
2. CDRH Premarket Review Submission Cover Sheet (Form 3514) ...................... 10
3. 510(k) Cover Letter ...........................................................................................18
4. Indications for Use Statement (Form 3881) .....................................................21
5. 510(k) Summary............................................................................................... 23
6. Truth and Accuracy Statement ........................................................................ 44
7. Class III Summary Certification ........................................................................ 46
8. Financial Certification or Disclosure Statement (Form 3454) .......................... 47
9. Certification of Compliance (Form 3674) ......................................................... 49
10. Executive Summary ......................................................................................... 54
10.1 Executive Summary 54
10.2 Pre-Submission Q202674 65
11. Device Description ........................................................................................... 66

11.1 Description 66
11.2 Procedure 68
11.3 Description of the Target Primer/Probe Sequence 69
11.4 Kit Components 70
11.5 Establishing the Cut-Off 71
11.6 LIAISON MDX Technology 75
11.6.1 LIAISON MDX System ............................................................................................ 75
12. Substantial Equivalence Discussion ................................................................. 76

12.1 Comparison to Predicate 76
13. Proposed Labeling ........................................................................................... 80

13.1 Instructions for Use 80
Page 2 of 250
13.2 Kit Component Labels 80

13.3 Candidate Device Kit Box Labels 80
13.4 Assay Specific QR Barcode 81
14. Sterilization and Shelf Life ............................................................................... 83

14.1 Sterilization 83
14.2 Shelf Life 83
14.3 Reaction Mix and Positive Control Open Vial (Room Temperature) Stability 87
15. Biocompatibility ............................................................................................... 97

16. Software .......................................................................................................... 98
16.1 LIAISON® MDX Software Studio 98
17. Electromagnetic Compatibility (EMC) and Electrical Safety .......................... 101

18. Performance Testing Bench ........................................................................... 102
18.1 Cross-Reactivity 102
18.2 Inhibition by Other Organisms (Microbial Inhibition) 109
18.3 Analytical Reactivity 122
18.4 Competitive Interference 146
18.5 Sample Stability (Fresh vs. Frozen) 150
a) Sample Stability (Fresh vs Frozen) Nasopharyngeal Swabs (NPS) in Universal
Transport Medium (UTM), Puritan UniTranz-RT and Saline (0.9% sodium chloride in
water) 150
b) Media Equivalency Nasopharyngeal Swabs (NPS) in Universal Transport Medium
(UTM), Puritan UniTranz-RT and Saline (0.9% sodium chloride in water) 158
18.6 Reproducibility 166
18.7 Inter-lot Precision – Reaction Mix 177
18.8 Inter-lot Precision – Positive Control 186
18.9 Amplification Carry-Over/Direct Amplification Disc Re-Usability 192
18.10 Limit of Detection (LoD) 192
a) Influenza A (Hong Kong/8/1968 and Michigan/45/2015), influenza B (Malaysia/2506/04
and Phuket/3073/2013) and COVID-19 2019-nCoV/USA-WA1/2020 192
Page 3 of 250
b) Limit of Detection using WHO International Standard (COVID-19) 213

18.11 Interference 219
18.12 No Template Control Specificity 228
18.13 Testing with a Moderate Complexity User 231
18.14 Direct Amplification Disc (DAD) Consumable – Disc Reusability 231
19. Performance Testing – Animal ....................................................................... 232

20. Performance Testing – Clinical ...................................................................... 233
20.1 Clinical Agreement 233
20.2 Expected Values 248
21. Miscellaneous .............................................................................................. 250

21.1 CLIA Complexity Categorization 250
Page 4 of 250
List of Attachments
Attachments (Documents) Attachment Number
Establishing the Cut-off Line data for NTC 11.5-1
Establishing the Cut-off Line data for LoD 11.5-2
Predicate IFU - BioFire Respiratory Panel 2.1 (RP2.1) (DEN200031) 12.1-1
Predicate IFU - Simplexa™ Flu A/B & RSV Direct Gen II (K201505) 12.1-2
Predicate IFU - Simplexa™ Flu A/B & RSV Positive Control Pack
12.1-3
(K201505)
Simplexa™ COVID-19 & Flu A/B Direct IFU (IFUK.US.MOL4250) 13-1
Simplexa™ COVID-19 & Flu A/B Positive Control Pack IFU

13-2
(IFUC.US.MOL4260)
Candidate Device Stability Memo 14.2-1
Room Temperature Reaction Mix Stability Protocol 14.3-1
Room Temperature Reaction Mix Stability Line Data pdf 14.3-2
Room Temperature Reaction Mix Stability Line Data xls 14.3-3
Room Temperature Positive Control Stability Protocol 14.3-4
Room Temperature Positive Control Stability Line Data pdf 14.3-5
Room Temperature Positive Control Stability Line Data xls 14.3-6
LIAISON MDX SW 1.1 to 2.1 Technical Report 16-1
LIAISON MDX SW 1.1 to 2.1 Technical Report Data 16-2
Cross Reactivity Protocol 18.1-1
Cross Reactivity BLAST Results 18.1-2
Cross Reactivity Line Data (pdf) 18.1-3
Cross Reactivity Line Data (xls) 18.1-4

Page 5 of 250
Microbial Inhibition Protocol 18.2-1
Microbial Inhibition in silico BLAST Analysis Results 18.2-2
Microbial Inhibition Line Data (pdf) 18.2-3
Microbial Inhibition Line Data (xls) 18.2-4
Analytical Reactivity Protocol 18.3-1
Analytical Reactivity in-silico BLAST Analysis Results 18.3-2
Analytical Reactivity Line Data (pdf) 18.3-3
Analytical Reactivity Line Data (xls) 18.3-4
Analytical Reactivity COVID-19 Strains Line Data (pdf) 18.3-5
Analytical Reactivity COVID-19 Strains Line Data (xls) 18.3-6
Simplexa™ COVID-19 Clinical Strain Monitoring Report 18.3-7
Competitive Interference Protocol 18.4-1
Competitive Interference Line Data (pdf) 18.4-2
Competitive Interference Line Data (xls) 18.4-3
Sample Stability Fresh Vs Frozen Protocol 18.5-1
Sample Stability Fresh Vs Frozen Report 18.5-2
Sample Stability Fresh Vs Frozen Report (Line Data) (xls) 18.5-3
Sample Stability - Media Equivalency Swab Protocol (Other Media) 18.5-4
Sample Stability - Media Equivalency Swab Line Data (pdf) 18.5-5
Sample Stability - Media Equivalency Swab Line Data (xls) 18.5-6
Reproducibility Protocol 18.6-1
Reproducibility Line Data (pdf) 18.6-2
Reproducibility Line Data (xls) 18.6-3
Inter-lot Precision – Reaction Mix Protocol 18.7-1

Page 6 of 250
Inter-lot Precision – Reaction Mix Line Data (pdf) 18.7-2
Inter-lot Precision – Reaction Mix Line Data (xls) 18.7-3
Inter-lot Precision – Positive Control Protocol 18.8-1
Inter-lot Precision – Positive Control Line Data (pdf) 18.8-2
Inter-lot Precision – Positive Control Line Data (xls) 18.8-3
Limit of Detection Study –Protocol 18.10-1
Limit of Detection Line Data (pdf) 18.10-2
Limit of Detection Line Data (xls) 18.10-3
Limit of Detection (WHO Standard) Study Protocol 18.10-4
Limit of Detection (WHO Standard) Line Data (pdf) 18.10-5
Limit of Detection (WHO Standard) Line Data (xls) 18.10-6
WHO Documentation 18.10-7
Interference Protocol 18.11-1
Interference Line Data (pdf) 18.11-2
Interference Line Data (xls) 18.11-3
Interference Study 2 Line Data (pdf) 18.11-4
Interference Study 2 Line Data (xls) 18.11-5
No Template Control Protocol 18.12-1
No Template Control Line Data (pdf) 18.12-2
No Template Control Line Data (xls) 18.12-3
Clinical Study Protocol 20-1
Clinical Study Line Data (pdf) 20-2
Clinical Study Line Data (xls) 20-3

Page 7 of 250
1. Medical Device User Fee Cover Sheet (FORM FDA 3601)

11/17/21, 3:01 PM Site: MDUFMA Cover Sheet
Form Approved: OMB No. 0910-0511 Expiration Date: August 31, 2022. See Instructions for OMB Statement.
DEPARTMENT OF HEALTH AND HUMAN SERVICES

PAYMENT IDENTIFICATION NUMBER: MD6127487
FOOD AND DRUG ADMINISTRATION
Write the Payment Identification number on your check.
MEDICAL DEVICE USER FEE COVER SHEET
A completed cover sheet must accompany each original application or supplement subject to fees. If payment is sent by U.S. mail
or courier, please include a copy of this completed form with payment. Payment and mailing instructions can be found at:
https://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/HowtoMarketYourDevice/ucm370879.htm
1. COMPANY NAME AND ADDRESS (include name, street 2. CONTACT NAME
address, city state, country, and post office code) Tara Viviani
2.1 E-MAIL ADDRESS
Diasorin Molecular LLC regulatoryaffairs_molecular@diasorin.com
11331 Valley View St
2.2 TELEPHONE NUMBER (include Area code)
Cypress 562-240-6133
CA 90630-5300 2.3 FACSIMILE (FAX) NUMBER (Include Area code)
US
1.1 EMPLOYER IDENTIFICATION NUMBER (EIN)
*****0354
3. TYPE OF PREMARKET APPLICATION (Select one of the following in each column; if you are unsure, please refer to the
application descriptions at the following web site:
http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/ucm345263.htm
Select an application type: 3.1 Select a center
[X] Premarket notification(510(k)); except for third party [X] CDRH
[ ] 513(g) Request for Information [ ] CBER
[ ] Biologics License Application (BLA) 3.2 Select one of the types below
[ ] Premarket Approval Application (PMA) [X] Original Application
[ ] Modular PMA Supplement Types:
[ ] Product Development Protocol (PDP) [ ] Efficacy (BLA)
[ ] Premarket Report (PMR) [ ] Panel Track (PMA, PMR, PDP)
[ ] 30-Day Notice [ ] Real-Time (PMA, PMR, PDP)
[ ] De Novo Request [ ] 180-day (PMA, PMR, PDP)
4. ARE YOU A SMALL BUSINESS? (See the instructions for more information on determining this status)
[ ] YES, I meet the small business criteria and have submitted the required [X] NO, I am not a small business
qualifying documents to FDA
4.1 If Yes, please enter your Small Business Decision Number:
5. FDA WILL NOT ACCEPT YOUR SUBMISSION IF YOUR COMPANY HAS NOT PAID AN ESTABLISHMENT REGISTRATION FEE THAT
IS DUE TO FDA. HAS YOUR COMPANY PAID ALL ESTABLISHMENT REGISTRATION FEES THAT ARE DUE TO FDA?
[X] YES (All of your establishments have registered and paid the fee, or this is your first device and you will register and pay the
fee within 30 days after entering into an operation that requires you to register and submit device listing information.)
[ ] NO (If you currently market a medical device and your establishment is required to register and submit device listing
information, FDA will not accept your submission until you have paid all fees due to FDA. See
http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/HowtoMarketYourDevice/RegistrationandListing/ucm053165.htm
for additional information)
6. IS THIS PREMARKET APPLICATION COVERED BY ANY OF THE FOLLOWING USER FEE EXCEPTIONS? IF SO, CHECK THE
APPLICABLE EXCEPTION.
[ ] This application is the first PMA submitted by a qualified small [ ] The sole purpose of the application is to support
business, including any affiliates conditions of use for a pediatric population
[ ] This biologics application is submitted under section 351 of the [ ] The application is submitted by a state or federal
Public Health Service Act for a product licensed for further government entity for a device that is not to be distributed
manufacturing use only commercially
7. IS THIS A SUPPLEMENT TO A PREMARKET APPLICATION FOR WHICH FEES WERE WAIVED DUE TO SOLE USE IN A PEDIATRIC
POPULATION THAT NOW PROPOSES CONDITION OF USE FOR ANY ADULT POPULATION? (If so, the application is subject to the
fee that applies for an original premarket approval application (PMA).
[ ] YES [X] NO
PAPERWORK REDUCTION ACT STATEMENT
Public reporting burden for this collection of information is estimated to average 18 minutes per response, including the time for
reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing
the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information,
https://userfees.fda.gov/OA_HTML/mdufmaCScdCfgItemsPopup.jsp?vcname=Tara Viviani&vcmpname=Diasorin Molecular LLC&vemail=regulatoryaff… 1/2

11/17/21, 3:01 PM Site: MDUFMA Cover Sheet
including suggestions for reducing this burden, to the address below.
Department of Health and Human Services

Food and Drug Administration
Office of Chief Information Officer
Paperwork Reduction Act (PRA) Staff
PRAStaff@fda.hhs.gov
[Please do NOT return this form to the above address, except as it pertains to comments on the burden estimate.]
8. USER FEE PAYMENT AMOUNT SUBMITTED FOR THIS PREMARKET APPLICATION
$12,745.00 17-Nov-2021
Form FDA 3601 (08/19)
"Close Window" Print Cover sheet
https://userfees.fda.gov/OA_HTML/mdufmaCScdCfgItemsPopup.jsp?vcname=Tara Viviani&vcmpname=Diasorin Molecular LLC&vemail=regulatoryaff… 2/2

Page 10 of 250
2. CDRH Premarket Review Submission Cover Sheet (Form 3514)

Page 18 of 250
3. 510(k) Cover Letter

CONFIDENTIAL
March 30, 2022
U.S. Food and Drug Administration

Center for Devices and Radiological Health
Document Mail Center – WO66-G609
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
Subject: Traditional 510(k) Pre-Market Notification for Simplexa™ COVID-19 & Flu A/B
Direct (MOL4250) and Simplexa™ COVID-19 & Flu A/B Positive Control Gen II
Pack (MOL4260)
Attention Virology Division
Attached please find the electronic copy of the DiaSorin Molecular Pre-market Notification for the
above named kits and components.
DiaSorin Molecular is prepared and committed to complete cooperation with the FDA to
accomplish the review.
The following documents are enclosed within the accompanying electronic file copy of the
510(k) Pre-Market Notification:
1. CDRH Pre-market Review Submission Cover Sheet

2. Premarket Notification Truthful and Accurate Statement
3. Medical Device User Fee Cover Sheet
4. 510(k) Pre-Market Notification
5. Financial Interests and Arrangements of Clinical Investigators (Form FDA 3454)
6. Certification of Compliance (FDA 3674)
7. Indications for Use (FDA 3881)
8. 510(k) Summary of Safety and Effectiveness
DiaSorin Molecular LLC 11331 Valley View Street, Cypress, CA 90630 Tel 562-240-6500
This project has been funded in whole or in part with Federal funds from the Department of Health
and Human Services; Office of the Assistant Secretary for Preparedness and Response;
Biomedical Advanced Research and Development Authority, under Contract No.
75A50121P00007. Dr. Chitra Edwin, or an alternate designated representative, is the BARDA
contact with permission to interact with FDA for this pre-market notification.
The existence of this 510(k) Pre-Market Notification and other information that it contains are
confidential, and the protection afforded to such confidential information by 21 CFR 807.95 is
hereby claimed.
We trust that the FDA will find this 510(k) Pre-Market Notification for the Simplexa COVID-19 &
Flu A/B Direct complete and look forward to a timely if not expeditious review to clearance decision
of the submission. Should you have any questions or concerns, please do not hesitate to contact
me at the coordinates provided below. In my absence, please contact Tara Viviani at the contact
coordinates also provided below.
• Primary contact: Sharon Young: phone (562) 240-6680 or e-mail

Sharon.Young@DiaSorin.com
• Secondary contact: Tara Viviani: phone (562) 240-6271 or e-mail

Tara.Viviani@DiaSorin.com
Sincerely,
Sharon Young
Manager, Regulatory Affairs
DiaSorin Molecular LLC.
Attachment/Enclosure:
o e-Copy disc (1)
cc: Tara Viviani, RAC, Senior Director, Molecular Regulatory Affairs, DiaSorin Molecular LLC.
Mari Meyer, Vice President, Regulatory & Clinical Affairs, North America, DiaSorin Inc.
DiaSorin Molecular LLC. 11331 Valley View Street, Cypress, CA 90630 Tel 562-240-6500
Page 21 of 250
4. Indications for Use Statement (Form 3881)

Page 23 of 250
5. 510(k) Summary
K
510(k) Summary
Simplexa™ COVID-19 & Flu A/B Direct MOL4250
Simplexa™ COVID-19 & Flu A/B Positive Control Pack. MOL4260
March 30, 2020
Page 1 of 20
Applicant DiaSorin Molecular LLC.

11331 Valley View Street
Cypress, California 90630
USA
Establishment Registration No. 2023365
Contact Person Sharon Young

Manager, Regulatory Affairs
Tel. 562.240.6680
Sharon Young@DiaSorin.com
Summary Date March 30, 2022
Proprietary Name Simplexa™ COVID-19 & Flu A/B Direct and

Simplexa™ COVID-19 & Flu A/B Positive Control Pack
US Product Codes/Names and QOF – Multi-Target Respiratory Specimen Nucleic Acid Test
Regulation Numbers Including Sars-Cov-2 And Other Microbial Agents 21 CFR §
866.3981
OOI - Real Time Nucleic Acid Amplification System 21 CFR §
862.2570
Classification Class II
Predicate Devices BioFire Respiratory Panel 2.1 (RP2.1) (DEN200031) and

Simplexa™ Flu A/B & RSV Direct Gen II (K201505)
Intended Use
Simplexa COVID-19 & Flu A/B Direct (Catalog Number: MOL4250):

The DiaSorin Molecular Simplexa™ COVID-19 & Flu A/B Direct real-time RT-PCR assay is intended for
use on the LIAISON® MDX instrument for the in vitro qualitative detection and differentiation of nucleic acid
from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus and influenza B
virus in nasopharyngeal swabs (NPS) from human patients with signs and symptoms of respiratory tract
infection in conjunction with clinical and epidemiological risk factors.
The Simplexa™ COVID-19 & Flu A/B Direct assay is intended for use as an aid in the differential diagnosis
of SARS-CoV-2, influenza A and influenza B infection.
Negative results do not preclude SARS-CoV-2, influenza A or influenza B infection and should not be used
as the sole basis for patient management decisions. Negative results should be combined with clinical
observations, patient history, and epidemiological information.
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.
K
510(k) Summary
March 30, 2020
Page 2 of 20
The Simplexa™ COVID-19 & Flu A/B Direct assay is intended for use by qualified and trained clinical
laboratory personnel specifically instructed and trained in the techniques of real-time PCR and in vitro
diagnostic procedures.
Simplexa COVID-19 & Flu A/B Positive Control Pack (Catalog Number: MOL4260):
The Simplexa™ COVID-19 & Flu A/B Positive Control Pack is intended to be used as a control with the
Simplexa™ COVID-19 & Flu A/B Direct kit for use on the LIAISON® MDX instrument. This control is not
intended for use with other assays or systems.
Device Description
The Simplexa™ COVID-19 & Flu A/B Direct assay system is a real-time RT-PCR system that enables the
direct amplification, detection and differentiation of SARS-CoV-2 RNA, human influenza A (Flu A) virus
RNA and human influenza B (Flu B) virus RNA from unprocessed nasopharyngeal swabs (NPS) that have
not undergone nucleic acid extraction. The system consists of the Simplexa™ COVID-19 & Flu A/B Direct
assay, the LIAISON® MDX (with LIAISON® MDX Studio Software), the Direct Amplification Disc and
associated accessories.
In the Simplexa™ COVID-19 & Flu A/B Direct assay, fluorescent probes are used together with
corresponding forward and reverse primers to amplify SARS-CoV-2, Flu A, Flu B and internal control RNA
targets. For COVID-19 detection, the assay targets two different regions specific to the SARS-CoV-2
genome; the S gene which encodes the spike glycoprotein and the ORF1ab region which encodes well-
conserved non-structural proteins and therefore is less susceptible to recombination. For Flu detection the
assay targets conserved regions of influenza A viruses (matrix gene) and influenza B viruses (matrix gene).
The assay provides three results; COVID-19 (ORF1ab and/or S gene detection), influenza A viruses (matrix
gene detection) and influenza B viruses (matrix gene detection). An RNA internal control is used to detect
RT-PCR failure and/or inhibition.
Simplexa™ COVID-19 &Flu A/B Direct REF MOL4250
Reactions
EC Symbol Abbreviated Cap Number Volume
Component Name REF per
on Label Name Color of Vials per Vial
Vial/Kit
Simplexa™ COVID-19
& Flu A/B Direct MOL4251 REAG C RM Blue 24 1/24 50 µL
Reaction Mix
K
510(k) Summary
March 30, 2020
Page 3 of 20
Simplexa™ COVID-19 &Flu A/B Direct Components and Descriptions
Kit
Contents
Component
DNA polymerase, Reverse Transcriptase, RNase inhibitor, buffer and dNTPs, encapsulated RNA
Template, primers and dye-labeled fluorescent probes specific for detection of SARS-CoV-2,
influenza A, influenza B and for the RNA Internal Control
Target Channel Excitation Emission Targeted Gene
Simplexa™ Flu A 520 445-505 507-533 matrix
COVID-19 &
Flu A/B Direct
Reaction Mix Flu B 560 505-543 547-573 matrix
(RM)
SARS-CoV-2
610 502-596 597-623 S and ORF1ab
“COVID”
Internal Control
690 622-658 652-708 N/A
“RNA IC”
Simplexa™
COVID-19 &
Assay specific parameters, lot number, expiration date
Flu A/B Direct
Barcode Card
Simplexa™ COVID-19 &Flu A/B Positive Control Pack REF MOL2260

Component and Description
Reactions
Cap Number Volume
Component Name REF Description per
Color of Vial per Vial
Vial/Kits
Simplexa™ COVID- Inactivated SARS-CoV-2 virus,
19 & Flu A/B Direct MOL4261 inactivated influenza A virus, Red 10 1/10 50µL
Positive Control inactivated influenza B virus
Materials Supplied Separately
Direct Amplification Disc Kit (REF MOL1455) Direct Amplification Discs for use on the LIAISON® MDX
Comparison to Predicate Device

Comparison to Predicate Device 1: Predicate Device 2: Candidate Device:
Predicate Device BioFire Respiratory Panel 2.1 Simplexa™ Flu A/B & RSV -
(RP2.1) (DEN200031) Direct Gen II (K201505)
Product Code QOF OCC QOF
OOI
Regulation 21 CFR 866.3981 21 CFR 866.3980 21 CFR 866.3981
Number
21 CFR 862.2570
Organism SARS-CoV-2 Influenza A (Flu A), SARS-CoV-2, Influenza A
Detected Influenza B (Flu B) and RSV (Flu A) and Influenza B (Flu
B)
Measurand RNA from SARS-CoV-2 RNA from Influenza A (Flu RNA from SARS-CoV-2,
A), Influenza B (Flu B) and Influenza A (Flu A) and
RSV Matrix gene (Scorpion Influenza B (Flu B) Matrix
Probes) gene
K
510(k) Summary
March 30, 2020
Page 4 of 20
Target FilmArray Torch Technology Well conserved region of the SARS-CoV-2 Well
matrix gene (Scorpion
conserved regions of the S
SARS-CoV-1 Well conserved Technology)
and ORF genes
region of the S gene
Flu A and B Targets Well
conserved regions of the
SARS-CoV-2 Well conserved matrix gene (TAQ Man
region of the M gene Technology)
Intended Use Kit The BioFire Respiratory Panel Simplexa Flu A/B & RSV Simplexa COVID-19 & Flu
2.1 (RP2.1) is a PCR-based Direct Gen II A/B Direct
multiplexed nucleic acid test The DiaSorin Molecular The DiaSorin Molecular
intended for use with the Simplexa™ Flu A/B & RSV Simplexa™ COVID-19 & Flu
BioFire FilmArray 2.0 or BioFire Direct Gen II assay is A/B Direct real-time RT-PCR
FilmArray Torch systems for the intended for use on the assay is intended for use on
simultaneous qualitative LIAISON® MDX instrument the LIAISON® MDX
detection and identification of for the in vitro qualitative instrument for the in vitro
multiple respiratory viral and detection and differentiation qualitative detection and
bacterial nucleic acids in of influenza A virus, differentiation of nucleic acid
nasopharyngeal swabs (NPS) influenza B virus, and from severe acute
obtained from individuals respiratory syncytial virus respiratory syndrome
suspected of respiratory tract (RSV) RNA in coronavirus 2 (SARS-CoV-
infections, including COVID-19. nasopharyngeal swabs 2), influenza A virus and
The following organism types (NPS) from human patients influenza B virus in
and subtypes are identified with signs and symptoms of nasopharyngeal swabs
using the BioFire RP2.1: respiratory tract infection in (NPS) from human patients
• Adenovirus, conjunction with clinical and with signs and symptoms of
• Coronavirus 229E, epidemiological risk factors. respiratory tract infection in
• Coronavirus HKU1, This test is intended for use conjunction with clinical and
• Coronavirus NL63, as an aid in the differential epidemiological risk factors.
• Coronavirus OC43, diagnosis of influenza A,
influenza B, and RSV viral The Simplexa™ COVID-19
• Severe Acute & Flu A/B Direct assay is
Respiratory Syndrome infections in humans.
Negative results do not intended for use as an aid in
• Coronavirus (SARS- the differential diagnosis of
CoV-2), preclude influenza virus or
RSV infection and should SARS-CoV-2, influenza A
• Human and influenza B infection.
not be used as the sole
Metapneumovirus,
basis for treatment or other Negative results do not
• Human
patient management preclude SARS-CoV-2,
Rhinovirus/Enterovirus,
decisions. Performance influenza A or influenza B
• Influenza A, including characteristics for influenza
subtypes H1, H1-2009, infection and should not be
A were established with used as the sole basis for
and H3, clinical specimens collected patient management
• Influenza B, during the 2010/2011 decisions. Negative results
• Parainfluenza Virus 1, influenza season when 2009 should be combined with
• Parainfluenza Virus 2, H1N1 influenza and H3N2 clinical observations, patient
• Parainfluenza Virus 3, were the predominant history, and epidemiological
• Parainfluenza Virus 4, influenza A viruses in information.
• Respiratory Syncytial circulation. When other
Virus, influenza A viruses are If infection with a novel
• Bordetella emerging, performance influenza A virus is
parapertussis characteristics may vary. If suspected based on current
(IS1001), infection with a novel clinical and epidemiological
• Bordetella pertussis influenza A virus is screening criteria
(ptxP), suspected based on current recommended by public
• Chlamydia clinical and epidemiological health authorities,
pneumoniae, and screening criteria specimens should be
recommended by public collected with appropriate
infection control precautions
K
510(k) Summary
March 30, 2020
Page 5 of 20
• Mycoplasma health authorities, for novel virulent influenza
pneumoniae specimens should be viruses and sent to the state
Nucleic acids from the collected with appropriate or local health department
respiratory viral and bacterial infection control precautions for testing. Viral culture
organisms identified by this test for novel virulent influenza should not be attempted in
are generally detectable in NPS viruses and sent to the state these cases unless a BSL
specimens during the acute or local health department 3+ facility is available to
phase of infection. The for testing. Viral culture receive and culture
detection and identification of should not be attempted in specimens.
specific viral and bacterial these cases unless a BSL
3+ facility is available to The Simplexa™ COVID-19
nucleic acids from individuals
receive and culture & Flu A/B Direct assay is
exhibiting signs and/or
specimens. intended for use by qualified
symptoms of respiratory
Simplexa™ Flu A/B & RSV and trained clinical
infection is indicative of the
Positive Control Pack laboratory personnel
presence of the identified
DiaSorin Molecular’s specifically instructed and
microorganism and aids in the
Simplexa ™Flu A/B & RSV trained in the techniques of
diagnosis of respiratory
Positive Control Pack is real-time PCR and in vitro
infection if used in conjunction
intended to be used as a diagnostic procedures.
with other clinical and
epidemiological information. control with the Simplexa™
The results of this test should Flu A/B & RSV Direct kit and Simplexa COVID-19 & Flu
not be used as the sole basis the Simplexa™ Flu A/B & A/B Positive Control Pack
for diagnosis, treatment, or RSV Direct Gen II kit for use The Simplexa™ COVID-19 &
other patient management on the LIAISON® MDX Flu A/B Positive Control Pack
decisions. instrument. This control is is intended to be used as a
Negative results in the setting of not intended for use with control with the Simplexa™
a respiratory illness may be due other assays or systems. COVID-19 & Flu A/B Direct
to infection with pathogens that kit for use on the LIAISON®
are not detected by this test, or MDX instrument. This control
lower respiratory tract infection is not intended for use with
that may not be detected by an other assays or systems.
NPS specimen. Positive results
do not rule out coinfection with
other organisms. The agent(s)
detected by the BioFire RP2.1
may not be the definite cause of
disease. Additional laboratory
testing (e.g. bacterial and viral
culture, immunofluorescence,
and radiography) may be
necessary when evaluating a
patient with possible respiratory
tract infection.
Automated Automated Same Same
System (Sample
to Answer)
Instrumentation BioFire® FilmArray® 2.0 or LIAISON® MDX LIAISON® MDX
BioFire® FilmArray® Torch
Systems
Sample Types Nasopharyngeal Swab (NPS) Same Same
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CLINICAL PERFORMANCE
The performance of Simplexa™ COVID-19 & Flu A/B Direct was evaluated using prospective, retrospective,
pre-selected positive and negative nasopharyngeal swab (NPS) specimens from human patients with signs
and symptoms of respiratory tract infection. The prospective samples were collected from four (4) external
sites, while retrospective samples were collected from five (5) external sites. Collection sites included five
(5) reference laboratories and four (4) biorepository banks, across four (4) different geographical locations.
Testing was performed from October 12th 2021 to February 18th 2022. The comparator for influenza A
and B targets was a FDA cleared RT-PCR assay. For discordant samples a second FDA cleared assay
and/or bidirectional sequencing assay was used. For the COVID-19 target a composite reference method
was utilized. Samples were tested using two (2) FDA Emergency Use Authorized (EUA) SARS-CoV-2 RT-
PCR Assays. Discordant results were tested by a third SARS-CoV-2 EUA RT-PCR assay and a two out of
three rule was applied to determine if a sample was positive or negative.
Tables 1-3 show the results of the Simplexa™ COVID-19 & Flu A/B Direct assay and comparator assay
results for their respective targets in the prospective and retrospective (pre-selected) study analysis. The
positive percent agreement (PPA) and negative percent agreement (NPA) is based on a total of 759
enrolled specimens.
Table 1. Simplexa™ COVID-19 & Flu A/B Direct vs FDA Cleared NAAT - Flu A
Simplexa™ COVID-19 & Flu FDA Cleared NAAT Assay
Total
A/B Direct Detected Not Detected
Detected 80 0 80
Not Detected 5a 674 674
Total 85 674 759

PPA: 94.1% (80/85) NPA: 100% (674/674)
95% CI: 87% to 97% 95% CI: 99.9% to 100%
a
Flu A was not detected in five (5) specimens, and three of the five (3/5) specimens were confirmed negative by a second FDA
cleared NAAT.
PPA = Positive Percent Agreement, NPA = Negative Percent Agreement, CI = Confidence Interval. The 95% confidence
intervals (CI) were calculated following the Wilson Score method.
Table 2. Simplexa™ COVID-19 & Flu A/B Direct vs FDA cleared NAAT- Flu B
Simplexa™ COVID-19 & Flu FDA Cleared NAAT Assay
Total
Detected 112 0 112
Total 112 640 759

PPA: 94.1.% (112/119) NPA: 100% (640/640)
95% CI: 88% - 97% 95% CI: 99% to 100%
a
Flu B was not detected in seven (7) specimens and five of the seven (5/7) specimens were confirmed negative by a second
FDA cleared NAAT.
PPA = Positive Percent Agreement, NPA = Negative Percent Agreement, CI = Confidence Interval. The 95% confidence
intervals (CI) were calculated following the Wilson Score method.
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Table 3. Simplexa™ COVID-19 & Flu A/B Direct vs Composite Reference Method (CRM) – COVID-19
Simplexa™ COVID-19 & Flu Composite Reference Method (CRM)
Total
Detected 68 11b 79
Total 69 684 753

PPA:98.6% (68/69)) NPA: 98.4% (673/684)
95% CI: 92% to100% 95% CI: 97% - 99%
a
One specimen was confirmed positive by a second FDA cleared NAAT.
b
Nine of the eleven specimens detected by Simplexa™ COVID-19 & Flu A/B Direct assay were confirmed positive with a FDA
cleared NAAT.
PPA = Positive Percent Agreement, NPA = Negative Percent Agreement, CI = Confidence Interval. The 95% confidence intervals (CI)
were calculated following the Wilson Score method.
REPRODUCIBILITY
The reproducibility study was performed by one (1) internal site and two (2) external sites. The panel
consisted of eight (8) reproducibility panel members, including six (6) contrived samples,one (1) negative
sample [UTM as No Template Control (NTC)] and one (1) positive sample, Positive Control (PC). The
contrived panel members were prepared by spiking each analyte at approximately two times the Limit of
Detection (2x LoD, low positive) and approximately four times (4x) LoD (medium positive) into native
negative nasopharyngeal swab matrix in UTM. Each panel member was tested in triplicate for five (5) days.
Each site had two (2) operators who each assayed the entire panel once per day, for a total of two (2) sets
of data per day. Agreement with expected results are presented in Table 4 with average Cts, standard
deviation (SD) and coefficient of variation (%CV).
Table 4. Simplexa™ COVID-19 & Flu A/B Direct Reproducibility
Site 1 Site 2 Site 3 All Sites

Agreement Agreement Agreement Agreement
Sample Avg. Ct ± Avg. Ct ± Avg. Ct ± Avg. Ct
with with with with
SD SD SD ± SD 95% CI
expected expected expected expected
(%CV) (%CV) (%CV) (%CV)
results results results results
32.3 ± 32.9 ± 32.8 ± 32.7 ± 95.9%
Flu A Hong 100.0% 100.0% 100.0% 100.0%
0.54 1.22 0.55 0.86 to
Kong - LP (30/30) (30/30) (30/30) (90/90)
(1.7%) (3.7%) (1.7%) (2.6%) 100.0%
31.7 ± 31.5 ± 31.9 ± 31.7 ± 95.9%
Flu A Hong 100.0% 100.0% 100.0% 100.0%
0.49 0.43 0.44 0.47 to
Kong - MP (30/30) (30/30) (30/30) (90/90)
(1.5%) (1.4%) (1.4%) (1.5%) 100.0%
Flu B Malaysia 30.8 ± 30.9 ± 31.5 ± 31.1 ± 95.9%
100.0% 100.0% 100.0% 100.0%
- 0.69 0.60 0.62 0.70 to
(30/30) (30/30) (30/30) (90/90)
LP (2.2%) (1.9%) (2.0%) (2.2%) 100.0%
Flu B Malaysia 30.0 ± 29.9 ± 30.4 ± 30.1 ± 95.9%
100.0% 100.0% 100.0% 100.0%
- 0.35 0.44 0.48 0.48 to
(30/30) (30/30) (30/30) (90/90)
MP (1.2%) (1.5%) (1.6%) (1.6%) 100.0%
30.2 ± 29.5 ± 30.2 ± 30.0 ± 95.9%
COVID-19 100.0% 100.0% 100.0% 100.0%
0.41 0.40 0.67 0.59 to
USA-WA1 - LP (30/30) (30/30) (30/30) (90/90)
(1.4%) (1.4%) (2.2%) (2.0%) 100.0%
COVID-19 29.0 ± 28.8 ± 29.4 ± 29.1 ± 95.9%
100.0% 100.0% 100.0% 100.0%
USA-WA1 - 0.46 0.45 0.61 0.56 to
(30/30) (30/30) (30/30) (90/90)
MP (1.6%) (1.6%) (2.1%) (1.9%) 100.0%
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Sample Avg. Ct ± Avg. Ct ± Avg. Ct ± Avg. Ct
with with with with
SD SD SD ± SD 95% CI
expected expected expected expected
(%CV) (%CV) (%CV) (%CV)
26.2 ± 26.1 ± 26.0 ± 26.1 ±
0.32 0.21 0.20 0.26
(1.2%) (0.8%) (0.8%) (1.0%)
27.6 ± 28.2 ± 27.1 ± 27.6 ± 95.9%
Positive Control 100.0% 100.0% 100.0% 100.0%
0.87 0.39 0.22 0.71 to
(PC) (30/30) (30/30) (30/30) (90/90)
(3.2%) (1.4%) (0.8%) (2.6%) 100.0%
27.8 ± 27.2 ± 26.8 ± 27.3 ±
1.18 0.23 0.22 0.80
(4.2%) (0.9%) (0.8%) (2.9%)
0.0 ± 0.0 ± 0.0 ± 0.0 ±
0.00 0.00 0.00 0.00
(N/A%) (N/A%) (N/A%) (N/A%)
0.0 ± 0.0 ± 0.0 ± 0.0 ± 95.9%
Negative 100.0% 100.0% 100.0% 100.0%
0.00 0.00 0.00 0.00 to
(UTM) (30/30) (30/30) (30/30) (90/90)
(N/A%) (N/A%) (N/A%) (N/A%) 100.0%
0.0 ± 0.0 ± 0.0 ± 0.0 ±
0.00 0.00 0.00 0.00
(N/A%) (N/A%) (N/A%) (N/A%)
99.5%
100.0% 100.0% 100.0% 100.0%
Total to
(240/240) (240/240) (240/240) (720/720)
100.0%
Ct. = Cycle threshold, SD = Standard Deviation, %CV = Percent Coefficient of Variation, CI = Confidence Interval, LP = Low
Positive, MP = Medium Positive
ANALYTICAL SENSITIVITY/LIMIT OF DETECTION

The Limit of Detection (LoD) of the Simplexa™ COVID-19 & Flu A/B Direct assay in nasopharyngeal swabs
(NPS) was determined to be the lowest detectable concentration of quantitated inactivated titered viral
stocks (copies/mL or International Units/mL) at which ≥ 95% of all replicates were detected. Two (2) strains
of influenza A, two (2) strains of influenza B and two (2) strains SARS-CoV-2 serially diluted in negative
nasopharyngeal swab (NPS) matrix were used to determine the LoD. The LoD results are shown in Table
5.
Table 5. Simplexa™ COVID-19 & Flu A/B Direct Limit of Detection
Virus Strain LoD (copies/mL)
Influenza A/Hong Kong/8/68 500
Influenza A/Michigan/45/2015 500
Influenza B/Phuket/3073/2013 750
Influenza B/Malaysia/2506/2004 250
SARS-CoV-2 (USA-WA1/2020) 500
Virus Strain IU/mL

WHO International Standard for SARS-
651
CoV-2 RNA (NIBSC code: 20/146)
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ANALYTICAL REACTIVITY/CROSS REACTIVITY
Analytical Reactivity – Influenza A and B
Analytical reactivity was evaluated with nasopharyngeal swab (NPS) matrix for the Simplexa™ COVID-19
& Flu A/B Direct assay. A total of 63 Flu A strains, 21 Flu B strains and five (5) COVID-19 strains were
tested. Quantified viral material was spiked into negative NPS matrix at the concentrations listed in Tables
6-8 below and assayed in triplicate. The results are shown in Tables 6-8. Additional testing of all influenza
strains in the CDC panels for 2018-2021 was performed. The results are also shown in Tables 6-7 and the
CDC panels tested highlighted in Tables 9 - 11. All strains and subtypes were 100% detected with the
Simplexa™ COVID-19 & Flu A/B Direct assay.
Table 6. Simplexa™ COVID-19 & FLU A/B Direct Analytical Reactivity Results - Flu A
Tested
Organism % Detected
Concentration*
A/Anhui/1/2013 1:100,000 Dilution 100.0% (3/3)
A/black-legged kittiwake/Quebec/02838-1/2009 100 CEID50/mL 100.0% (3/3)
A/Brisbane/02/2018 100 EID50/mL 100.0% (3/3)
A/Brisbane/10/07 100 U/mL 100.0% (3/3)
A/Brisbane/59/07 100 U/mL 100.0% (3/3)
A/American green-winged teal/Mississippi/300/2010 100 CEID50/mL 100.0% (3/3)
A/California/02/2014 100 TCID50/mL 100.0% (3/3)
A/chicken/Germany/N/49 100 CEID50/mL 100.0% (3/3)

A/chicken/Vietnam/NCVD-016/2008(H5N1)-PR8-IDCDC-
1:100,000 Dilution 100.0% (3/3)
RG12
A/Christ Church/16/2010 1,000 EID50/mL 100.0% (3/3)
A/duck/Chabarovsk/1610/1972 100 CEID50/mL 100.0% (3/3)
A/duck/Czechoslovakia/1956 5,000 CEID50/mL 100.0% (3/3)
A/duck/Wisconsin/480/1979 100 CEID50/mL 100.0% (3/3)
A/Egypt/N03072/2010(H5N1)-PR8-IDCDC-RG29 1:100,000 Dilution 100.0% (3/3)
A/Guangdong-Maonan/1536/2019 100 EID50/mL 100.0% (3/3)
A/Hawaii/15/2001 100 CEID50/mL 100.0% (3/3)
A/Hong Kong/2671/2019 100 EID50/mL 100.0% (3/3)
A/Hong Kong/4801/2014 100 TCID50/mL 100.0% (3/3)
A/Hong Kong/33982/2009(H9N2)-PR8-IDCDC_RG26 100 CEID50/mL 100.0% (3/3)
A/Hubei/1/2010(H5N1)-PR8-IDCDC-RG30 1:100,000 Dilution 100.0% (3/3)
A/India/NIV/2006(H5N1)-PR8-IBCDC-RG7 1:100,000 Dilution 100.0% (3/3)

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Tested
Organism % Detected
Concentration*
A/Indiana/08/2011 100 TCID50/mL 100.0% (3/3)
A/Kansas/14/2017 100 EID50/mL 100.0% (3/3)
A/mallard/Netherlands/12/2000(H7N7)/PR8-IBCDC-1 1:100,000 Dilution 100.0% (3/3)
A/mallard/Illinois/10OS4334/2010 100 CEID50/mL 100.0% (3/3)
A/mallard/Wisconsin/4218/2009 100 CEID50/mL 100.0% (3/3)
A/mallard/Wisconsin/4230/2009 100 CEID50/mL 100.0% (3/3)
A/Massachusetts/15/2013 100 CEID50/mL 100.0% (3/3)
A/Mexico/4108/2009 100 CEID50/mL 100.0% (3/3)
A/Minnesota/11/2010 100 CEID50/mL 100.0% (3/3)
A/Minnesota/19/2011 100 CEID50/mL 100.0% (3/3)
A/New Caledonia/20/99 100 TCID50/mL 100.0% (3/3)
A/New York/18/2009 100 CEID50/mL 100.0% (3/3)
A/New York/55/2004 100 CEID50/mL 100.0% (3/3)
A/NY/02/09 100 TCID50/mL 100.0% (3/3)
A/Ohio/02/2012 100 CEID50/mL 100.0% (3/3)
A/Perth/16/2009 100 EID50/mL 100.0% (3/3)
A/pheasant/New Jersey/1355/1998(H5N2)-PR8-IBCDC-4 1:100,000 Dilution 100.0% (3/3)
A/Port Chalmers/1/1973 100 TCID50/mL 100.0% (3/3)
A/PR/8/34 100 TCID50/mL 100.0% (3/3)
A/quail/Italy/1117/1965 100 CEID50/mL 100.0% (3/3)
A/red knot/Delaware Bay/240/1994 100 CEID50/mL 100.0% (3/3)
A/red knot/Delaware/541/1988 1,000 CEID50/mL 100.0% (3/3)
A/redhead/Alberta/192/2002 100 CEID50/mL 100.0% (3/3)
A/Rhode Island/01/2010 100 CEID50/mL 100.0% (3/3)
A/Santiago/7981/2006 100 CEID50/mL 100.0% (3/3)
A/shorebird/Delaware Bay/211/1994 1,000 CEID50/mL 100.0% (3/3)
A/shorebird/Delaware/172/2006 1,000 CEID50/mL 100.0% (3/3)
A/Singapore/INFIMH-16-0019/2016 100 TCID50/mL 100.0% (3/3)
A/Solomon Island/3/2006 100 TCID50/mL 100.0% (3/3)
A/Swine/1976/31 100 U/mL 100.0% (3/3)
A/Swine/Iowa/15/30 100 U/mL 100.0% (3/3)
A/swine/Ohio/09SW1477/2009 100 TCID50/mL 100.0% (3/3)
A/swine/Ohio/09SW83E/2009 100 CEID50/mL 100.0% (3/3)

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Tested
Organism % Detected
Concentration*
A/Switzerland/9715293/2013 100 CEID50/mL 100.0% (3/3)
A/Taiwan/42/06 100 U/mL 100.0% (3/3)
A/turkey/Massachusetts/3740/1965 2,000 CEID50/mL 100.0% (3/3)
A/turkey/Virginia/4529/2002 (H7N2)xPR8-IBCDC-5 1:100,000 Dilution 100.0% (3/3)
A/Texas/50/2012 100 TCID50/mL 100.0% (3/3)
A/Wisconsin/67/05 100 TCID50/mL 100.0% (3/3)
A/WS/33 100 TCID50/mL 100.0% (3/3)

*TCID50/mL = Tissue Culture Infectious Dose, CEID50/mL = Chicken Embryo Infectious Dose, EID50/mL = Egg
Infectious Dose, U/mL = Units/milliliter
Table 7. Simplexa™ COVID-19 & Flu A/B Direct Analytical Reactivity Results - Flu B
Tested
Organism % Detection
Concentration*
B/Brisbane/33/2008 100 CEID50/mL 100% (3/3)
B/Brisbane/60/2008 100 U/mL 100% (3/3)
B/Christchurch/33/2004 100 TCID50/mL 100% (3/3)
B/Colorado/06/2017 100 TCID50/mL 100% (3/3)
B/Florida/02/2006 100 U/mL 100% (3/3)
B/Florida/04/2006 100 U/mL 100% (3/3)
B/Florida/07/04 100 U/mL 100% (3/3)
B/Great Lakes/1739/54 100 U/mL 100% (3/3)
B/Guangdong-Liwan/1133/2014 1,000 CEID50/mL 100% (3/3)
B/Maryland/1/59 100 TCID50/mL 100% (3/3)
B/Massachusetts/02/2012 100 TCID50/mL 100% (3/3)
B/Michigan/09/2011 100 EID50/mL 100% (3/3)
B/Nevada/03/2011 100 CEID50/mL 100% (3/3)
B/New Hampshire/01/2016 100 EID50/mL 100% (3/3)
B/Panama/45/90 100 U/mL 100% (3/3)
B/Texas/02/2013 100 TCID50/mL 100% (3/3)
B/Texas/81/2016 100 EID50/mL 100% (3/3)
B/Utah/09/2014 100 CEID50/mL 100% (3/3)

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Tested
Concentration*
B/Victoria/304/2006 100 CEID50/mL 100% (3/3)
B/Washington/02/2019 100 EID50/mL 100% (3/3)
B/Wisconsin/01/2010 100 CEID50/mL 100% (3/3)
*TCID50/mL = Tissue Culture Infectious Dose, CEID50/mL = Chicken Embryo Infectious Dose, EID50/mL = Egg Infectious
Dose, U/mL = Units/milliliter
Table 8. Simplexa™ COVID-19 & Flu A/B Direct Analytical Reactivity Results – SARS-CoV-2
Tested
Concentration*
England/204820464/2020 1000 copies/mL 100% (3/3)
hCoV19/USA/PHC658/2021 1500 copies/mL 100% (3/3)
HongKong/VM200001061/2020 1000 copies/mL 100% (3/3)
Japan/TY7-503/2021 1000 copies/mL 100% (3/3)
South Africa/KRISP-EC-K005325/2020 1000 copies/mL 100% (3/3)
Table 9 2018-2019 CDC panel Flu A and Flu B Strains Tested with Simplexa™ COVID-19 & Flu A/B Direct
Virus Subtype Organism

A A/Perth/16/2009
(H3N2) A/Singapore/INFIMH-16-0019/2016*
Flu A
A A/California/07/2009
(H1N1) pdm09 A/Michigan/45/2015*
B B/Brisbane/60/2008
(Victoria lineage) B/Colorado/06/2017*
Flu B
B B/Wisconsin/01/2010
(Yamagata lineage) B/Phuket/3073/2013*
*WHO recommended vaccine strains
Table10. 2019-2020 CDC panel Flu A and Flu B Strains Tested with Simplexa™ COVID-19 & Flu A/B Direct

A A/Perth/16/2009
(H3N2) A/Kansas/14/2017*
Flu A
A A/Christ Church/16/2010
(H1N1) pdm09 A/Brisbane/02/2018*
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B B/Michigan/09/2011
(Victoria lineage) B/Colorado/06/2017*
Flu B
B B/New Hampshire/01/2016
*WHO recommended vaccine strains
Table 11. 2020-2021 CDC panel Flu A and Flu B Strains Tested with Simplexa™ COVID-19 & Flu A/B Direct

A A/Perth/16/2009
(H3N2) A/Hong Kong/2671/2019*
Flu A
A/Christ Church/16/2010
A
(H1N1) pdm09 A/Guangdong-
Maonan/1536/2019*
B B/Michigan/09/2011
(Victoria lineage) B/Washington/02/2019*
Flu B
B B/Texas/81/2016
*WHO recommended vaccine strain
Analytical Reactivity/Inclusivity – COVID-19

An in-silico inclusivity analysis of the COVID-19 target primers and probes in the Simplexa™ COVID-19 &
Flu A/B Direct assay was performed. All primer and probe sets designed for detection of the ORF1ab and
S gene were tested against the complete SARS-CoV-2 genome sequences available in the GISAID
database submitted from November 01, 2021 to January 31, 2022. The analysis included 2,170,584
sequences in the amplicon regions of the ORF1ab and S gene primer/probe regions. Only target sequences
with full coverage of all three ORF1ab and S gene forward and reverse primer as well as probe region were
included in the analyses. The analysis showed that the Simplexa™ COVID-19 & Flu A/B Direct target
regions had no mismatch to 2,170,382 sequences (~99.99%) and were predicted to be detected by the
assay based on sequence homology. There were 202 sequences (~0.01%) with mismatches in at least one
primer or probe binding region, region in either ORF1ab or S gene target region. A melting temperature
(Tm) analysis was conducted for those sequences with mismatches in the binding sites of both gene assay
target regions. A Tm calculation was performed with assay-specific conditions using a Tm Mismatch
Bioinformatics Tool. Tm values observed above their respective annealing temperature had mismatches
that were not located at the 3’ end for the primers; as such, detection of these sequences are not affected
by the mismatches. Table 12 below summarizes the Tm analysis results.
Table 12. Summary of Tm Analysis Results
No. seq. where at least one gene oligo set meets Tm criteria 2,170,584
No. seq. where no gene oligo set meets Tm criteria 0

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Cross-Reactivity (Analytical Specificity)

Cross-reactivity of the Simplexa™ COVID-19 & Flu A/B Direct assay was evaluated by testing whole
organisms or purified nucleic acid from other organisms. Specimens for laboratory testing were prepared
by spiking cultured isolates/inactivated organisms/purified nucleic acids (whole genome) into negative
matrix (NPS) and determining cross reactivity based on three replicates. RNasin® was added to NPS for
specimens containing extracted RNA. Results from cross-reactivity testing are summarized in Table 13.
Table 13. Simplexa™ COVID-19 & Flu A/B Direct Cross-Reactivity Results
Tested
Organism COVID-19 Flu A Flu B
Concentration1
0.0% 0.0% 0.0%

Adenovirus Type 1 1 x 105 TCID50/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Adenovirus Type 7A 1 x 105 TCID50/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Bordetella pertussis 1 x 106 CFU/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Candida albicans 1 x 106 CFU/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Chlamydia pneumoniae 1 x 106 IFU/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Corynebacterium diphtheriae 1 x 106 CFU/mL
(0/3) (0/3) (0/3)
1 x 106 genome 0.0% 0.0% 0.0%
Coxiella burnetii
copies/mL (0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Cytomegalovirus 1 x 105 U/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Enterovirus Type 68 1 x 105 U/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Enterovirus Type 71 1 x 105 TCID50/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Epstein-Barr Virus 1 x 105 copies/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Escherichia coli O157:H7 1 x 106 CFU/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Haemophilus influenzae 1 x 106 CFU/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Human Coronavirus 229E* 1 x 104 TCID50/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Human Coronavirus NL63* 1 x 104 U/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Human Coronavirus OC43 1 x 105 TCID50/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Human Metapneumovirus 9* 1 x 104 TCID50/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Lactobacillus plantarum,17-5 1 x 106 CFU/mL
(0/3) (0/3) (0/3)
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Tested
Concentration1
0.0% 0.0% 0.0%

Legionella longbeachae 1 x 106 CFU/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Legionella pneumophila 1 x 106 CFU/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Leptospira interrogans 1 x 106 copies/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Measles 1 x 105 TCID50/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
MERS-Coronavirus 1 x 105 TCID50/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Moraxella catarrhalis 1 x 106 CFU/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Mumps 1 x 105 U/mL
(0/3) (0/3) (0/3)
Mycobacterium tuberculosis Genomic 0.0% 0.0% 0.0%
1 x 106 copies/mL
DNA (0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Mycoplasma pneumoniae 1 x 106 CCU/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Neisseria elongata 1 x 106 CFU/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Neisseria meningitidis 1 x 106 CFU/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Parainfluenza Type 1 1 x 105 U/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Parainfluenza Type 2 1 x 105 TCID50/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Parainfluenza Type 3 1 x 105 TCID50/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Parainfluenza Type 4 1 x 105 U/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Parechovirus Type 3 1 x 105 U/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Pseudomonas aeruginosa 1 x 106 CFU/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Rhinovirus 1A* 1 x 104 U/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
RSV-A 1 x 105 TCID50/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
RSV-B 1 x 105 TCID50/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Staphylococcus aureus 1 x 106 CFU/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Staphylococcus epidermidis 1 x 106 CFU/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Streptococcus pneumoniae 1 x 106 CFU/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Streptococcus pyogenes 1 x 106 CFU/mL
(0/3) (0/3) (0/3)
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Page 16 of 20
Tested
Concentration1
0.0% 0.0% 0.0%

Streptococcus salivarius 1 x 106 CFU/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Human Coronavirus RNA HKU1 1 x 105 U/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Human Genomic DNA (Leukocytes) 1 x 106 cells/mL
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
Pooled Human Nasal Wash 1:1 dilution
(0/3) (0/3) (0/3)
0.0% 0.0% 0.0%
SARS-COV1 Synthetic RNA 1 x 105 U/mL
(0/3) (0/3) (0/3)
A lower concentration was tested due to inability to obtain stock material with high titer
*
1
CCU/mL = Color changing units/milliliter, CFU/mL= Colony forming units/milliliter, IFU/mL = Infectious units/milliliter,
U/mL = Units/milliliter, TCID50/mL = Tissue Culture Infectious Dose
INTERFERING SUBSTANCES
Potentially interfering substances from respiratory specimens were tested for ability to generate false
negative results. Samples were prepared by spiking each potentially interfering substance into a baseline
sample consisting of pooled negative nasopharyngeal swab specimens and COVID-19 inactivated viral
particles (2019-nCoV/USA-WA1/2020 strain), Influenza A/Hong Kong/8/68 and Influenza
B/Malaysia/2506/2004. The test samples contained each of the three (3) viruses at a concentration of 3X
LoD. The results are shown in Table 14. None of the substances tested interfere with the detection of
COVID-19, influenza A or influenza B at the concentrations tested. The FluMist nasal vaccine was not
tested as an interfering substance due to its unavailability at the time of this study.
Table 14. Simplexa™ COVID-19 & Flu A/B Direct – Potentially Interfering Substances Results
Potentially Interfering Tested

Active Ingredient COVID-19 Flu A Flu B
Substance Concentration*
Afrin Nasal spray Oxymetazoline 15% (v/v) 100.0% (3/3) 100.0% (3/3) 100.0% (3/3)
Antibacterial, systemic Tobramycin 4 µg/mL 100.0% (3/3) 100.0% (3/3) 100.0% (3/3)
Antibiotic, nasal ointment Mupirocin 6.6 mg/mL 100.0% (3/3) 100.0% (3/3) 100.0% (3/3)
Whole Blood N/A 2% (v/v) 100.0% (3/3) 100.0% (3/3) 100.0% (3/3)
Cold Eeze
(Throat lozenges, Oral N/A 1.25% (w/v) 100.0% (3/3) 100.0% (3/3) 100.0% (3/3)
anesthetic and analgesic)
Nasal corticosteroid
Beclomethasone 5% (v/v) 100.0% (3/3) 100.0% (3/3) 100.0% (3/3)
(Beconase AQ)
Nasal corticosteroid
Fluticasone 5% (v/v) 100.0% (3/3) 100.0% (3/3) 100.0% (3/3)
(Flonase)
Relenza Antiviral Drug Zanamivir 3.3 mg/mL 100.0% (3/3) 100.0% (3/3) 100.0% (3/3)
Tamiflu Antiviral drug Oseltamivir 1 µM 100.0% (3/3) 100.0% (3/3) 100.0% (3/3)
Luffa operculata,
Galphimia glauca,
Zicam Nasal Gel 5% (w/v) 100.0% (3/3) 100.0% (3/3) 100.0% (3/3)
histaminum
hydrochloricum
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Page 17 of 20
Potentially Interfering Tested
Active Ingredient COVID-19 Flu A Flu B
Substance Concentration*
Zicam Nasal Spray
(Homeopathic allergy relief N/A 10% (v/v) 100.0% (3/3) 100.0% (3/3) 100.0% (3/3)
medicine)
Bovine submaxillary gland Purified Mucin
5mg/mL 100.0% (3/3) 100.0% (3/3) 100.0% (3/3)
mucin, type I-S** Protein
µg/mL = Micrograms/milliliter. mg/mL = Milligrams/milliliter, µM = Micromolar, v/v = Volume per Volume, w/v = Weight/Volume,
*
**Influenza strains (Influenza A/Michigan/45/2015 and Influenza B/Phuket/3073/2013)
COMPETITIVE INTERFERENCE
Competitive Interference was performed to assess the ability of the assay to detect low concentration of
one (1) target analyte in the presence of high concentration of another target analyte. Samples were
prepared by spiking one (1) assay target analyte at a low concentration (4X LoD) into negative
nasopharyngeal swab (NPS) matrix in the presence of a high concentration (1000X LoD) of one (1) of the
other two (2) assay target analytes. All the possible assay target combinations were tested. Each contrived
sample was tested in triplicate. The results are shown in Table 15. All of the combinations tested showed
no competitive interference for the detection of low concentrations of COVID-19, Flu A or Flu B in the
presence of high concentrations of another assay target analyte.
Table 15. Simplexa™ COVID-19 & Flu A/B Direct – Competitive Interference Results
Low Positive Baseline Sample Competitive Interferent % Detection

Copies/ Copies/m COVID-
Strain mL Strain L Flu A Flu B 19
Influenza B/
7.5 x 105 100% 100% 0.0%
Influenza A/Hong Phuket/3073/2013
2000
Kong/8/68 COVID-19/USA-
5.0 x 105 100% 0.0% 100%
WA1/2020
Influenza A/Hong
5.0 x 105 100% 100% 0.0%
Influenza B/ Kong/8/68
3000
Phuket/3073/2013 COVID-19/USA-
5.0 x 105 0.0% 100% 100%
WA1/2020
Influenza A/Hong
5.0 x 105 100% 0.0% 100%
COVID-19/USA- Kong/8/68
2000
WA1/2020 Influenza B/
7.5 x 105 0.0% 100% 100%
Phuket/3073/2013
INHIBITION BY OTHER MICROORGANISMS

The Simplexa™ COVID-19 & Flu A/B Direct assay was evaluated by testing the ability to identify SARS-
CoV-2, influenza A virus, and influenza B virus, when other potentially inhibitory organisms were present.
Specimens were prepared by spiking cultured isolates/inactivated organisms/purified nucleic acids (whole
genome) at a minimum of 106 CFU/ml (or higher) for bacteria, and 105 TCID50/mL or PFU/mL (or higher)
for viruses into negative nasopharyngeal swab (NPS) matrix in the presence of a low concentration (2X
LoD) of the three (3) targets (COVID-19, Flu A and Flu B viral particles) and determining microbial inhibition
based on three (3) replicates. For organisms not titered in CFU/mL or TCID50/mL, other industry acceptable
units were used as indicated. RNasin® was added to UTM for specimens containing extracted RNA. The
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March 30, 2020
Page 18 of 20
panel of 47 potentially inhibitory organisms was individually spiked into a pool with a low concentration
influenza A (Influenza A/Hong Kong/8/68), influenza B
(Influenza B/Malaysia/2506/2004), and COVID-19 (2019-nCoV/USA-WA1/2020). No inhibition by other

organisms was observed for COVID-19, influenza A or influenza B at the concentrations indicated in Table
16.
Table 16. Simplexa™ COVID-19 & Flu A/B Direct – Microbial Inhibition Results
Tested % Detection
Organism
Concentration1 COVID-19 Flu A Flu B
1 x 105
Adenovirus Type 1 100% 100% 100%
TCID50/mL
1 x 105
Adenovirus Type 7A 100% 100% 100%
TCID50/mL
Bordetella pertussis 1 x 106 CFU/mL 100% 100% 100%
Candida albicans 1 x 106 CFU/mL 100% 100% 100%
Chlamydia pneumoniae 1 x 106 IFU/mL 100% 100% 100%
Corynebacterium diphtheriae 1 x 106 CFU/mL 100% 100% 100%

1x 106
Coxiella burnetii genome 100% 100% 100%
copies/mL
Cytomegalovirus 1 x 105 U/mL 100% 100% 100%
Enterovirus Type 68 1 x 105 U/mL 100% 100% 100%

1x 105
Enterovirus Type 71 100% 100% 100%
TCID50/mL
1 x 105
Epstein-Barr Virus 100% 100% 100%
copies/mL
Escherichia coli O157:H7 1 x 106 CFU/mL 100% 95% 100%
Haemophilus influenzae 1 x 106 CFU/mL 100% 100% 100%

1x 104
Human Coronavirus 229E* 100% 100% 100%
TCID50/mL
Human Coronavirus NL63* 1 x 104 U/mL 100% 100% 100%
1x 105
Human Coronavirus OC43 100% 100% 100%
TCID50/mL
1 x 104
Human Metapneumovirus 9* 100% 100% 100%
TCID50/mL
Lactobacillus plantarum,17-5 1 x 106 CFU/mL 100% 100% 100%
Legionella longbeachae 1 x 106 CFU/mL 100% 100% 100%
Legionella pneumophila 1 x 106 CFU/mL 100% 100% 100%

1x 106
Leptospira interrogans 100% 100% 100%
copies/mL
1 x 105
Measles 100% 100% 100%
TCID50/mL
1 x 105
MERS-Coronavirus 100% 100% 100%
TCID50/mL
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March 30, 2020
Page 19 of 20
Tested % Detection
Organism
Concentration1 COVID-19 Flu A Flu B
Moraxella catarrhalis 1 x 106 CFU/mL 100% 100% 100%
Mumps 1 x 105 U/mL 100% 100% 100%

Mycobacterium tuberculosis Genomic 1x 106
100% 100% 100%
DNA copies/mL
Mycoplasma pneumoniae 1 x 106 CCU/mL 100% 100% 100%
Neisseria elongata 1 x 106 CFU/mL 100% 100% 100%
Neisseria meningitidis 1 x 106 CFU/mL 100% 100% 100%
Parainfluenza Type 1 1 x 105 U/mL 100% 100% 100%

1 x 105
Parainfluenza Type 2 100% 100% 100%
TCID50/mL
1 x 105
Parainfluenza Type 3 100% 100% 100%
TCID50/mL
Parainfluenza Type 4 1 x 105 U/mL 100% 100% 100%
Parechovirus Type 3 1 x 105 U/mL 100% 100% 100%
Pseudomonas aeruginosa 1 x 106 CFU/mL 100% 100% 100%
Rhinovirus 1A* 1 x 104 U/mL 95% 100% 100%

1x 105
RSV-A 100% 100% 100%
TCID50/mL
1 x 105
RSV-B 100% 100% 100%
TCID50/mL
Staphylococcus aureus 1 x 106 CFU/mL 100% 100% 100%
Staphylococcus epidermidis 1 x 106 CFU/mL 100% 100% 100%
Streptococcus pneumoniae 1 x 106 CFU/mL 100% 100% 100%
Streptococcus pyogenes 1 x 106 CFU/mL 100% 100% 100%
Streptococcus salivarius 1 x 106 CFU/mL 100% 100% 100%
Human Coronavirus RNA HKU1 1 x 105 U/mL 100% 100% 100%
Human Genomic DNA (Leukocytes) 1 x 106 cells/mL 100% 100% 100%
Pooled Human Nasal Wash 1:1 dilution 100% 100% 100%
SARS-COV1 Synthetic RNA 1 x 105 U/mL 100% 100% 100%

*A lower concentration was tested due to inability to obtain stock material with high titer
1
CCU/mL = Color Changing Units/milliliter, CFU/mL = Colony Forming Units/milliliter, IFU/mL = Infectious units/milliliter, U/mL =
Units/milliliter, TCID50/mL = Tissue Culture Infectious Dose/milliliter
CARRY-OVER CONTAMINATION
Amplification carry-over for the Simplexa™ assays has been assessed against existing assays that use the
same sample matrices, workflow and specimen type, and therefore no carry-over is anticipated. The study
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March 30, 2020
Page 20 of 20
was designed by alternately placing high positive and negative samples on each disc. No evidence of
carry-over contamination was observed.
CONCLUSION
The analytical and method comparison studies have demonstrated that the Simplexa™ COVID-19 & Flu
A/B Direct is substantially Equivalent to the predicate devices (DEN200031 and K201505). The device
labeling is compliant with 21 CFR § 809.10.
Page 44 of 250
6. Truth and Accuracy Statement

PREMARKET NOTIFICATION
TRUTHFUL AND ACCURATE STATEMENT
[As required by 21 CFR 807.87(k)]
I certify that, in my capacity as Manager, Regulatory Affairs, DiaSorin Molecular LLC., I believe to the
best of my knowledge, that all data and information submitted in the premarket notification are truthful
and accurate and that no material fact has been omitted.
Signature
Sharon Young
Name
3-30-2022
Date
Page 46 of 250
7. Class III Summary Certification

Not Applicable. This Device is not a Class III device.
Page 47 of 250
8. Financial Certification or Disclosure Statement (Form 3454)

Form Approved: 0MB No. 0910-0396
DEPARTMENT OF HEALTH AND HUMAN SERVICES
Food and Drug Administration Expiration Date: April 30, 2022
CERTIFICATION: FINANCIAL INTERESTS AND

ARRANGEMENTS OF CLINICAL INVESTIGATORS
TO BE COMPLETED BYAPPLICANT
With respect to all covered clinical studies (or specific clinical studies listed below (if appropriate)) submitted in
support of this application, I certify to one of the statements below as appropriate. I understand that this
certification is made in compliance with 21 CFR part 54 and that for the purposes of this statement, a clinical
investigator includes the spouse and each dependent child of the investigator as defined in 21 CFR 54.2(d).
I Please mark the applicable check box. I

� (1) As the sponsor of the submitted studies, I certify that I have not entered into any financial arrangement
with the listed clinical investigators (enter names of clinical investigators below or attach list of names to
this form) whereby the value of compensation to the investigator could be affected by the outcome of the
study as defined in 21 CFR 54.2(a). I also certify that each listed clinical investigator required to disclose
to the sponsor whether the investigator had a proprietary interest in this product or a significant equity in
the sponsor as defined in 21 CFR 54.2(b) did not disclose any such interests. I further certify that no
listed investigator was the recipient of significant payments of other sorts as defined in 21 CFR 54.2(f).
� Morgan A. Pence, Ph.D., Cooks Children's Hospital Janet Farhang, Ph.D. ,Luminex Corp.
·� Stephen Young, Ph.D., Tricore Louis Geller, MS. DiaSorin Molcular

"'
·s Omai Garner, Ph.D. UCLA NA
□ (2) applicant,
As the applicant who is submitting a study or studies sponsored by a firm or party other than the
I certify that based on information obtained from the sponsor or from participating clinical
investigators, the listed clinical investigators (attach list of names to this form) did not participate in any
financial arrangement with the sponsor of a covered study whereby the value of compensation to the
investigator for conducting the study could be affected by the outcome of the study (as defined in 21
CFR 54.2(a)); had no proprietary interest in this product or significant equity interest in the sponsor of
the covered study (as defined in 21 CFR 54.2(b)); and was not the recipient of significant payments of
other sorts (as defined in 21 CFR 54.2(f)).
□ (3) applicant,
As the applicant who is submitting a study or studies sponsored by a firm or party other than the
I certify that I have acted with due diligence to obtain from the listed clinical investigators
(attach list of names) or from the sponsor the information required under 54.4 and it was not possible to
do so. The reason why this information could not be obtained is attached.
NAME TITLE
Ryan Snellings Vice President and General Manager
FIRM/ORGANIZATION
DiaSorin Molecular LLC.
SIGNATURE DATE (mmldd/yyyy)
03/30/2022
This section applies only to the requirements of the Paperwork Reduction Act of 1995. Do NOT send your completed form to
An agency may not conduct or sponsor, and a person is not required to respond to, a collection of the PRA Staff email address below.
information unless it displays a currently valid 0MB control number. Public reporting burden for this Department of Health and Human Services
collection of information is estimated to average I hour per response, including time for reviewing Food and Drug Administration
instructions, searching existing data sources, gathering and maintaining the necessary data, and Office of Operations
completing and reviewing the collection of information. Send comments regarding this burden estimate PRAStaff@fda.hhs.gov
or any other aspect of this collection of information to the address to the right:
"An agency may not conduct or sponsor, and a person is not required to respond to, a collection of information unless it displays a currently valid 0MB number."
FORM FDA 3454 (4/21) PSCPublishing ,Sen.ices (301) 443-6740 EF
Page 49 of 250
9. Certification of Compliance (Form 3674)

CERTIFICATION STATEMENT/ INFORMATION (Continued)
10. If you checked box C, in number 9, provide the National Clinical Trial (NCT) Number(s) for any "applicable clinical trial(s)," for which you (the
sponsor/applicanUsubmitter) are the "responsible party'' under 42 U.S.C. § 2820)(1)(a)(i), section 4020)(1)(a)(i) of the Public Health Service
Act referenced in the application/ submission which this Certification accompanies. (Add continuation page as necessary.)
NCT Number(s):
I Continuation Page for#10 I

The undersigned declares, to the best of her/his knowledge, that this is an accurate, true, and complete submission of information.
I understand that the failure to submit the certification required by 42 U.S.C. § 282U)(5)(B), section 402U)(5)(B) of the Public Health
Service Act, and the knowing submission of a false certification under such section are prohibited acts under 21 U.S.C. § 331, section
301 of the Federal Food, Drug, and Cosmetic Act.
Warning: A willfully and knowingly false statement is a criminal offense, U.S. Code, title 18, section 1001.
11. Name and Title of the Person who Signs Number 15

Name Title
Ryan Snellings Vice President and General Manager
12. Address 13. Telephone and Fax Numbers
Address 1 (Street address, P.O. box, company name clo) (Include country code if applicable and
11331 ValleyViewStreet area code)
I
Address 2 (Apartment, suite, unit, building, floor, etc.) (Tel): (562) 240-6500
City State/Province/Region (Fax):
I
Cypress CA
Country ZIP or Postal Code
U.S.A. 90630
14. Date of Certification 15. Signature of Sponsor/ApplicanUSubmitter or an Authorized
03/30/2022
Representative (Sign)
I Sign
I
This section applies only to requirements of the Paperwork Reduction Act of 1995.
···Do NOT SEND YOUR COMPLETED FORM TO THE PRA STAFF EMAIL ADDRESS BELOW....
The burden time for this collection of information is estimated to average 15 minutes and 45 minutes (depending on the type of application/
submission) per response, including the time to review instructions, search existing data sources, gather and maintain the data needed and
complete and review the collection of information. Send comments regarding this burden estimate or any other aspect of this information collection,
including suggestions for reducing this burden to:
Department of Health and Human Services "An agency may not conduct or sponsor, and a person is not
Food and Drug Administration required to respond to, a collection of information unless it
Office of Chief Information Officer displays a currently valid 0MB number."
Paperwork Reduction Act (PRA) Staff
PRAStaff@fda.hhs.gov
FORM FDA 3674 (11/21) Page 2 of 4

Page 54 of 250
10. Executive Summary

10.1 Executive Summary
Applicant DiaSorin Molecular LLC.

11331 Valley View Street
Cypress, California 90630 USA
FDA 2023365
Establishment
Registration No.
Primary Contact Sharon Young, Manager, Regulatory Affairs
Tel 562-240-6680 Fax 562-240-6530 Sharon.Young@DiaSorin.com
Alternate Contact Tara Viviani, Senior Director, Molecular Regulatory Affairs
Tel 562-240-6271 Fax 562-240-6530 Tara.Viviani@DiaSorin.com
Proprietary Name Simplexa™ COVID-19 & Flu A/B Direct
Simplexa™ COVID-19 & Flu A/B Positive Control Pack
Generic Name COVID-19 & Flu A/B Real-time polymerase chain reaction (real-time RT PCR)
Product Code QOF – Multi-Target Respiratory Specimen Nucleic Acid Test Including Sars-Cov-2 And
Other Microbial Agents
OOI - Real Time Nucleic Acid Amplification System
US Regulation 21 CFR § 866.3981

Number
21 CFR § 862.2570
Classification Class II (Special Controls)
CLIA Complexity Moderate
Analytes SARS CoV-2, Influenza A and Influenza B viruses

Page 55 of 250
Specimen Types Nasopharyngeal Swabs (NPS)
Special LIAISON® MDX Instrument with LIAISON® MDX Studio Software

Instrument
Requirements
Predicate BioFire Respiratory Panel 2.1 (RP2.1) (DEN200031) and Simplexa™ Flu A/B & RSV Direct
Devices Gen II (K201505)
Background and
The Simplexa™ COVID-19 & Flu A/B Direct assay system is a real-time RT-PCR system
Device
that enables the direct amplification, detection and differentiation of SARS-CoV-2 RNA,
Description
human influenza A (Flu A) virus RNA and human influenza B (Flu B) virus RNA from
unprocessed nasopharyngeal swabs (NPS) that have not undergone nucleic acid
extraction. The system consists of the Simplexa™ COVID-19 & Flu A/B Direct assay, the
LIAISON® MDX (with LIAISON® MDX Studio Software), the Direct Amplification Disc and
associated accessories.
In the Simplexa™ COVID-19 & Flu A/B Direct assay, fluorescent probes are used together
with corresponding forward and reverse primers to amplify SARS-CoV-2, Flu A, Flu B and
internal control RNA targets. For COVID-19 detection, the assay targets two different
regions specific to the SARS-CoV-2 genome; the S gene which encodes the spike
glycoprotein and the ORF1ab region which encodes well-conserved non-structural proteins
and therefore is less susceptible to recombination. For Flu detection the assay targets
conserved regions of influenza A viruses (matrix gene) and influenza B viruses (matrix
gene). The assay provides three results; COVID-19 (ORF1ab and/or S gene detection),
influenza A viruses (matrix gene detection) and influenza B viruses (matrix gene detection).
An RNA internal control is used to detect RT-PCR failure and/or inhibition.
Diagnostic Need SARS-CoV-2 (also called COVID-19 virus) is a beta coronavirus belonging to the family of
coronaviruses, named for the crown-like spikes on their surface. There are four main sub-
groupings of coronaviruses, known as alpha, beta, gamma, and delta. Common human
coronaviruses include 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta
coronavirus) and HKU1 (beta coronavirus), and these usually cause mild to moderate
upper-respiratory tract illnesses, like the common cold.1,2,3 Other human coronaviruses
such as MERS-CoV (the beta coronavirus that causes Middle East Respiratory Syndrome,
or MERS) and SARS-CoV (the beta coronavirus that causes severe acute respiratory
syndrome, or SARS) have caused more severe respiratory illness with higher rates of
morbidity and mortality. The SARS-CoV-2 is a novel coronavirus that causes coronavirus
disease 2019, or COVID-19. SARS-CoV-2 caused an outbreak beginning in December
2019 in Wuhan City, Hubei Province, China and has spread globally, being consequently
declared a pandemic by the World Health Organization (WHO).2,4 Patients with COVID-19
Page 56 of 250
have had mild to severe respiratory illness with symptoms of fever, cough and shortness of
breath, and many patients have had complications including pneumonia in both lungs.5
Influenza is caused by three immunologic types (A, B, and C) of RNA viruses within the
Orthomyxoviridae family. Influenza A is classified further by describing two viral proteins
expressed on its surface, hemagglutinin and neuraminidase. Hemagglutinin facilitates
binding of the virus to respiratory epithelial cells, whereas neuraminidase functions to break
those bonds with the host cell so that new virions can be released. Seasonal influenza is
typically caused by viruses that contain one of three major subtypes of hemagglutinin (H1,
H2, or H3) and one of two subtypes of neuraminidase (N1 or N2). Influenza B is not
classified into subtypes.6
Influenza classically presents with a combination of upper and lower respiratory signs and
symptoms, fever, headache, myalgia, and general malaise. Illness can take on a variety of
appearances, ranging from isolated respiratory findings that resemble the common cold, to
severe pneumonia requiring hospitalization. Persons at higher risk for hospitalization from
seasonal influenza include children <2 years of age, adults >65 years of age, and those
with significant comorbidities. Influenza may cause exacerbation of underlying medical
conditions, such as asthma or congestive heart failure. The duration of illness is typically 2-
5 days, but symptoms may last for a week or longer.7, 8
REFERENCES
1. Cui J, Li F, Shi ZL. Nat Rev Microbiol. 2019 Mar;17(3):181-192. doi:

10.1038/s41579-018-0118-9.
2. World Health Organization. Coronavirus. https://www.who.int/health-
topics/coronavirus
3. Centers for Disease Control and Prevention. Coronavirus.
https://www.cdc.gov/coronavirus/general-information.html
4. Cheng, Z.J., Shan, J. 2019 Novel coronavirus: where we are and what we know.
Infection (2020). https://doi.org/10.1007/s15010-020-01401-y
5. Centers for Disease Control and Prevention. Coronavirus Disease 2019 (COVID-
19). https://www.cdc.gov/coronavirus/2019-ncov/about/index.html
6. http://www.cdc.gov/flu/about/viruses/types.html
7. http://www.cdc.gov/flu/weekly/pastreports.htm
8. http://www.health.gov.au/internet/main/publishing.nsf/Content/cda-ozflu-no44-
10.htm
Page 57 of 250
Intended Use Simplexa COVID-19 & Flu A/B Direct (Catalog Number: MOL4250):
The DiaSorin Molecular Simplexa™ COVID-19 & Flu A/B Direct real-time RT-PCR assay
is intended for use on the LIAISON® MDX instrument for the in vitro qualitative detection
and differentiation of nucleic acid from severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2), influenza A virus and influenza B virus in nasopharyngeal swabs (NPS)
from human patients with signs and symptoms of respiratory tract infection in conjunction
with clinical and epidemiological risk factors.
The Simplexa™ COVID-19 & Flu A/B Direct assay is intended for use as an aid in the
differential diagnosis of SARS-CoV-2, influenza A and influenza B infection.
Negative results do not preclude SARS-CoV-2, influenza A or influenza B infection and

should not be used as the sole basis for patient management decisions. Negative results
should be combined with clinical observations, patient history, and epidemiological
information.
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.
The Simplexa™ COVID-19 & Flu A/B Direct assay is intended for use by qualified and
trained clinical laboratory personnel specifically instructed and trained in the techniques of
real-time PCR and in vitro diagnostic procedures.
Simplexa COVID-19 & Flu A/B Positive Control Pack (Catalog Number: MOL4260):
The Simplexa™ COVID-19 & Flu A/B Positive Control Pack is intended to be used as a
control with the Simplexa™ COVID-19 & Flu A/B Direct kit for use on the LIAISON® MDX
instrument. This control is not intended for use with other assays or systems.
Indications The Simplexa™ COVID-19 & Flu A/B Direct assay is intended for use as an aid in the
differential diagnosis of SARS-CoV-2, influenza A and influenza B infection from virus in
nasopharyngeal swabs (NPS) from human patients with signs and symptoms of respiratory
tract infection in conjunction with clinical and epidemiological risk factors.
Summary of • Reproducibility: The reproducibility study was performed by one (1) internal site
Studies and two (2) external sites. The panel consisted of eight (8) reproducibility panel
Performed and members, including six (6) contrived samples, one (1) negative sample [UTM as
Results No Template Control (NTC)] and one (1) positive sample (PC used “as is”). The
Page 58 of 250
contrived panel members were prepared by spiking each analyte at approximately

two times (2x) the Limit of Detection (LoD, low positive) and approximately four
times (4x) LoD (medium positive) into native negative nasopharyngeal swab matrix
in UTM. Each panel member was tested in triplicate for five (5) days. Each site had
at least two (2) operators who each assayed the entire panel once per day, for a
total of two (2) sets of data per day. The results demonstrated the reproducibility of
the Candidate Device, and the percent coefficient of variation (% CV) ranged
between 0.8 - 4.2%.
• Limit of Detection: The Limit of Detection (LoD) of the Candidate Device in

nasopharyngeal swabs (NPS) was determined to be the lowest detectable
concentration of quantitated inactivated tittered viral stocks (copies/mL or IU/mL)
at which ≥ 95% of all replicates were detected. Two (2) strains of influenza A, two
(2) strains of influenza B and two (2) strains SARS-CoV-2 serially diluted in
negative nasopharyngeal swab (NPS) matrix were used to determine the LoD.
Candidate Device Limit of Detection

Virus Strain LoD (copies/mL)
Influenza A/Hong Kong/8/68 500
Influenza A/Michigan/45/2015 500
Influenza B/Phuket/3073/2013 750
Influenza B/Malaysia/2506/2004 250
SARS-CoV-2 (USA-WA1/2020) 500
Virus Strain IU/mL
WHO International Standard for

SARS-CoV-2 RNA (NIBSC code:
651
20/146) England/02/2020 isolate of
SARS-CoV-2
• Analytical Reactivity: The Candidate Device was evaluated for analytical reactivity
with additional influenza A, influenza B and SARS-CoV-2 strains that were not
tested during the LOD study. Additional 63 Influenza A (Flu A) strains, 21 Influenza
B (Flu B) strains and five (5) strains of COVID-19 were tested. The study was
performed at DiaSorin Molecular, Cypress, CA for Influenza A and Influenza B
strains using eight (8) LIAISON MDX Instruments one (1) lot of Candidate Device
Reaction Mix and two (2) lots of Candidate Device Positive Control. TAnother study
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was performed at DiaSorin Molecular, Gerenzano, IT for COVID-19 strains using

three (3) LIAISON® MDX Instruments, one (1) lot of Candidate Device Reaction
Mix and one (1) lot of Candidate Device Positive Control.The Analytical Reactivity
study demonstrated the Candidate Device can detect an additional 63 Flu A strains,
21 Flu B strains and five (5) COVID-19 strains. All strains had 100% detection at
the initial concentration that was tested or at a higher concentration.
• Analytical Inclusivity: An in-silico inclusivity analysis of the COVID-19 target

primers and probes in the Simplexa™ COVID-19 & Flu A/B Direct assay
was performed. All primer and probe sets designed for detection of the
ORF1ab and S gene were tested against the complete SARS-CoV-2
genome sequences available in the GISAID database submitted from
November 01, 2021 to January 31, 2022. The analysis included 2,170,584
sequences in the amplicon regions of the ORF1ab and S gene primer/probe
regions. Only target sequences with full coverage of all three ORF1ab and
S gene forward and reverse primer and probe regions were included in the
analyses. The results showed that there were 2,170,382 sequences
(~99.99%) with no mismatches in at least one gene oligo set and therefore
are predicted to be detected by the assay based on sequence homology.
There were 202 sequences (~0.01%) with mismatches in at least one
primer or probe binding region in either ORF1ab or S gene. A melting
temperature (Tm) analysis was conducted for those sequences with
mismatches in the binding sites of both gene assay target regions. The Tm
calculation was performed with assay-specific conditions using a Tm
Mismatch Bioinformatics Tool. The analysis showed that there was at least
one oligo gene set where primers and probes exhibited Tm values above
their respective annealing temperature, and the mismatches were not
located at the 3’ end for the primers; as such, detection of these sequences
are not affected by the mismatches. Table 12 below summarizes the Tm
analysis results. Candidate Device In-silico Inclusivity
No. seq. where at least one gene oligo set meets Tm criteria
2,170,584
• Cross-Reactivity: Cross-reactivity of the Candidate Device was evaluated by

testing whole organisms or purified nucleic acid from other organisms. Specimens
for laboratory testing were prepared by spiking cultured isolates/inactivated
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organisms/purified nucleic acids (whole genome) into negative matrix (NPS) and
determining cross-reactivity based on three replicates. RNasin® was added to NPS
for specimens containing extracted RNA. The samples were assayed in triplicate.
Cross-reactivity was not observed with the Candidate Device and the 47 organisms
tested.
• Competitive Interference: Competitive Interference was performed to assess the

ability of the assay to detect low concentration of one (1) target analyte in the
presence of high concentration of another target analyte. Samples were prepared
by spiking one (1) assay target analyte at a low concentration (4X LoD) into
negative nasopharyngeal swab (NPS) matrix in the presence of a high
concentration (1000X LoD) of one (1) of the other two (2) assay target analytes. All
the possible assay target combinations were tested. Each contrived sample was
tested in triplicate. All of the combinations tested showed no competitive
interference for the detection of low concentrations of COVID-19, Flu A or Flu B in
the presence of high concentrations of another assay target analyte.
• Microbial Inhibition: The Candidate Device was evaluated for microbial inhibition
by testing 44 different viruses, bacteria and fungi that could be found in
nasopharyngeal swab specimens. All samples were prepared by spiking each
potentially inhibiting organism into a baseline consisting of Flu A, Flu B and COVID-
19 viral particles at 2x LoD, in pooled negative NPS matrix. RNAsin was added at
a concentration of 1.6 U/µL for samples that had viral RNA. For potentially inhibiting
bacteria or fungi, the testing concentration was 1x106 CFU/mL; for viruses, 1x105
TCID50/mL. For organisms that were not able to be acquired at these
concentrations they were tested via in silico (BLAST) analysis. For organisms not
titered in CFU/mL or TCID50/mL, other industry acceptable units were used.
For Rhinovirus 1A at a concentration of 1x104 U/mL, there was 100% positivity for
Flu B and COVID-19 targets but there was one replicate out of three (1/3) not
detected for Flu A. Upon testing an additional 17 replicates, the number of
replicates detected for Flu A was 19 out of 20 replicates total. Therefore, there was
no microbial inhibition with the 47 organisms tested with the Candidate Device.
• Interference: The Candidate Device was evaluated for the effects of 13 potentially
interfering endogenous and exogenous substances that may be present in
nasopharyngeal swabs. All samples were prepared by spiking each potentially
interfering substance into a baseline sample consisting of pooled negative
nasopharyngeal swab specimens and COVID-19 inactivated viral particles and
influenza A and B spiked with 3x LoD. Each interferent was spiked into the 3x LoD
baseline sample.
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None of the substances tested caused interference with the detection of Influenza
A, Influenza B or COVID-19 at 3x LoD, in the Simplexa™ COVID-19 & Flu A/B
Direct (MOL4250) assay, with the exception of Cold Eeze Throat Lozenges at a
concentration of 2.5% (w/v). At a concentration of 1.25% (w/v) Cold Eeze Throat
Lozenges was not found to cause interference. All Controls performed as
expected.
• Inter-Lot Precision Reaction Mix: The Candidate Device was evaluated for
potential lot-to-lot variability. The study was performed at DiaSorin Molecular,
Cypress, CA using four (4) LIAISON MDX Instruments, three (3) lots of Candidate
Device kit, and one (1) lot of Candidate Device Positive Control (PC) run as a
sample. Two (2) lots of Candidate Device Positive Control (PC) were run as daily
controls. A total of 144 evaluable replicates for each sample panel member were
performed by four (4) operators over the course of 13 non-consecutive days (22
and 30 June and 01, 07, 28, 29 and 30 July and 03, 04, 05, 06, 10, 11 August,
2021).
The Candidate Device Reaction Mix overall %CV for all three (3) lots was <4% for
Flu A; <3%, for Flu B and COVID-19; and <4% for the Internal Control (IC). The
Candidate Device Reaction Mix demonstrated a robust precision across the three
(3) lots of Reaction Mix and performed as intended. All of the controls performed
as expected.
• Inter-lot Precision Positive Control: The Candidate Device Positive Control was
evaluated for potential lot-to-lot variability (precision). A total of three (3) Candidate
Device Positive Control (PC) lots were used in a total of six (6) runs were performed
by three (3) operators across twelve non-consecutive days (30 June, 01, 02, 06,
07, 22, 23, 26, 27, 28, 29 and 30 July 2021).
Forty-eight (48) replicates were tested from each of the three (3) lots of the
Simplexa™ COVID-19 & Flu A/B Direct Positive Control over the course of twelve
(12) days. All targets Flu A (FAM), Flu B, (JOE), COVID-19 (CFR610) and Internal
Control (Q670) were detected in each of the 144 total replicates.
The Candidate Device Positive Control overall %CV for all three (3) lots of the
Simplexa™ COVID-19 & Flu A/B Direct Positive Control was <3% for Flu A and
<4% for Flu B Ct and <5% for COVID-19. For the internal control Ct, the %CV was
<3% The Candidate Device Positive Control demonstrated a robust precision
across the three (3) lots of Positive Control and performed as intended.
• Carry-over Contamination/Disc Reusability: Swab samples have previously

been successfully validated on the DAD/LIAISON MDX in K120413. In the study,
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high positive samples were placed next to negative samples in an alternating

pattern. No carry-over contamination was observed. For DAD reusability each
wedge was set up using a high positive sample and alternating with setting up a
negative sample. The wedges were run one at a time until all eight wedges were
used. The study demonstrated the DAD disc could be successfully used up to eight
(8) times without evidence of false positivity or carry-over with the swab sample
type.
• Fresh/Frozen and Media Equivalency Sample Stability: Fresh vs. Frozen and
Media Equivalency stability studies were conducted. The samples were stored at
varying temperatures and durations and tested on the Candidate Device. None of
the conditions or media types tested negatively impacted the performance of the
samples with the Candidate Device.
• Clinical Performance: The performance of Simplexa™ COVID-19 & Flu A/B Direct
was evaluated using prospective, retrospective, pre-selected positive and negative
nasopharyngeal swab (NPS) specimens from human patients with signs and
symptoms of respiratory tract infection. The prospective samples were collected
from four (4) external sites, while retrospective samples were collected from five (5)
external sites. Collection sites included five (5) reference laboratories and four (4)
biorepository banks, across four (4) different geographical locations. Testing was
performed from October 12th 2021 to February 18th 2022. The comparator for
influenza A and B targets was a FDA cleared RT-PCR assay. For discordant
samples a second FDA cleared assay and/or bidirectional sequencing assay was
used. For the COVID-19 target a composite reference method was utilized.
Samples were tested using two (2) FDA Emergency Use Authorized (EUA) SARS-
CoV-2 RT-PCR Assays. Discordant results were tested by a third SARS-CoV-2
EUA RT-PCR assay and a two out of three rule was applied to determine if a
sample was positive or negative.
The following tables show the results of the Candidate Device and the comparator
assay results for their respective targets in the prospective and retrospective (pre-
selected) study analysis. The positive percent agreement (PPA) and negative
percent agreement (NPA) is based on a total of 759 enrolled specimens.
Page 63 of 250
Candidate Device vs FDA cleared NAAT- Flu A
Positive Percent Negative Percent

Agreement Agreement
Candidate Device Target
TP / PPA 95% TN / NPA 95%
(TP+FN) (%) CI (TN+FP) (%) CI
0% - 99% -
Fresh 0/2 0% 323/323 100%
66% 100%
0% - 98% -
Prospective Frozen 0/1 0% 175/175 100%
79% 100%
Influenza A 0% - 99% -
Combined 0/3 0% 498/498 100%
56% 100%
92% - 98% -
Pre-Selected 80/82 97.6% 176/176 100%
99% 100%
87% - 99% -
Overall 80/85a 94.1% 674/674 100%
97% 100%
a
Four of the five Influenza A False Negatives were tested with Biofire RP2.1 and three of these were negative
for Influenza A.
Page 64 of 250
Candidate Device vs FDA cleared NAAT- Flu B
Negative Percent
Positive Percent Agreement
Agreement
0% - 99% -
Fresh 0/3 0% 322/322 100%
56% 100%
0% - 98% -
66% 100%
0% - 99% -
Influenza B Combined 0/5 0% 496/496 100%
43% 100%
94% - 97% -
Pre-Selected 112/114 98.2% 144/144 100%
100% 100%
88% -
Overall 112/119b 94.1% 640/640 100%
97% 99% -
100%
b
Six of the seven Influenza B False Negatives were tested with Biofire RP2.1 and five of these were negative
for Influenza B.
Page 65 of 250
Candidate Device vs Composite Reference Method (CRM) – SARS-CoV-2
Positive Percent Negative Percent

Agreement Agreement
97%
86% - -
Fresh 24/24 100% 298/301 99%
100% 100
%
88%
Prospective 88% -
Frozen 44/45 97.8% 123/131 93.9% -
100%
97%
SARS-CoV-2 95%
92% -
Combined 68/69 98.6% 421/432 97.5% -
100%
99%
98%
Pre-Selected 0/0 N/A N/A 252/252 100% -
100%
97%
92% -
Overall 68/69c 98.6% 673/684d 98.4% -
100%
99%
c
The one SARS-CoV-2 False Negative was positive by Biofire RP2.1.
d
Nine of the eleven SARS-CoV-2 False Positives were positive by BioFire RP 2.1.
PPA = Positive Percent Agreement, NPA = Negative Percent Agreement, CI = Confidence Interval, TP = True
Positive, FN = False Negative, TN = True Negative
• Kit Stability: Ongoing.
10.2 Pre-Submission Q202674
DiaSorin Molecular’s pre-submission Q202674 was not reviewed by the FDA due to FDA
resource limitations. DiaSorin Molecular received notification of this decision February 23, 2021.
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11. Device Description

11.1 Description
.....................................................................................................................................................................................................................
The Candidate Device assay system is a real-time RT-PCR system that enables the direct
amplification, detection and differentiation of SARS-CoV-2 RNA, human influenza A (Flu A) virus
RNA and human influenza B (Flu B) virus RNA from unprocessed nasopharyngeal swabs (NPS)
that have not undergone nucleic acid extraction. The system consists of the Candidate Device
assay, the LIAISON® MDX (with LIAISON® MDX Studio Software), the Direct Amplification Disc
and associated accessories.
In the Candidate Device assay, fluorescent probes are used together with corresponding forward
and reverse primers to amplify SARS-CoV-2, Flu A, Flu B and internal control RNA targets. For
COVID-19 detection the assay targets two different regions of the SARS-CoV-2 genome; the S
gene encodes the spike glycoprotein of the SARS-CoV-2 (COVID-19 virus) and is also targeted
to specifically detect the presence of SARS-CoV-2. The ORF1ab region encodes well-conserved
non-structural proteins and therefore is less susceptible to recombination. For Flu detection the
assay targets conserved regions of influenza A viruses (matrix gene) and influenza B viruses
(matrix gene). The assay provides three results; COVID-19 (ORF1a/b and/or S gene detection),
influenza A viruses (matrix gene) and influenza B viruses (matrix gene) are targeted to identify
these viruses in the specimen. An RNA internal control is used to detect RT-PCR failure and/or
inhibition
The LIAISON MDX system was previously cleared under K102314 as the 3M Integrated Cycler.
Specific functions of the software and hardware, as they apply to the DAD, are as follows. The
LIAISON MDX instrument is a real-time Polymerase Chain Reaction (PCR) thermocycler used
for the identification of nucleic acid from biological specimens. The DAD can process up to eight
(8) specimens or controls. The LIAISON MDX instrument uses real-time fluorescence detection
to identify targets within the sample wells. The LIAISON MDX instrument is controlled by an
external computer (laptop) running the LIAISON MDX Studio Software.
The DAD consumable is compartmentalized into eight (8) separate wedges and up to eight (8)
separate specimens or controls may be processed on each disc. The DAD may be reused until
all wedges have been utilized. Each wedge contains sample and reagent input wells, microfluidic
channels and laser activated valves to control the fluid flow, and a reaction chamber. This disc is
specifically designed to meter the amount of reagent (Reaction Mix) and sample that are placed
into specific input wells in the disc. To start processing a patient sample, a foil seal is lifted and
the user adds 50 µL of Reaction Mix to the reagent input well (R) using a fixed volume pipette
(Figure 11.1-1). Next, the user adds 50 µL of unextracted specimen to the sample input well
(SAMPLE).
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Figure 11.1-1: Direct Amplification Disc (input wells)
After loading the reagent and sample(s), the wells of the individual wedge are resealed with the
original top foil and the tab is removed at the perforation. Centrifugal force moves the fluid into
the metering chamber. The reagent chamber is specifically designed to measure 40 µL of reagent
and the sample chamber is specifically designed to measure 10 µL of sample. Excess reagent
and sample are forced into the waste chambers by centrifugal force (Figure 11.1-2).
Figure 11.1-2: Direct Amplification Disc (from back with

under side foil and PET film removed)
Page 68 of 250
At a specific point in the assay protocol, the software triggers the laser to open the sample laser
valve. This allows the fluid to pass from the sample metering well into the reaction chamber.
The system verifies that a sufficient volume of specimen has been delivered to the detection
chamber. The LIAISON MDX Studio software will perform a check to detect absence or presence
of the sample. This check is done before the Reaction Mix is moved into the detection chamber.
The LIAISON MDX Studio software will use acceptance criteria to determine whether those
criteria are met. Once this check is performed, at a specific point in the assay protocol the
software triggers the laser to open the Reaction Mix laser valve. This allows the reagent fluid to
pass from the Reaction Mix metering well into the reaction chamber.
After the centrifugal force has mixed the sample and reagent within the reaction chamber and
PCR amplification cycles begin. A sample is considered positive for a particular target if intensity
of the optical reading crosses a particular threshold before a predetermined cut-off cycle.
11.2 Procedure
Table 11.2-1. Procedure (condensed version)
1. Program assay definition by scanning barcode into LIAISON MDX Studio software (upon
initial use or if instructed to do so with a new lot of reagent).
2. Scan the barcode on the Reaction Mix Vial or on the barcode label containing lot specific
information.
3. Scan or enter your sample ID.
4. Lift the foil cover over the desired wedge of the DAD.
5. Add 50 µL of Reaction Mix to appropriate well of DAD.
6. Add 50 µL of sample to DAD.
7. Reseal the wedge with the original foil and tear off tab.
8. Repeat steps 4-7 for additional specimens or controls.
9. Load DAD onto LIAISON® MDX instrument and start run.
10. Obtain results by using the LIAISON® MDX Studio software.
Please see the Instructions for Use in Section 13 (Attachments 13-1 and 13-2) for the detailed
procedure.
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11.3 Description of the Target Primer/Probe Sequence
Table 11.3-1 shows the target, component and the sequence of the Candidate Device.
Table 11.3-1. Target, Component and Sequence Detail of the Candidate Device
Target Description Sequence
FLU A Forward Primer 5’ d-GGCATTTTGGACAAAGCGTCTA 3’
FLU A Reverse Primer 5’ d-TCTTGTCACCTCTGACTAAGGGGAT 3’

Flu A
5’ FAM-ACGCTCACCGTGCCCAGTGAGCG-BHQ1
FLU A Probe
3’
FLU B Forward Primer 5' d-CTGAATTTCCCATIGAGCTCTGCT 3’
FLU B Reverse Primer 5’ d-CTGCAAAGCAACATTGGAGT 3’

Flu B
5’ JOE-ATTGCAAAGGATGTAATGGAAGTG-BHQ1
FLU B Probe
3’
COVID-19 Forward
5’ d-ATGGTAGAGTTGATGGTCAA 3’
Primer ORF1ab
COVID-19 Reverse
5’ d-TAAGACTAGCTTGTTTGGGA 3’
Primer ORF1ab
COVID-19 Probe 5’ Texas Red-

ORF1ab TGCCCGTAATGGTGTTCTTATTACAGA-BHQ2 3’
COVID-19
COVID-19 Forward
5’ d-CTAACCAGGTTGCTGTTCTT 3’
Primer gene S
COVID-19 Reverse
5’ d-CCTGTAGAATAAACACGCCA 3’
Primer gene S
COVID-19 Probe gene 5’ Texas Red-TGCACAGAAGTCCCTGTTGCT-

S BHQ2 3’
Forward Primer IC
5’ d-CTCGTCGACAATGGCGGAA 3’
RNA Taqman
Internal Reverse Primer IC

5’ d-TTCAGCGACCCCGTTAGC 3’
Control RNA Taqman
Probe IC RNA 5’ Quasar 670-

Taqman GCTTGGGGCGACAGTCACGTCGC-BHQ2 3’
Page 70 of 250
11.4 Kit Components
The Candidate Device consists of three separate products:
1) Assay kit (Simplexa COVID-19 & Flu A/B Direct, REF MOL4250),
2) Control pack (Simplexa COVID-19 & Flu A/B Positive Control Pack, REF MOL4260) and
3) Consumable disc (Simplexa Direct Amplification Disc Kit, REF MOL1455).
Descriptions of the Candidate Device are provided below in Tables 11.4-1, 11.4-2 and 11.4-3.
Table 11.4-1. Candidate Device Kit REF MOL4250
Component EC Symbol Abbreviated Cap Number Reactions Volume

REF
Name on Label Name Color of Vials per Vial/Kit per Vial
Simplexa™ MOL4251 REAG C RM Blue 24 1/24 50 µL

COVID-19 & Flu
A/B Direct
Reaction Mix
Table 11.4-2. Candidate Device Kit Components and Description

Kit
Contents
Component
DNA polymerase, Reverse Transcriptase, RNase inhibitor, buffer and dNTPs,

encapsulated RNA Template, primers and dye-labeled fluorescent probes
specific for detection of COVID-19, influenza A, influenza B and for the Internal
Control
Targeted
Simplexa™ Target Channel Excitation Emission
Gene
COVID-19 &
Flu A/B Direct
Reaction Mix Flu A 520 445-505 507-533 matrix
(RM)
Flu B 560 505-543 547-573 matrix
SARS-CoV-2 610 502-596 597-623 S and

ORF1a/b
Internal Control “RNA 690 622-658 652-708 N/A
IC”
Simplexa™
COVID-19 &
Assay specific parameters, lot number, expiration date
Flu A/B Direct
Barcode Card
Page 71 of 250
Table 11.4-3. Candidate Device Positive Control Pack REF MOL4260

Component and Description
Reactions
Component Cap Number Volume
REF Description per
Name Color of Vials per Vial
Vial/Kit
Simplexa™ MOL4261 Inactivated SARS- Blue 10 1/10 50 µL
COVID-19 & CoV-2 virus,
Flu A/B Direct inactivated
Positive influenza A virus,
Control inactivated
influenza B virus
Candidate Device Direct Amplification Disc Kit, REF MOL1455
One (1) box containing three (3) Direct Amplification Discs (DADs) discs individually pouch-
sealed and labeled with REF MOL1452 (Individual DAD).
11.5 Establishing the Cut-Off
The development of the Candidate Device took place starting from the thermal protocol/assay
definition previously optimized and implemented for two previously validated DiaSorin Molecular
devices, namely the Simplexa™ Flu A/B & RSV Gen II Direct (MOL2655) and the Simplexa™
COVID-19 Direct (MOL4150) devices. The cycle threshold (Ct) cut-off used for the Candidate
Device is identical to these two (2) devices.
The RT-PCR Ct cut-off was validated using No Template Control (NTC) testing and contrived
positive samples in the Limit of Detection (LoD) study.
Feasibility data and verification data (see sections 11.5 and 18) demonstrates the Candidate
Device reaches the required sensitivity and specificity within the established Ct cut-off.
The approach is summarized below.
No Template Control (NTC)Testing
The No Template Control (NTC) study assessed sub-amplifications or the risk of false positive
results using NTC. The NTC used for the test consisted of negative nasopharyngeal (NPS) matrix
and the study utilized three (3) lots of reaction mix. Twenty-two replicates of NTC (NPS) were
loaded with each of the three (3) reaction mix lots. Qualitative results and amplification curves
were evaluated for any unacceptable amplification.
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Results - No Template Control (NTC)Testing
A total of 66 replicates of NPS were tested across three (3) reaction mix lots. The results were
as expected yielding 100% of NTC replicates as Not Detected for COVID-19, Flu A and B and
with valid IC amplification. The details of the testing are shown in Table 11.5.1.
Table 11.5.1. NTC study
Conclusion - No Template Control (NTC)Testing
Using the Candidate Device’s RT-PCR cycle threshold (Ct) cut-off, no non-specific signals were
detected. One hundred percent analytical specificity was observed with the NPS matrix.
LoD testing
The Limit of Detection (LoD) study determined the lowest detectable concentration of viral
genomic RNA (copies/mL) at which ≥ 95% of all replicates tested were positive. The study used
two (2) strains for Flu A, two (2) strains for Flu B and one (1) strain for COVID-19. Serial dilutions
of each of the viral stocks were tested for five (5) replicates using reaction mix lot 1 and the lowest
concentration at which all five (5) replicates were positive was interpreted as the tentative LoD
(Screening) for the test.
The LoD of the test was then confirmed by testing 20 replicates at the tentative limit of detection
concentration for all the three (3) reaction mix feasibility lots. The final LoD (Confirmation) was
determined to be the lowest concentration at which ≥ 95% of all replicates were detected.
Results – LoD Testing
The results showed 100% detection of ≤1,000 copies/mL of viral particles diluted in NPS matrix
for all the three (3) assay targets. This is shown in Tables 11.5.2, 11.5.3 and 11.5.4.
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Table 11.5.2. LoD Confirmation-Flu A
Flu A LoD was determined to be 500 copies/mL for Michigan strain (H1N1) and 1000
copies/mL for Hong Kong strain (H3N2) in NPS matrix.
Table 11.5.3. LoD Confirmation-Flu B
Flu B LoD was determined to be 750 copies/mL for Phuket strain (Yamagata lineage) and 500
copies/mL for Malaysia strain (Victoria lineage) in NPS matrix.
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Table 11.5.4. LoD Confirmation-SARS-CoV-2
SARS-CoV-2 LoD (COVID-19 Target) was determined to be 500 copies/mL in NPS matrix.
Conclusion – LoD Testing
Using the Candidate Device RT-PCR cycle threshold (Ct) cut-off, the assay met analytical
sensitivity requirements as demonstrated by the results of the NTC and LoD testing.
The Candidate Device assay target Ct cut-offs and cycling protocol are stored in the Candidate
Device’s assay definition.
Attachment
Section 11.5 Attachments (Documents)
number
Establishing the Cut-off Line data for NTC 11.5-1
Establishing the Cut-off Line data for LoD 11.5-2

Page 75 of 250
11.6 LIAISON MDX Technology
11.6.1 LIAISON MDX System
The LIAISON MDX instrument is a thermocycler that is capable of heating, cooling, mixing of
sample and reagents, and real-time detection of fluorophores at four (4) distinct wavelengths.
Sensors on the instrument monitor the status of the primary functions of the system. The LIAISON
MDX and software (LIAISON MDX Studio Software) were reviewed for direct amplification with
the clearance of Simplexa Flu A/B & RSV Direct (K120413).
B
A
C
Figure 11.6-1: LIAISON MDX system including the: A) LIAISON MDX Instrument, B)
laptop with LIAISON MDX Studio Software and barcode scanner and C) Direct
Amplification Disc (DAD).
Page 76 of 250
12. Substantial Equivalence Discussion
12.1 Comparison to Predicate
Table 12.1-1. Comparison to Predicate Device

Predicate
Device BioFire Respiratory Panel 2.1 Simplexa™ Flu A/B & RSV -
Product Code QOF OCC QOF
OOI
Regulation 21 CFR 866.3981 21 CFR 866.3980 21 CFR 866.3981

Number
21 CFR 862.2570
Organism SARS-CoV-2 Influenza A (Flu A), Influenza SARS-CoV-2, Influenza A (Flu

Detected B (Flu B) and RSV A) and Influenza B (Flu B)
Measurand RNA from SARS-CoV-2 RNA from Influenza A (Flu A), RNA from SARS-CoV-2,
Influenza B (Flu B) and RSV Influenza A (Flu A) and
Matrix gene (Scorpion Probes) Influenza B (Flu B) Matrix gene
Target FilmArray Torch Technology Well conserved region of the SARS-CoV-2 Well conserved
matrix gene (Scorpion regions of the S and ORF
SARS-CoV-1 Well conserved Technology) genes
region of the S gene
Flu A and B Targets Well
SARS-CoV-2 Well conserved conserved regions of the matrix
region of the M gene gene (TAQ Man Technology)
Intended Use The BioFire Respiratory Panel Simplexa Flu A/B & RSV Simplexa COVID-19 & Flu A/B
Kit 2.1 (RP2.1) is a PCR-based Direct Gen II Direct
multiplexed nucleic acid test
intended for use with the BioFire The DiaSorin Molecular The DiaSorin Molecular
FilmArray 2.0 or BioFire Simplexa™ Flu A/B & RSV Simplexa™ COVID-19 & Flu A/B
FilmArray Torch systems for the Direct Gen II assay is intended Direct real-time RT-PCR assay
simultaneous qualitative for use on the LIAISON® MDX is intended for use on the
detection and identification of instrument for the in vitro LIAISON® MDX instrument for
multiple respiratory viral and qualitative detection and the in vitro qualitative detection
bacterial nucleic acids in differentiation of influenza A and differentiation of nucleic acid
nasopharyngeal swabs (NPS) virus, influenza B virus, and from severe acute respiratory
Page 77 of 250
obtained from individuals respiratory syncytial virus syndrome coronavirus 2 (SARS-

suspected of respiratory tract (RSV) RNA in nasopharyngeal CoV-2), influenza A virus and
infections, including COVID-19. swabs (NPS) from human influenza B virus in
patients with signs and nasopharyngeal swabs (NPS)
The following organism types symptoms of respiratory tract from human patients with signs
and subtypes are identified using infection in conjunction with and symptoms of respiratory
the BioFire RP2.1: clinical and epidemiological tract infection in conjunction with
risk factors. This test is clinical and epidemiological risk
• Adenovirus, intended for use as an aid in factors.
• Coronavirus 229E, the differential diagnosis of
• Coronavirus HKU1, The Simplexa™ COVID-19 &
influenza A, influenza B, and
Flu A/B Direct assay is intended
• Coronavirus NL63, RSV viral infections in
for use as an aid in the
• Coronavirus OC43, humans. Negative results do
differential diagnosis of SARS-
• Severe Acute not preclude influenza virus or
CoV-2, influenza A and influenza
Respiratory Syndrome RSV infection and should not
B infection.
Coronavirus (SARS- be used as the sole basis for
CoV-2), treatment or other patient Negative results do not preclude
• Human management decisions. SARS-CoV-2, influenza A or
Metapneumovirus, Performance characteristics influenza B infection and should
• Human for influenza A were not be used as the sole basis for
Rhinovirus/Enterovirus, established with clinical patient management decisions.
• Influenza A, including specimens collected during the Negative results should be
subtypes H1, H1-2009, 2010/2011 influenza season combined with clinical
and H3, when 2009 H1N1 influenza observations, patient history,
• Influenza B, and H3N2 were the and epidemiological information.
• Parainfluenza Virus 1, predominant influenza A
If infection with a novel influenza
• Parainfluenza Virus 2, viruses in circulation. When
A virus is suspected based on
• Parainfluenza Virus 3, other influenza A viruses are
current clinical and
emerging, performance
• Parainfluenza Virus 4, epidemiological screening
characteristics may vary. If
• Respiratory Syncytial criteria recommended by public
infection with a novel influenza
Virus, health authorities, specimens
A virus is suspected based on
• Bordetella parapertussis should be collected with
current clinical and
(IS1001), appropriate infection control
epidemiological screening
• Bordetella pertussis precautions for novel virulent
criteria recommended by
(ptxP), influenza viruses and sent to the
public health authorities,
• Chlamydia pneumoniae, state or local health department
specimens should be collected
and for testing. Viral culture should
with appropriate infection
• Mycoplasma not be attempted in these cases
control precautions for novel
pneumoniae unless a BSL 3+ facility is
virulent influenza viruses and
available to receive and culture
sent to the state or local health
Nucleic acids from the specimens.
department for testing. Viral
respiratory viral and bacterial
culture should not be The Simplexa™ COVID-19 &
organisms identified by this test
attempted in these cases Flu A/B Direct assay is intended
are generally detectable in NPS
unless a BSL 3+ facility is for use by qualified and trained
specimens during the acute
available to receive and culture clinical laboratory personnel
phase of infection. The detection
specimens. specifically instructed and
and identification of specific viral
trained in the techniques of real-
and bacterial nucleic acids from
individuals exhibiting signs
Page 78 of 250

Predicate
Device BioFire Respiratory Panel 2.1 Simplexa™ Flu A/B & RSV -
and/or symptoms of respiratory Simplexa™ Flu A/B & RSV time PCR and in vitro diagnostic
infection is indicative of the Positive Control Pack procedures.
presence of the identified
microorganism and aids in the DiaSorin Molecular’s Simplexa
diagnosis of respiratory infection ™Flu A/B & RSV Positive
Simplexa COVID-19 & Flu A/B
if used in conjunction with other Control Pack is intended to be
Positive Control Pack
clinical and epidemiological used as a control with the
information. The results of this Simplexa™ Flu A/B & RSV The Simplexa™ COVID-19 &
test should not be used as the Direct kit and the Simplexa™ Flu A/B Positive Control Pack is
sole basis for diagnosis, Flu A/B & RSV Direct Gen II kit intended to be used as a control
treatment, or other patient for use on the LIAISON® MDX with the Simplexa™ COVID-19
management decisions. instrument. This control is not & Flu A/B Direct kit for use on
intended for use with other the LIAISON® MDX instrument.
Negative results in the setting of assays or systems. This control is not intended for
a respiratory illness may be due use with other assays or
to infection with pathogens that systems.
are not detected by this test, or
lower respiratory tract infection
that may not be detected by an
NPS specimen. Positive results
do not rule out coinfection with
other organisms. The agent(s)
detected by the BioFire RP2.1
may not be the definite cause of
disease. Additional laboratory
testing (e.g. bacterial and viral
culture, immunofluorescence,
and radiography) may be
necessary when evaluating a
patient with possible respiratory
tract infection.
Automated Automated Same Same

System (Sample
to Answer)
Instrumentation BioFire® FilmArray® 2.0 or LIAISON® MDX LIAISON® MDX

BioFire® FilmArray® Torch
Systems
Sample Types Nasopharyngeal Swab (NPS) Same Same
Page 79 of 250
Section 12.1 Attachment (Document) Attachment number
Predicate IFU - BioFire Respiratory Panel 2.1 (RP2.1) (DEN200031) 12.1-1
Predicate IFU - Simplexa™ Flu A/B & RSV Direct Gen II (K201505) 12.1-2
Predicate IFU - Simplexa™ Flu A/B & RSV Positive Control Pack (K201505) 12.1-3
Page 80 of 250
13. Proposed Labeling
13.1 Instructions for Use
The Instructions for Use (IFU) for the Candidate Device Catalog number MOL4250 and the
Candidate Device Positive Control Pack Catalog number MOL4260 are provided as attachments
(reference Attachments 13-1 and 13-2).
13.2 Kit Component Labels
Facsimiles of the labels are provided below.
Lot specific information (such as the product lot number and expiration date) is found in the Data
Matrix (DM) barcode on the labels below.
Candidate Device Reaction Mix Candidate Device Positive Control
13.3 Candidate Device Kit Box Labels
Facsimile of the Candidate Device kit label is provided below. An enlarged image of the
example Unique Device Identifier (UDI) barcode is shown to the right of the label facsimile.
Label Detail of the UDI Barcode (Enlarged)

Page 81 of 250
Candidate Device Positive Control Pack Label
Facsimile of the Candidate Device Positive Control Pack kit label is provided below. An
enlarged image of the example UDI barcode is shown to the right of the label facsimile.
Label Detail of the UDI Barcode (enlarged)*
13.4 Assay Specific QR Barcode
The Quick Response/Reference (QR) barcode card provided below contains a digital
representation of all the assay parameters, including lot specific information required to load the
assay definition onto the system. The Assay Definition information is scanned into the software
by using the card or by importing the Assay Definition file into the database. Valid assay definition
files may be pre-loaded into the database or loaded at the time of installation by DiaSorin
Molecular Field Service Engineers or the Field Application Scientists.
Page 82 of 250
Attachment
Section 13 Attachments (Documents)
number
Simplexa™ COVID-19 & Flu A/B Direct IFU (IFUK.US.MOL4250) 13-1
Simplexa™ COVID-19 & Flu A/B Positive Control Pack IFU

13-2
(IFUC.US.MOL4260)
Page 83 of 250
14. Sterilization and Shelf Life

14.1 Sterilization
Not applicable. The Candidate Devices are not sterilized nor do the devices contain reprocessed
single use devices.
14.2 Shelf Life
The Candidate Device and the Candidate Device Positive Control Pack will initially have a twelve
(12) month shelf life when stored at -20°C ± 10°C. Initial stability for the Candidate Device was
leveraged based on previously completed stability study information that was in support of the
FDA 510(k) cleared Simplexa™ Flu A/B & RSV Direct (K120413) which has a 12 month shelf
life, Simplexa™ Bordetella Direct (K173498) which has shelf life of 24 months and SimplexaTM
COVID-19 Direct which has shelf life of twelve (12) months (EUA200026).
The reference to these previously cleared products in support of the Candidate Device kit and
the Candidate Device Positive Control Pack is based on the fact that the reagent formulations,
as well as intended specimen type (i.e., NPS) of Simplexa™ Flu A/B & RSV Direct and
Simplexa™ Bordetella Direct products are similar to the Candidate Device kit and the Candidate
Device Positive Control Pack.
The polymerase, oligos, buffer system, salts and dNTPs format of the Candidate Device is
common to all Simplexa™ products using the TaqMan chemistry including, but not limited to,
SimplexaTM Bordetella Direct (MOL2750, shelf life 24 months) and Simplexa™ COVID-19 Direct
(MOL4151, shelf life 12 months). Simplexa™ COVID-19 Direct is the most similar because it
includes the same reverse transcriptase and RNAse inhibitors.
The Candidate Device Positive Control (MOL4261) uses inactivated viral particles diluted in
transport media which is common to all SimplexaTM virus based positive controls which all have
twelve (12) months stability. Simplexa™ Flu A/B & RSV Direct Positive Control (MOL2661, shelf
life 12 months) is the most relevant due to it being an RNA virus based positive control.
Four (4) lots of Candidate Device Reaction Mix (the only component of the Kit) and three (3) lots
of Candidate Device Positive Control (the only component of the Positive Control Pack) are under
assessment in the real time stability program. The data collected so far was analyzed using
MINITABs stability regression algorithm. The software predicts stability based on the regression,
its single sided 95% confidence interval and the specified limits. The minimum predicted shelf life
is eighteen (18) months based on the minimum shelf life derived from the threshold cycle (Ct)
upper limit of 40 for the Flu B channel for one lot (see Figures 14.2.1 and 14.2.2).
Page 84 of 250
Figure 14.2.1. MINITAB graphical output (left) and prediction (right) for MOL4250
SimplexaTM COVID-19 & Flu A/B Direct Reaction Mix for Flu A, Flu B, and COVID-19
when running the positive control and internal control when running non-template
control
Flu A
Flu B
Page 85 of 250
COVID-19
Internal Control
Figure 14.2.2. MINITAB graphical output (left) and prediction (right) for MOL4260
SimplexaTM COVID-19 & Flu A/B Positive Control for Flu A, Flu B, and COVID-19.
Flu A
Page 86 of 250
Flu B
COVID-19
Section 14.2 Attachments (Documents) Attachment number
Candidate Device Stability Memo 14.2-1

Page 87 of 250
14.3 Reaction Mix and Positive Control Open Vial (Room Temperature) Stability
Reaction Mix Open Vial Stability
The Candidate Device Reaction Mix was evaluated for stability after room temperature storage
for 35 and 60 minutes. The study was performed at DiaSorin Molecular, Cypress, CA using six
(6) LIAISON MDX Instruments (100074, 100090, 100091, 100222, 100332, 100336), one (1) lot
of Candidate Device Reaction Mix (Lot V11909N), one (1) lot of Candidate Device Positive
Control (Lot V12441N) and one (1) lot of Direct Amplification Discs (11855N). A total of 32
experimental runs were performed by two (2) operators over the course of two (2) days (15 July
2021 and 16 July 2021).
The Candidate Device Reaction Mix was evaluated for stability at room temperature by using the
Candidate Device Positive Control as a test sample.
Results - Reaction Mix Open Vial Stability Study
Seven (7) vials of Candidate Device Positive Control (MOL4261) were diluted 1:10 with UTM and
stored at -60°C until prior to testing.
Sixteen vials of Candidate Device Reaction Mix vials (lot V11909N) were removed from -20 °C
storage and left at room temperature (18-25 °C) for 60 minutes (T60). After 25 minutes from the
time T60 vials had thawed, an additional 16 vials of Candidate Device Reaction Mix of same lot
were removed from -20 °C storage and left at room temperature for 35 minutes (T35). When 35
minutes of room temperature was complete, another set of 16 Candidate Device Reaction Mix
vials of same lot were thawed for the zero (0) minute (immediate use) time point (T0). All vials
were assayed. The reaction mix stability results are summarized in Table 14.3.1.
Table 14.3.1. Summary of Reaction Mix Stability for all time points
% Detection (# Detected/#Tested) Internal Control (IC)
Qualitative Results:
Flu A Qualitative Flu B Qualitative COVID-19 Qualitative % Detection
Results: Results: Results: (# Detected /#Tested)
Room
Temperature
0 35 60 0 35 60 0 35 60 0 35 60
Storage Duration
(Minutes)
% Detected 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0
(# Detected/# % % % % % % % % % % % %
Tested) (16/16) (16/16) (16/16) (16/16) (16/16) (16/16) (16/16) (16/16) (16/16) (16/16) (16/16) (16/16)
Page 88 of 250
Results for Flu A when testing bulk positive control, with Candidate Device Reaction Mix that had
been stored at room temperature for zero (0), 35 and 60 minutes prior to testing are presented
in Table 14.3.2. All replicates for all time points were detected.
Table 14.3.2. Reaction Mix Stability Results for Flu A

Room Flu A
Temperature
% Detection % Difference of
Sample Storage Mean Ct ± SD
Duration (# Detected/ # Mean Ct from Time
(%CV)
(Minutes) Tested) 0
0 100.0% (16/16) 28.9 ± 0.13 (0.4%) N/A

1:10 Pooled
Positive 35 100.0% (16/16) 28.8 ± 0.26 (0.9%) -0.35%
Control
60 100.0% (16/16) 28.7 ± 0.29 (1.0%) -0.69%
Ct = Cycle Threshold, SD = Standard Deviation, %CV = Percent Coefficient of Variation, N/A = Not Applicable
Results for Flu B when testing bulk positive control, with Candidate Device Reaction Mix that has
been stored at room temperature for zero (0), 35 and 60 minutes prior to testing are presented
in Table 14.3.3. All replicates for all time points were detected.
Table 14.3.3. Reaction Mix Stability Results for Flu B
Room Flu B
Temperature
Duration (# Detected/ # Mean of Ct from
(%CV)
(Minutes) Tested) Time 0
Pooled 1:10 0 100.0% (16/16) 28.9 ± 0.38 (1.3%) N/A

Pooled 35 100.0% (16/16) 28.9 ± 0.39 (1.3%) 0%
Positive
Control 60 100.0% (16/16) 28.9 ± 0.55 (1.9%) 0%
Results for COVID-19 when testing bulk positive control, with Candidate Device Reaction Mix
that had been stored at room temperature for zero (0), 35 and 60 minutes prior to testing are
presented in Table 14.3.4. All replicates for all time points were detected.
Page 89 of 250
Table 14.3.4. Reaction Mix Stability Results for COVID-19
Room COVID-19
Temperature
(%CV)
1:10 Pooled 0 100.0% (16/16) 29.2 ± 0.36 (1.2%) N/A

Positive 35 100.0% (16/16) 29.0 ± 0.48 (1.7%) -0.68%
Control
60 100.0% (16/16) 28.8 ± 0.61 (2.1%) -1.37%
Results for the Internal Control when testing bulk Candidate Device Positive Control, with
Candidate Device Reaction Mix that has been stored at room temperature for zero (0), 35 and
60 minutes prior to testing are presented in Table 14.3.5. All replicates for all time points were
detected.
Table 14.3.5. Reaction Mix Stability Results for RNA Internal Control
Room Internal Control
Temperature
(%CV)
1:10 Pooled 0 100.0% (16/16) 30.1 ± 0.34 (1.1%) N/A

Positive 35 100.0% (16/16) 30.1 ± 0.33 (1.1%) 0%
Control
60 100.0% (16/16) 29.8 ± 0.77 (2.6%) -1.00%
Summary of Control Results - Reaction Mix Open Vial Stability
Table 14.3.6 shows the results of the Positive Control (PC) and No Template Control (NTC).
Controls were run once per day on each instrument used in the study. All controls performed as
expected.
Page 90 of 250
Table 14.3.6. Results for Daily Controls

Flu A Flu B COVID-19 IC
% % %
%
Detection Detection Detection
Instrument Mean Mean Ct Detection Mean Mean
Control
SN (% Ct ± SD (% ± SD Ct ± SD (% Ct ± SD
(%
Detection (%CV) Detection (%CV) (%CV) Detection (%CV)
Detection/
/% /% /%
% Tested)
Tested) Tested) Tested)
100074 0.0% (0/1) N/A ± 0.0% (0/1) N/A ± 0.0% (0/1) N/A ± 100.0% 30.2 ±
N/A N/A N/A (1/1) N/A
N/A ± N/A ± N/A ± 29.9 ±
100.0%
100090 0.0% (0/1) N/A 0.0% (0/1) N/A 0.0% (0/1) N/A N/A
(1/1)
(N/A%) (N/A%) (N/A%) (N/A%)
N/A ± N/A ± N/A ± 30.1 ±

100.0%
100091 0.0% (0/1) N/A 0.0% (0/1) N/A 0.0% (0/1) N/A N/A
(1/1)
(N/A%) (N/A%) (N/A%) (N/A%)
N/A ± N/A ± N/A ± 30.6 ±

100.0%
100222 0.0% (0/1) N/A 0.0% (0/1) N/A 0.0% (0/1) N/A N/A
NTC (1/1)
(N/A%) (N/A%) (N/A%) (N/A%)
N/A ± N/A ± N/A ± 30.7 ±

100.0%
100332 0.0% (0/2) N/A 0.0% (0/2) N/A 0.0% (0/2) N/A 0.2
(2/2)
(N/A%) (N/A%) (N/A%) (0.7%)
N/A ± N/A ± N/A ± 29.0 ±

100.0%
100336 0.0% (0/1) N/A 0.0% (0/1) N/A 0.0% (0/1) N/A N/A
(1/1)
(N/A%) (N/A%) (N/A%) (N/A%)
N/A ± N/A ± N/A ± 30.2 ±

100.0%
ALL 0.0% (0/7) N/A 0.0% (0/7) N/A 0.0% (0/7) N/A 0.6
(7/7)
(N/A%) (N/A%) (N/A%) (2.0%)
100074 100.0% 25.6 ± 100.0% 26.3 ± 100.0% 26.1 ± 100.0% 30.1 ±

(1/1) N/A (1/1) N/A (1/1) N/A (1/1) N/A
25.6 ± 25.9 ± 25.9 ± 30.1 ±
100.0% 100.0% 100.0% 100.0%
100090 N/A N/A N/A N/A
(1/1) (1/1) (1/1) (1/1)
(N/A%) (N/A%) (N/A%) (N/A%)
PC 25.7 ± 25.5 ± 26.2 ± 31.0 ±

100.0% 100.0% 100.0% 100.0%
100091 N/A N/A N/A N/A
(1/1) (1/1) (1/1) (1/1)
(N/A%) (N/A%) (N/A%) (N/A%)
25.8 ± 26.7 ± 26.5 ± 32.2 ±

100.0% 100.0% 100.0% 100.0%
100222 N/A N/A N/A N/A
(1/1) (1/1) (1/1) (1/1)
(N/A%) (N/A%) (N/A%) (N/A%)
Page 91 of 250
% % %
%
Instrument Mean Mean Ct Detection Mean Mean
Control
SN (% Ct ± SD (% ± SD Ct ± SD (% Ct ± SD
(%
Detection (%CV) Detection (%CV) (%CV) Detection (%CV)
Detection/
/% /% /%
% Tested)
26.0 ± 26.6 ± 26.6 ± 31.8 ±

100.0% 100.0% 100.0% 100.0%
100332 0.1 0.1 0.1 1.0
(2/2) (2/2) (2/2) (2/2)
(0.5%) (0.5%) (0.5%) (3.1%)
25.5 ± 25.9 ± 25.4 ± 29.0 ±

100.0% 100.0% 100.0% 100.0%
100336 N/A N/A N/A N/A
(1/1) (1/1) (1/1) (1/1)
(N/A%) (N/A%) (N/A%) (N/A%)
25.7 ± 26.2 ± 26.2 ± 30.9 ±

100.0% 100.0% 100.0% 100.0%
ALL 0.2 0.5 0.4 1.2
(7/7) (7/7) (7/7) (7/7)
(0.8%) (1.8%) (1.7%) (4.0%)
PC = Positive Control, NTC = No Template Control, Ct = Cycle Threshold, SD = Standard Deviation, %CV = Percent Coefficient of
Variation, N/A = Not Applicable
Conclusion – Reaction Mix Open Vial Stability
The Candidate Device Reaction Mix showed 100% detection of Flu A, Flu B and COVID-19 at all
time points. The storage requirements in the Candidate Device instructions for use (IFU)
prescribe the Candidate Device Reaction Mix can remain at room temperature for 30 minutes
after thawing before use. Thirty minutes is well within the validated 60 minutes room temperature
storage DiaSorin Molecular demonstrated the Candidate Device Reaction Mix could be stored at
with no determent to the performance of the device. Controls run during the study performed as
expected.
Please see attachment list at the conclusion of section 14.3 for a listing of study protocols and
the associated line data.
Positive Control Open Vial Stability
The Candidate Device Positive Control Pack was evaluated for open vial stability after storage
at room temperature for 35 and 60 minutes. The study was performed at DiaSorin Molecular,
Cypress, CA using six (6) LIAISON MDX Instruments (100074, 100090, 100091, 100222, 100332
and 100336) one (1) lot of Candidate Device Reaction Mix (Lot V11909N) and one (1) lot of
Candidate Device Positive Control (Lot V12441N) and one (1) lot of Direct Amplification Discs
Page 92 of 250
(11855N). A total of 14 experimental runs were performed by two (2) operators on one (1) day
(15 July 2021).
Results - Positive Control Open Vial Stability
Sixteen vials of Candidate Device Positive Control (lot V12441N) were removed from -20 °C
storage and kept at room temperature for 60 minutes (T60). After 25 minutes from when the time
T60 vials had thawed, 16 vials of Candidate Device Positive Control of the same lot were
removed from -20 °C storage and left at room temperature for 35 minutes (T35). When the
additional 35 minutes of room temperature storage was complete, 16 vials of Candidate Device
Positive Control vials of same lot were thawed for the zero (0) time point (T0). All vials were
assayed using Candidate Device Reaction Mix lot V11909N. The Candidate Device Positive
Control stability results are summarized in Table 14.3.7.
Table 14.3.7. Summary of Positive Control Stability for all time points
% Detection (# Detected/#Tested) Internal Control (IC)
Qualitative Results:
Flu A Qualitative Flu B Qualitative COVID-19 Qualitative % Detection
Results: Results: Results: (# Detected/#Tested)
Room
Temperature
0 35 60 0 35 60 0 35 60 0 35 60
Storage Duration
(Minutes)
% Detected 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0
(# Detected/# % % % % % % % % % % % %
Tested) (16/16) (16/16) (16/16) (16/16) (16/16) (16/16) (16/16) (16/16) (16/16) (16/16) (16/16) (16/16)
Results for Flu A testing of the Candidate Device Positive Control that had been stored at room
temperature for zero (0), 35 and 60 minutes prior to testing are presented in Table 14.4.8. All
replicates for all time points were detected.
Page 93 of 250
Table 14.4.8. Positive Control Stability Results for Flu A
Room Flu A
Temperature
% Detection %
Sample Storage
Mean Ct ± SD Difference
Duration (# Detected/ # (%CV) of Mean Ct
(Minutes) Tested) from Time 0
0 100.0% (16/16) 25.7 ± 0.14 (0.5%) N/A

Positive Control 35 100.0% (16/16) 25.8 ± 0.28 (1.1%) 0.39%
60 100.0% (16/16) 25.6 ± 0.32 (1.3%) -0.39%
Results for Flu B testing of the Candidate Device Positive Control that had been stored at room
temperature for zero (0), 35 and 60 minutes prior to testing are presented in Table 14.4.9. All
replicates for all time points were detected.
Table 14.4.9. Positive Control Stability Results for Flu B
Room Flu B
Temperature
Sample Storage
Mean Ct ± SD
Duration (# Detected/ # Mean Ct from
(%CV)
26.2 ± 0.30
0 100.0% (16/16) N/A
(1.1%)
26.2 ± 0.34
Positive Control 35 100.0% (16/16) 0.00%
(1.3%)
26.6 ± 0.25
60 100.0% (16/16) 1.53%
(0.9%)
Results for COVID-19 testing of the Candidate Device Positive Control that had been stored at
room temperature for zero (0), 35 and 60 minutes prior to testing are presented in Table 14.4.10.
All replicates for test points zero (0),35, and 60 minutes were detected.
Page 94 of 250
Table 14.4.10. Positive Control Stability Results for COVID-19
Room COVID-19
Temperature
Sample Storage
Mean Ct ± SD
(%CV)
26.2 ± 0.21
0 100.0% (16/16) N/A
(0.8%)
26.2 ± 0.36
Positive Control 35 100.0% (16/16) 0.00%
(1.4%)
26.0 ± 0.46
60 100.0% (16/16) -0.76%
(1.8%)
Results for the Internal Control (IC) testing Candidate Device Positive Control that had been
stored at room temperature for zero (0), 35 and 60 minutes prior to testing are presented in Table
14.4.11. All replicates for test points zero (0), 35 and 60 minutes were detected.
Table 14.4.11. Positive Control Stability Results for RNA Internal Control (Q670)
Room Internal Control (IC)

Temperature
Sample Storage
Mean Ct ± SD
(%CV)
30.6 ± 0.59
0 100.0% (16/16) N/A
(1.9%)
30.5 ± 0.60
Positive Control 35 100.0% (16/16) -0.33%
(2.0%)
30.8 ± 1.57
60 100.0% (16/16) 0.65%
(5.1%)
Summary of Control Results – Positive Control Open Vial Stability
Table 14.4.12 shows the results of the Positive Control (PC) and No Template Control (NTC).
Controls were run once per day on each instrument used in the study. All controls performed as
expected.
Page 95 of 250
Table 14.4.12. Results for Daily Controls

Flu A Flu B COVID-19 Internal Control (IC)
Instrument % Detection Mean Ct % Detection Mean Ct % Detection Mean Ct % Detection Mean

Control
SN ± SD ± SD ± SD Ct ± SD
(% Detection (% Detection (% Detection (% Detection
/% Tested) (%CV) /% Tested) (%CV) /% Tested) (%CV) /% Tested) (%CV)
100074 0.0% (0/1) N/A ± 0.0% (0/1) N/A ± 0.0% (0/1) N/A ± 100.0% (1/1) 30.2 ±
N/A N/A N/A N/A
N/A ± N/A ± N/A ± 29.9 ±
100090 0.0% (0/1) N/A 0.0% (0/1) N/A 0.0% (0/1) N/A 100.0% (1/1) N/A
(N/A%) (N/A%) (N/A%) (N/A%)
N/A ± N/A ± N/A ± 30.1 ±

100091 0.0% (0/1) N/A 0.0% (0/1) N/A 0.0% (0/1) N/A 100.0% (1/1) N/A
(N/A%) (N/A%) (N/A%) (N/A%)
N/A ± N/A ± N/A ± 30.6 ±

100222 0.0% (0/1) N/A 0.0% (0/1) N/A 0.0% (0/1) N/A 100.0% (1/1) N/A
NTC (N/A%) (N/A%) (N/A%) (N/A%)
N/A ± N/A ± N/A ± 30.5 ±

100332 0.0% (0/1) N/A 0.0% (0/1) N/A 0.0% (0/1) N/A 100.0% (1/1) N/A
(N/A%) (N/A%) (N/A%) (N/A%)
N/A ± N/A ± N/A ± 29.0 ±

100336 0.0% (0/1) N/A 0.0% (0/1) N/A 0.0% (0/1) N/A 100.0% (1/1) N/A
(N/A%) (N/A%) (N/A%) (N/A%)
N/A ± N/A ± N/A ± 30.1 ±

All 0.0% (0/6) N/A 0.0% (0/6) N/A 0.0% (0/6) N/A 100.0% (6/6) 0.58
(N/A%) (N/A%) (N/A%) (1.9%)
100074 100.0% (1/1) 25.6 ± 100.0% (1/1) 26.3 ± 100.0% (1/1) 26.1 ± 100.0% (1/1) 30.1 ±
N/A N/A N/A N/A
25.6 ± 25.9 ± 25.9 ± 30.1 ±
100090 100.0% (1/1) N/A 100.0% (1/1) N/A 100.0% (1/1) N/A 100.0% (1/1) N/A
(N/A%) (N/A%) (N/A%) (N/A%)
25.7 ± 25.5 ± 26.2 ± 31.0 ±

100091 100.0% (1/1) N/A 100.0% (1/1) N/A 100.0% (1/1) N/A 100.0% (1/1) N/A
PC (N/A%) (N/A%) (N/A%) (N/A%)
25.8 ± 26.7 ± 26.5 ± 32.2 ±

100222 100.0% (1/1) N/A 100.0% (1/1) N/A 100.0% (1/1) N/A 100.0% (1/1) N/A
(N/A%) (N/A%) (N/A%) (N/A%)
25.9 ± 26.5 ± 26.5 ± 31.1 ±

100332 100.0% (1/1) N/A 100.0% (1/1) N/A 100.0% (1/1) N/A 100.0% (1/1) N/A
(N/A%) (N/A%) (N/A%) (N/A%)
Page 96 of 250
Instrument % Detection Mean Ct % Detection Mean Ct % Detection Mean Ct % Detection Mean

Control
SN ± SD ± SD ± SD Ct ± SD
(% Detection (% Detection (% Detection (% Detection
/% Tested) (%CV) /% Tested) (%CV) /% Tested) (%CV) /% Tested) (%CV)
25.5 ± 25.9 ± 25.4 ± 29.0 ±

100336 100.0% (1/1) N/A 100.0% (1/1) N/A 100.0% (1/1) N/A 100.0% (1/1) N/A
(N/A%) (N/A%) (N/A%) (N/A%)
25.7 ± 26.1 ± 26.1 ± 30.6 ±

All 100.0% (6/6) 0.15 100.0% (6/6) 0.45 100.0% (6/6) 0.41 100.0% (6/6) 1.10
(0.6%) (1.7%) (1.6%) (3.6%)
SN = Serial Number, NTC = No Template Control, PC = Positive Control, Ct = Cycle Threshold, SD = Standard Deviation, %CV = Percent
Coefficient of Variation, N/A = Not Applicable
Conclusions - Positive Control Open Vial (Room Temperature) Stability
The Candidate Device Positive Control showed 100% detection in 16/16 replicates across all
time points. The storage requirements in the Candidate Device instructions for use (IFU)
prescribe the Candidate Device Positive Control can remain at room temperature for 30 minutes
after thawing before use. Thirty minutes is well within the validated 60 minutes room temperature
storage DiaSorin Molecular demonstrated the Candidate Device Positive Control could be stored
at with no determent to the performance of the device. Controls run during the study performed
as expected.
Section 14.3 (b)Attachments (Documents) Attachment number
Room Temperature Reaction Mix Stability Protocol 14.3-1
Room Temperature Reaction Mix Stability Line Data pdf 14.3-2
Room Temperature Reaction Mix Stability Line Data xls 14.3-3
Room Temperature Positive Control Stability Protocol 14.3-4
Room Temperature Positive Control Stability Line Data pdf 14.3-5
Room Temperature Positive Control Stability Line Data xls 14.3-6

Page 97 of 250
15. Biocompatibility
Not applicable. The Candidate Device does not contain components which have direct contact with
patients; therefore, no biocompatibility testing is required.
Page 98 of 250
16. Software
16.1 LIAISON® MDX Software Studio
Not Applicable. The Candidate Device does not contain software.
The LIAISON MDX Studio Software that is used with the Candidate Device was reviewed for
direct amplification with the clearance of Simplexa Flu A/B & RSV Direct (K120413) and recently
with clearance of Simplexa Flu A/B & RSV Direct Gen II (K201505).
Documentation for Software versions 1.1 and 2.1 have been included with this Pre-Market
Notification. The verification and validation for the Candidate Device was performed using
LIAISON MDX Studio software version 2.1, however as some of our customers have LIAISON
MDX Studio software version 1.1 the Candidate Device Instructions for Use state LIAISON MDX
Studio software version 1.1 or greater. DiaSorin Molecular took the data generated in LIAISON
MDX Studio software version 2.1 and reprocessed the data with LIAISON MDX Studio software
version 1.1. The data was found to be comparable. The report and the data can be found as
Attachments 16-1 and 16-2.
The study compared 5019 Candidate Device results of which 5010 had concordant results.
There were nine (9) discordant results. There were a total of 20,076 analytes processed (four
analytes per sample – COVID-19, Flu A, Flu B and Internal Control), of which 20,067 had
concordant results. Of the nine (9) discordant results, one (1) was Flu A, seven (7) were Flu B
and one (1) was COVID-19. There were no discordant results for Internal Control (IC). This
excluded samples that were valid for the IC but had a zero Ct value. Table 16.1.1 shows the
discordant results for Flu A, Table 16.1.2 shows the discordant results for Flu B, and Table 16.1.3
shows the discordant results for COVID-19, Table 16.1.4 shows the concordant results summary
and Table 16.1.5 shows the regression analysis summary.
Table 16.1.1. Flu A Discordant Results
Instrument
Well Sample SW v1.1 SW v2.1 SW SW v2.1
Run Serial
# Name Result Result* v1.1 Ct Ct *
Number
FABC_CA_NPS_R17_P21
Not
5_EYA_05-17-2021 At 100215 1 FABC-6046 Detected 40 0
Detected
0944
*original result., # = number, SW v = Software Version, Ct. = Cycle threshold

Page 99 of 250
Table 16.1.2. Flu B Discordant Results
Instrument
Well Sample SW v1.1 SW v2.1 SW SW v2.1
Run Serial
# Name Result Result* v1.1 Ct Ct *
Number
FABC_AR_113_114_P170
FABC-AR- Not
_MXR_07-09-2021 At 100170 3 Detected 36.1 0
113 Detected
1336
FABC_AR_121_P90_CXM FABC-AR-
100090 1 Detected EC500 29.7 32.5
_07-12-2021 At 1034 121
FABC_DSI_CA_2083-
2085_SR Run 08-04-2021 200151 3 DIAV_2085 Detected EC500 33.6 36.3
At 1133
FABC_LoD_002_RM1_P1
FABC-LOD-
22_MXR_ 04-20-2021 At 100122 2 Detected EC500 31.9 34.9
002
1306
FABC_OpenKit 4 RM T35 Detected EC500 28.8 29.4

RM_T35_P332_EB_07-15- 100332
2021 At 1356 8 RM T35 Detected EC500 28.7 29.1
FABC_PrelimLoD_P215_M FABC-LOD-
100215 4 Detected EC500 34.7 35.6
XX_04-20-2021 At 0820 S5-010
Table 16.1.3. COVID-19 (610) Discordant Results
Instrument SW
Wel Sample SW v1.1 SW SW v2.1
Run Serial v2.1
l# Name Result v1.1 Ct Ct *
Number Result*
COVID-19
COVID 1E3 Not
Flu_V11908N_Conf_WHO 2D0055 5 Detected 38.30 0
IU/mL Detected
_COVID_1
Table 16.1.4. Result Concordance Summary
Discordant Concordant Total Percent

Assay
Results Results Results Concordance
Candidate Device 9 20,067 20,076 99.96%

Page 100 of 250
Regression analysis was performed on all valid samples that were detected.
Table 16.1.5. Regression Analysis Summary
Slope Intercept
95% Confidence 95% Confidence
Assay Coefficient of Interval Interval
Determination
(R2) Lower Upper Lower Upper
Candidate Device 0.9975 1.00020 1.00199 -0.02341 0.03011
The concordance between results generated with LIAISON MDX Studio software version 2.1 and
version 1.1 was 99.96%. Both LIAISON MDX Studio software versions produce equivalent
detection results for the Candidate Device.
Section 17 Attachments (Documents) Attachment Number
LIAISON MDX SW 1.1 to 2.1 Technical Report 16-1
LIAISON MDX SW 1.1 to 2.1 Technical Report Data 16-2

Page 101 of 250
17. Electromagnetic Compatibility (EMC) and Electrical Safety

EMC, Electrical Safety and Electrical Emissions evaluations have been completed. The LIAISON MDX
and software (LIAISON MDX Studio Software) were reviewed for direct amplification with the clearance
of Simplexa Flu A/B & RSV Direct (K120413).
Page 102 of 250
18. Performance Testing Bench
All analytical studies for the Candidate Device were performed using the Candidate Device with the
LIAISON MDX using LIAISON MDX Studio Software versions 2.1.
All protocols and data are provided in the attachments to each section.
18.1 Cross-Reactivity
The Candidate Device was evaluated for analytical specificity/cross-reactivity by testing 47

different viruses, bacteria and fungi that could be found in nasopharyngeal swab (NPS)
specimens. The study was performed at DiaSorin Molecular, Cypress, CA using five (5) LIAISON
MDX Instruments (100122, 100170, 100215, 100305 and 100445), one (1) lot of Candidate
Device kit Lot V11909N), two (2) lots of Candidate Device Positive Control Packs (Lots V11808N
and V11923N) and three (3) lots of Direct Amplification Discs (Lots R9211NA, 11747N and
11855N). A total of 37 experimental runs were performed by two (2) operators over the course
of six (6) non-consecutive days (17-18, 21 and 23 June 2021, 6 and 15 July 2021).
All samples were prepared by spiking each potentially cross-reacting organism into Flu A, Flu B
and COVID-19 negative pooled nasopharyngeal swab (NPS) in UTM matrix.
The viral particles of human coronavirus HKU1 were not commercially available at the time of
the study, therefore the potentially inhibiting effect of human coronavirus HKU1 was evaluated
through the use of its viral RNA genome.
The components of commercial transport media could have an adverse impact on the ability to
detect viral RNA when the RNA nucleic acid is directly diluted into the transport media, without
being packaged into a nucleocapsid1.
1
. Pathology. 2020 Dec; 52(7): 811–814. Published online 2020 Oct “The impact of viral transport
media on PCR assay results for the detection of nucleic acid from SARS-CoV-2”
P.D.Kirkland and M.J. Frost
In order to prevent the degradation of viral RNA genome, HKU1 RNA was spiked in pooled
negative NPS matrix in UTM with the addition of RNasin at a concentration of 1.6 U/µL. RNasin
is a ribonuclease inhibitor that works inhibiting RNase activity, avoiding the degradation of viral
RNA genome and ensuring its integrity.
DiaSorin Molecular contacted suppliers of viral particles (ATCC and Microbiologics) again at the
end of January 2022, to no avail as the suppliers were still unable to provide human coronavirus
HKU1 viral particles.
For potentially cross-reacting bacteria or fungi, the testing concentration was 1x106 CFU/mL; for
viruses, 1x105 TCID50/mL. For organisms not titered in CFU/mL or TCID50/mL, other industry
acceptable units were used.
Page 103 of 250
DiaSorin Molecular also tested pooled nasal human fluid to represent a diverse microbial flora
and leucocytes.
Stocks of Bacillus anthracis, Chlamydophila psittaci, Influenza C and Pneumocystis jirovecii were
not available. For these organisms, in silico analysis using BLAST (Basic Local Alignment Search
Tool) provided by NCBI (National Center for Biotechnology Information) was performed. Human
Coronavirus 229E, Human Coronavirus NL63, Human Metapneumovirus 9 and Rhinovirus 1A
could not be tested at 1x105 TCID50/mL due to not being able to obtain a stock of sufficient
concentration. These organisms were tested at a lower concentration (1x104 U/mL) and
additionally BLAST analysis was performed. The results of the BLAST analyses showed no risk
of cross-reactivity.
Table 18.1.1 shows the results for the Flu A, Flu B and COVID-19 and Internal Control for each
potentially cross-reacting organism. No cross-reactivity was observed for any of the organisms
tested.
Table 18.1.1. Cross-Reactivity Results
Internal
Flu A Flu B COVID-19
Control
% % %
Test % Summary
Organism Concentra Detection of
tion Statistics
(#Detected (#Detected (#Detected
(#Detected of IC Ct
/#Tested) /#Tested) /#Tested)
/#Tested)
Mean + Ct
31 ± 0.2
0.0% (0/5) 0.0% (0/5) 0.0% (0/5)
CR_Baseline_1 N/A 100% (5/5) (0.5%)
30.9 ± 0.3
N/A 0.0% (0/5) 0.0% (0/5) 0.0% (0/5)
CR_Baseline_1 100% (5/5) (1.1%)
30.6 ± 0.3
N/A 0.0% (0/5) 0.0% (0/5) 0.0% (0/5)
CR_Baseline_1 100% (5/5) (0.9%)
31 ± 0.3
N/A 0.0% (0/5) 0.0% (0/5) 0.0% (0/5)
CR_Baseline_1 100% (5/5) (0.8%)
31.2 ± 0.2
N/A 0.0% (0/5) 0.0% (0/5) 0.0% (0/5)
CR_Baseline 2 100% (5/5) (0.6%)
30.4 ± 0.2
N/A 0.0% (0/5) 0.0% (0/5) 0.0% (0/5)
CR-Baseline-3 100% (5/5) (0.7%)
Page 104 of 250
Microorganisms
1 x 105 30.8 ± 0.2

Adenovirus Type 1 TCID50/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) 100% (3/3) (0.7%)
1 x 105 30.7 ± 0.3

Adenovirus Type 7A TCID50/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) 100% (3/3) (1.0%)
1 x 106 31.3 ± 0.4

Bordetella pertussis CFU/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) 100% (3/3) (1.2%)
1 x 106 30.9 ± 0.2

Candida albicans CFU/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) 100% (3/3) (0.7%)
1 x 106 30.6 ± 0.2

Chlamydia pneumoniae IFU/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) 100% (3/3) (0.7%)
Corynebacterium 1 x 106 30.7 ± 0.3

diphtheriae CFU/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) 100% (3/3) (1.0%)
1 x 106
31.2 ± 0.1
Coxiella burnetii 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) 100% (3/3)
genome (0.3%)
copies/mL
1 x 105 30.9 ± 0.3

Cytomegalovirus 100% (3/3)
U/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (0.8%)
1 x 105 30.9 ± 0.2

Enterovirus Type 68 100% (3/3)
U/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (0.5%)
1 x 105 30.9 ± 0.2

Enterovirus Type 71 100% (3/3)
TCID50/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (0.7%)
1 x 105 31.1 ± 0.5

Epstein-Barr Virus 100% (3/3)
copies/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (1.6%)
Escherichia coli 1 x 106 31.1 ± 0.3

100% (3/3)
O157:H7 CFU/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (1.0%)
1 x 106 30.7 ± 0.1

Haemophilus influenzae 100% (3/3)
CFU/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (0.2%)
Human Coronavirus 1 x 104 30.6 ± 0.1

100% (3/3)
229E* TCID50/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (0.2%)
Human Coronavirus 1 x 104 31 ± 0.2

100% (3/3)
NL63* U/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (0.7%)

100% (3/3)
OC43 TCID50/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (1.4%)
Page 105 of 250
Human 1 x 104 31 ± 0.3

100% (3/3)
Metapneumovirus 9* TCID50/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (1.0%)
Lactobacillus 1 x 106 31.1 ± 0.3

100% (3/3)
plantarum,17-5 CFU/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (0.8%)
1 x 106 30.8 ± 0.4

Legionella longbeachae 100% (3/3)
CFU/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (1.1%)
1 x 106 30.6 ± 0.4

Legionella pneumophila 100% (3/3)
CFU/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (1.1%)
1 x 106 30.6 ± 0.2

Leptospira interrogans 100% (3/3)
copies/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (0.5%)
1 x 105 31 ± 0.3
Measles 100% (3/3)
TCID50/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (0.8%)
1 x 105 30.7 ± 0.3

MERS-Coronavirus 100% (3/3)
TCID50/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (1.0%)
1 x 106 30.7 ± 0.3

Moraxella catarrhalis 100% (3/3)
CFU/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (0.8%)
1 x 105 30.5 ± 0.3

Mumps 100% (3/3)
U/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (0.8%)
Mycobacterium
1 x 106 31 ± 0.2
tuberculosis Genomic 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) 100% (3/3)
copies/mL (0.7%)
DNA
Mycoplasma 1 x 106 30.9 ± 0.2

100% (3/3)
pneumoniae CCU/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (0.6%)
1 x 106 30.6 ± 0.0

Neisseria elongata 100% (3/3)
CFU/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (0.0%)
1 x 106 30.4 ± 0.1

Neisseria meningitidis 100% (3/3)
CFU/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (0.2%)
1 x 105 31 ± 0.2
Parainfluenza Type 1 100% (3/3)
U/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (0.7%)
1 x 105 30.8 ± 0.2

TCID50/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (0.5%)
1 x 105 30.9 ± 0.1

TCID50/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (0.3%)
1 x 105 30.5 ± 0.3

U/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (0.8%)
Page 106 of 250
1 x 105 30.7 ± 0.2

Parechovirus Type 3 100% (3/3)
U/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (0.7%)
Pseudomonas 1 x 106 30.7 ± 0.1

100% (3/3)
aeruginosa CFU/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (0.4%)
1 x 104 30.8 ± 0.3

Rhinovirus 1A* 100% (3/3)
U/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (0.8%)
1 x 105 30.8 ± 0.1

RSV-A 100% (3/3)
TCID50/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (0.2%)
1 x 105 30.9 ± 0.2

RSV-B 100% (3/3)
TCID50/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (0.7%)
1 x 106 30.9 ± 0.2

Staphylococcus aureus 100% (3/3)
CFU/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (0.6%)
Staphylococcus 1 x 106 30.8 ± 0.2

100% (3/3)
epidermidis CFU/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (0.7%)
Streptococcus 1 x 106 31 ± 0.1

100% (3/3)
pneumoniae CFU/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (0.2%)
Streptococcus 1 x 106 32 ± 0.1

100% (3/3)
pyogenes CFU/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (0.4%)
1 x 106 30.8 ± 0.3

Streptococcus salivarius 100% (3/3)
CFU/mL 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) (0.8%)

0.0% (0/3) 0.0% (0/3) 0.0% (0/3) 100% (3/3)
RNA HKU1 U/mL (1.1%)
Other
Human Genomic DNA 1 x 106 31 ± 0.1

0.0% (0/3) 0.0% (0/3) 0.0% (0/3) 100% (3/3)
(Leukocytes) cells/mL (0.3%)
Pooled Human Nasal 30.2 ± 0.1

1:1 dilution 0.0% (0/3) 0.0% (0/3) 0.0% (0/3) 100% (3/3)
Wash (0.4%)
SARS-COV1 Synthetic 1 x 105 31 ± 0.1

0.0% (0/3) 0.0% (0/3) 0.0% (0/3) 100% (3/3)
RNA U/mL (0.4%)
*Unable to obtain stock material with high titer

N/A = Not applicable, Ct = Cycle threshold, %CV = Percent Coefficient of Variation, SD = standard deviation, U/mL =
Units/mL, TCID50/mL = Tissue culture infectious dose, CCU/mL = Color changing units/mL, CFU/mL = Colony forming
units/mL, IFU/mL = Infectious units/mL
Page 107 of 250
Summary of Control Results – Cross-Reactivity
Table 18.1.2 shows the results of the Positive Control (PC) and No Template Control (NTC) for
each assay run. All controls performed as expected.
Table 18.1.2. Results for Controls

% % % %
Instrument
Control Detection Mean Detection Mean Ct Detection Mean Ct Detection Mean
SN
Ct ± SD ± SD ± SD Ct ± SD
(#Detected/ (%CV) (#Detected/ (%CV) (#Detected/ (%CV) (#Detected/ (%CV)
#Tested) #Tested) #Tested) #Tested)
N/A ± N/A ± N/A ± 30.7 ±

100122 0% (0/2) N/A 0% (0/2) N/A 0% (0/2) N/A 100% (2/2) 0.4
(N/A%) (N/A%) (N/A%) (1.4%)
N/A ± N/A ± N/A ± 30.2 ±

100170 0% (0/1) N/A 0% (0/1) N/A 0% (0/1) N/A 100% (1/1) N/A
(N/A%) (N/A%) (N/A%) (N/A)
N/A ± N/A ± N/A ± 30.1 ±

100215 0% (0/4) N/A 0% (0/4) N/A 0% (0/4) N/A 100% (4/4) 0.2
(N/A%) (N/A%) (N/A%) (0.8%)
NTC
N/A ± N/A ± N/A ± 30.8 ±
100305 0% (0/1) N/A 0% (0/1) N/A 0% (0/1) N/A 100% (1/1) N/A
(N/A%) (N/A%) (N/A%) (N/A)
N/A ± N/A ± N/A ± 30.8 ±

100445 0% (0/4) N/A 0% (0/4) N/A 0% (0/4) N/A 100% (4/4) 0.2
(N/A%) (N/A%) (N/A%) (0.7%)
N/A ± N/A ± N/A ± 30.5 ±

100%
All 0% (0/12) N/A 0% (0/12) N/A 0% (0/12) N/A 0.4
(12/12)
(N/A%) (N/A%) (N/A%) (1.3%)
26.9 ± 27.7 ± 27.8 ± 30.9 ±

100122 100% (2/2) 0.3 100% (2/2) 0.9 100% (2/2) 1.5 100% (2/2) 0.7
(1.0%) (3.1%) (5.3%) (2.3%)
PC
26.2 ± 27.6 ± 28.4 ± 29.8 ±
100170 100% (1/1) N/A 100% (1/1) N/A 100% (1/1) N/A 100% (1/1) N/A
(N/A) (N/A) (N/A) (N/A)
Page 108 of 250
% % % %
Instrument
Control Detection Mean Detection Mean Ct Detection Mean Ct Detection Mean
SN
Ct ± SD ± SD ± SD Ct ± SD
(#Detected/ (%CV) (#Detected/ (%CV) (#Detected/ (%CV) (#Detected/ (%CV)
27.3 ± 27.1 ± 26.6 ± 30.8 ±

100215 100% (4/4) 0.1 100% (4/4) 0.1 100% (4/4) 0.3 100% (4/4) 0.3
(0.5%) (0.4%) (0.9%) (1.0%)
27.1 ± 26.9 ± 26.5 ± 30.4 ±

100305 100% (1/1) N/A 100% (1/1) N/A 100% (1/1) N/A 100% (1/1) N/A
(N/A) (N/A) (N/A) (N/A)
26.7 ± 27.1 ± 26.6 ± 30.7 ±

100445 100% (4/4) 0.2 100% (4/4) 0.2 100% (4/4) 0.2 100% (4/4) 0.2
(0.9%) (0.8%) (0.9%) (0.8%)
26.9 ± 27.2 ± 26.9 ± 30.7 ±

100% 100% 100% 100%
All 0.4 0.4 0.8 0.4
(12/12) (12/12) (12/12) (12/12)
(1.4%) (1.5%) (3.1%) (1.4%)
Det = Detected, Ct = Cycle threshold, SD = Standard Deviation, %CV = Percent Coefficient of Variation, N/A = Not applicable
Conclusions – Cross-Reactivity
Cross-reactivity was not observed with the Candidate Device and the 47 organisms tested and
all controls performed as expected.
Section 18.1 Attachments (Documents) Attachment Number
Cross Reactivity Protocol 18.1-1
Cross Reactivity BLAST Results 18.1-2
Cross Reactivity Line Data (pdf) 18.1-3
Cross Reactivity Line Data (xls) 18.1-4

Page 109 of 250
18.2 Inhibition by Other Organisms (Microbial Inhibition)
The Candidate Device was evaluated for microbial inhibition by testing 44 different viruses,
bacteria and fungi that could be found in nasopharyngeal swab specimens. The study was
performed at DiaSorin Molecular, Cypress, CA using eight (8) LIAISON MDX Instruments
(100090, 100122, 100157, 100215, 100222, 100305, 100336 and 100445), one (1) lot of
Candidate Device kit (Lot V11909N), three (3) lots of Candidate Device Positive Control Packs
Lots V11808N, V11923N and V12441N) and two (2) lots of Direct Amplification Discs (Lots
11474N and R9211NA). A total of 51 experimental runs were performed by two (2) operators
over the course of eight (8) non-consecutive days (18 and 21-24 June 2021, and 06, 09 and 15
July 2021).
All samples were prepared by spiking each potentially inhibiting organism into a baseline
consisting of Flu A, Flu B and COVID-19 viral particles at 2x LoD, in pooled negative
nasopharyngeal swab (NPS) in UTM matrix. RNAsin was added at a concentration of 1.6 U/µL
for samples that used viral RNA. For potentially inhibiting bacteria or fungi, the testing
concentration was 1x106 CFU/mL; for viruses, 1x105 TCID50/mL. For organisms that were not
able to be acquired at these concentrations they were tested via in silico analysis using BLAST
(Basic Local Alignment Search Tool) provided by NCBI (National Center for Biotechnology
Information). For organisms not titered in CFU/mL or TCID50/mL, other industry acceptable units
were used.
DiaSorin Molecular added one (1) additional sample to the panel which was a pooled nasal
human fluid was added to the panel to represent a diverse microbial flora.
Stocks of Bacillus anthracis, Chlamydophila psittaci, Influenza C and Pneumocystis jirovecii were
not available for testing. For these organisms, in silico (BLAST) analysis was performed. In
addition, Human Coronavirus 229E, Human Coronavirus NL63, Human Metapneumovirus 9 and
Rhinovirus 1A could not be tested at 1x105 TCID50/mL due to not being able to obtain a stock of
sufficient concentration. These organisms were tested at a lower concentration (1x104
TCID50/mL) and also tested via in silico (BLAST) analysis. The results of the BLAST analyses
showed no risk of microbial inhibition.
The viral particles of human coronavirus HKU1 were not commercially available at the time of
the study, therefore the potentially inhibiting effect of human coronavirus HKU1 was evaluated
through the use of its viral RNA genome.
The components of commercial transport media could have an adverse impact on the ability to
detect viral RNA when the RNA nucleic acid is directly diluted into the transport media, without
being packaged into a nucleocapsid1.
1
. Pathology. 2020 Dec; 52(7): 811–814. Published online 2020 Oct “The impact of viral transport
media on PCR assay results for the detection of nucleic acid from SARS-CoV-2”
P.D.Kirkland and M.J. Frost
Page 110 of 250
In order to prevent the degradation of viral RNA genome, HKU1 RNA was spiked in pooled
negative NPS matrix in UTM with the addition of RNasin at a concentration of 1.6 U/µL in the
presence of low concentrations of Flu A, Flu B and COVID-19 viral particles. RNasin is a
ribonuclease inhibitor that works inhibiting RNase activity, avoiding the degradation of viral RNA
genome and ensuring its integrity.
DiaSorin Molecular contacted suppliers of viral particles (ATCC and Microbiologics) again at the
end of January 2022, to no avail as the suppliers were still unable to provide human coronavirus
HKU1 viral particles.
Table 18.2.1 presents a summary of the microbial inhibition results. At a concentration of 1x104
U/mL, Rhinovirus 1A had one (1) replicate not detected for Flu A. Upon testing an additional 17
replicates, the total number of replicates detected for Flu A was 19 out of 20 replicates. Therefore,
no microbial inhibition with Rhinovirus 1A was observed with the Candidate Device.
Table 18.2.1. Summary of Microbial Inhibition Results
Flu A Flu B Internal

COVID-19 Control
% Detection % Detection % Detection % Detection
Organism Tested Concentration
(#Detected/ (#Detected/ (#Detected/ (#Detected/
Baseline 1 N/A 100% (5/5) 100% (5/5) 100% (5/5) 100% (5/5)
Baseline 1 N/A 100% (5/5) 100% (5/5) 100% (5/5) 100% (5/5)
Baseline 1 N/A 100% (5/5) 100% (5/5) 100% (5/5) 100% (5/5)
Baseline 1 N/A 100% (5/5) 100% (5/5) 100% (5/5) 100% (5/5)
Baseline 1 N/A 100% (5/5) 100% (5/5) 100% (5/5) 100% (5/5)
Baseline 2 N/A 100% (5/5) 100% (5/5) 100% (5/5) 100% (5/5)
Baseline 2.5 N/A 100% (5/5) 100% (5/5) 100% (5/5) 100% (5/5)
Baseline 2.5 N/A 100% (5/5) 100% (5/5) 100% (5/5) 100% (5/5)
Baseline 2.5 N/A 100% (5/5) 100% (5/5) 100% (5/5) 100% (5/5)
Baseline 3 N/A 100% (5/5) 100% (5/5) 100% (5/5) 100% (5/5)
Microorganisms
Adenovirus Type 1 1 x 105 TCID50/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Page 111 of 250

COVID-19 Control
Adenovirus Type 7A 1 x 105 TCID50/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Bordetella pertussis 1 x 106 CFU/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Candida albicans 1 x 106 CFU/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Chlamydia pneumoniae 1 x 106 IFU/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Corynebacterium diphtheriae 1 x 106 CFU/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
1 x 106
Coxiella burnetii 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
genome copies/mL
Cytomegalovirus 1 x 105 U/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Enterovirus Type 68 1 x 105 U/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Enterovirus Type 71 1 x 105 TCID50/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Epstein-Barr Virus 1 x 105 copies/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Escherichia coli O157:H7 1 x 106 CFU/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Haemophilus influenzae 1 x 106 CFU/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Human Coronavirus 229E* 1 x 104 TCID50/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Human Coronavirus NL63* 1 x 104 U/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Human Coronavirus OC43 1 x 105 TCID50/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Human Metapneumovirus 9* 1 x 104 TCID50/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Lactobacillus plantarum,17-5 1 x 106 CFU/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Legionella longbeachae 1 x 106 CFU/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Legionella pneumophila 1 x 106 CFU/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Leptospira interrogans 1 x 106 copies/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Measles 1 x 105 TCID50/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
MERS-Coronavirus 1 x 105 TCID50/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Page 112 of 250

COVID-19 Control
Moraxella catarrhalis 1 x 106 CFU/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Mumps 1 x 105 U/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Mycobacterium tuberculosis
1 x 106 copies/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Genomic DNA
Mycoplasma pneumoniae 1 x 106 CCU/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Neisseria elongata 1 x 106 CFU/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Neisseria meningitidis 1 x 106 CFU/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Parainfluenza Type 1 1 x 105 U/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Parainfluenza Type 2 1 x 105 TCID50/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Parainfluenza Type 3 1 x 105 TCID50/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Parainfluenza Type 4 1 x 105 U/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Parechovirus Type 3 1 x 105 U/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Pseudomonas aeruginosa 1 x 106 CFU/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
100% 100% 100%

Rhinovirus 1A* 1 x 104 U/mL 95% (19/20)
(20/20) (20/20) (20/20)
RSV-A 1 x 105 TCID50/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
RSV-B 1 x 105 TCID50/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Staphylococcus aureus 1 x 106 CFU/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Staphylococcus epidermidis 1 x 106 CFU/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Streptococcus pneumoniae 1 x 106 CFU/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Streptococcus pyogenes 1 x 106 CFU/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Streptococcus salivarius 1 x 106 CFU/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Human Coronavirus RNA HKU1 1 x 105 U/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
Page 113 of 250

COVID-19 Control
Other
Human Genomic DNA

1 x 106 cells/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
(Leukocytes)
Pooled Human Nasal Wash 1:1 dilution 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
SARS-COV1 Synthetic RNA 1 x 105 U/mL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
*Unable to obtain stock material with high titer

N/A = Not applicable, Ct = Cycle threshold, %CV = Percent Coefficient of Variation, SD = standard deviation, U/mL = Units/mL,
TCID50/mL = Tissue culture infectious dose, CCU/mL = Color changing units/mL,CFU/mL = Colony forming units/mL, IFU/mL =
Infectious units/mL
Table 18.2.2 shows the results from Table 18.2.1 showing the Mean Ct ± Standard Deviation
(SD) and the percent coefficient of variation for the microbial inhibition panel tested replicates for
each assay target.
Table 18.2.2. Mean Ct ± Standard Deviation (SD) and the percent coefficient of
variation (%CV) for the Microbial Inhibition Panel Tested Replicates for Each
Assay Target
Flu A Flu B COVID-19 Internal Control
Tested % Det Mean % Det Mean % Det Mean % Det Mean

Organism
Concentration Ct ± Ct ± Ct ± Ct ±
(# Det/ SD (# Det/ SD (# Det/ SD (# Det/ SD
# Test) (%CV) # Test) (%CV) # Test) (%CV) # Test) (%CV)
31.9 ± 29.9 ± 28.6 ± 30.1 ±

100% 100% 100% 100%
Baseline 1 N/A 0.22 0.26 0.39 0.23
(5/5) (5/5) (5/5) (5/5)
(0.7%) (0.9%) (1.4%) (0.8%)
31.9 ± 30.4 ± 29.2 ± 30.6 ±

100% 100% 100% 100%
Baseline 1 N/A 0.33 0.33 0.16 0.15
(5/5) (5/5) (5/5) (5/5)
(1.0%) (1.1%) (0.6%) (0.5%)
31.7 ± 30.8 ± 29.1 ± 30.8 ±

100% 100% 100% 100%
Baseline 1 N/A 0.41 0.70 0.05 0.11
(5/5) (5/5) (5/5) (5/5)
(1.3%) (2.3%) (0.2%) (0.4%)
Page 114 of 250

Organism
32.2 ± 30.3 ± 28.7 ± 30.3 ±

100% 100% 100% 100%
Baseline 1 N/A 0.34 0.17 0.23 0.32
(5/5) (5/5) (5/5) (5/5)
(1.1%) (0.6%) (0.8%) (1.1%)
31.9 ± 30.6 ± 29.4 ± 30.8 ±

100% 100% 100% 100%
Baseline 1 N/A 0.57 0.59 0.44 0.29
(5/5) (5/5) (5/5) (5/5)
(1.8%) (1.9%) (1.5%) (0.9%)
33.1 ± 31.3 ± 29.9 ± 30.7 ±

100% 100% 100% 100%
Baseline 2 N/A 0.83 0.72 0.41 0.18
(5/5) (5/5) (5/5) (5/5)
(2.5%) (2.3%) (1.4%) (0.6%)
33.2 ± 31 ± 30.1 ± 30.2 ±

100% 100% 100% 100%
Baseline 2.5 N/A 0.74 0.65 0.30 0.19
(5/5) (5/5) (5/5) (5/5)
(2.2%) (2.1%) (1.0%) (0.6%)
32.4 ± 30.7 ± 29.3 ± 29.6 ±

100% 100% 100% 100%
Baseline 2.5 N/A 0.32 0.58 0.26 0.10
(5/5) (5/5) (5/5) (5/5)
(1.0%) (1.9%) (0.9%) (0.3%)
33.1 ± 31.3 ± 30 ± 30.5 ±

100% 100% 100% 100%
Baseline 2.5 N/A 0.53 0.80 0.42 0.19
(5/5) (5/5) (5/5) (5/5)
(1.6%) (2.6%) (1.4%) (0.6%)
33.2 ± 31.6 ± 30.2 ± 30.5 ±

100% 100% 100% 100%
Baseline 3 N/A 0.44 1 0.39 0.29
(5/5) (5/5) (5/5) (5/5)
(1.3%) (3.2%) (1.3%) (1.0%)
Microorganisms
33.6 ± 31.4 ± 30 ± 30.6 ±

1 x 105 100% 100% 100% 100%
Adenovirus Type 1 0.70 0.29 0.15 0.06
TCID50/mL (3/3) (3/3) (3/3) (3/3)
(2.1%) (0.9%) (0.5%) (0.2%)
33.1 ± 32 ± 29.9 ± 30.6 ±

1 x 105 100% 100% 100% 100%
Adenovirus Type 7A 0.90 0.35 0.15 0.06
TCID50/mL (3/3) (3/3) (3/3) (3/3)
(2.7%) (1.1%) (0.5%) (0.2%)
34.2 ± 31.3 ± 30.4 ± 30.9 ±

1 x 106 100% 100% 100% 100%
Bordetella pertussis 1.50 0.36 0.15 0.36
CFU/mL (3/3) (3/3) (3/3) (3/3)
(4.4%) (1.2%) (0.5%) (1.2%)
Page 115 of 250

Organism
32.8 ± 31 ± 30.1 ± 31 ±
1 x 106 100% 100% 100% 100%
Candida albicans 0.36 0.40 0.26 0.15
CFU/mL (3/3) (3/3) (3/3) (3/3)
(1.1%) (1.3%) (0.9%) (0.5%)
33.7 ± 31.9 ± 30.6 ± 30.8 ±

Chlamydia 100% 100% 100% 100%
1 x 106 IFU/mL 0.06 0.35 0.42 0.00
pneumoniae (3/3) (3/3) (3/3) (3/3)
(0.2%) (1.1%) (1.4%) (0.0%)
33.2 ± 31.5 ± 30 ± 30.8 ±

Corynebacterium 1 x 106 100% 100% 100% 100%
0.40 0.06 0.12 0.47
diphtheriae CFU/mL (3/3) (3/3) (3/3) (3/3)
(1.2%) (0.2%) (0.4%) (1.5%)
1 x 106
33.1 ± 31.4 ± 29.7 ± 31.2 ±
100% 100% 100% 100%
Coxiella burnetii 0.56 0.60 0.32 0.06
genome (3/3) (3/3) (3/3) (3/3)
(1.7%) (1.9%) (1.1%) (0.2%)
copies/mL
33.6 ± 31.1 ± 30 ± 30.4 ±

100% 100% 100% 100%
Cytomegalovirus 1 x 105 U/mL 0.45 0.47 0.29 0.21
(3/3) (3/3) (3/3) (3/3)
(1.3%) (1.5%) (1.0%) (0.7%)
33.1 ± 31.2 ± 30.4 ± 30.7 ±

100% 100% 100% 100%
Enterovirus Type 68 1 x 105 U/mL 0.65 0.30 0.40 0.36
(3/3) (3/3) (3/3) (3/3)
(2.0%) (1.0%) (1.3%) (1.2%)
33.6 ± 31.4 ± 29.8 ± 30.6 ±

1 x 105 100% 100% 100% 100%
Enterovirus Type 71 0.31 0.45 0.40 0.12
TCID50/mL (3/3) (3/3) (3/3) (3/3)
(0.9%) (1.4%) (1.3%) (0.4%)
33.4 ± 31.4 ± 30.4 ± 30.8 ±

1 x 105 100% 100% 100% 100%
Epstein-Barr Virus 0.42 0.15 0.32 0.38
copies/mL (3/3) (3/3) (3/3) (3/3)
(1.3%) (0.5%) (1.1%) (1.2%)
33.7 ± 30.5 ± 29.9 ± 30.4 ±

Escherichia coli 1 x 106 100% 100% 100% 100%
1.70 0.40 0.36 0.06
O157:H7 CFU/mL (3/3) (3/3) (3/3) (3/3)
(5.0%) (1.3%) (1.2%) (0.2%)
33.2 ± 31.2 ± 30.2 ± 30.7 ±

1 x 106 100% 100% 100% 100%
Haemophilus influenza 0.12 0.36 0.31 0.06
CFU/mL (3/3) (3/3) (3/3) (3/3)
(0.4%) (1.2%) (1.0%) (0.2%)
Page 116 of 250

Organism
32.7 ± 31.6 ± 29.2 ± 30.6 ±

Human Coronavirus 1 x 104 100% 100% 100% 100%
0.72 1.70 0.93 0.15
229E* TCID50/mL (3/3) (3/3) (3/3) (3/3)
(2.2%) (5.4%) (3.2%) (0.5%)
33.9 ± 31.1 ± 29.6 ± 30.6 ±

Human Coronavirus 100% 100% 100% 100%
1 x 104 U/mL 0.72 0.21 0.70 0.10
NL63* (3/3) (3/3) (3/3) (3/3)
(2.1%) (0.7%) (2.4%) (0.3%)
33.2 ± 31.5 ± 29.9 ± 30.7 ±

Human Coronavirus 1 x 105 100% 100% 100% 100%
0.31 0.44 0.49 0.40
OC43 TCID50/mL (3/3) (3/3) (3/3) (3/3)
(0.9%) (1.4%) (1.6%) (1.3%)
33.3 ± 31.9 ± 29.6 ± 30.8 ±

Human 1 x 104 100% 100% 100% 100%
0.15 0.87 0.57 0.20
Metapneumovirus 9* TCID50/mL (3/3) (3/3) (3/3) (3/3)
(0.5%) (2.7%) (1.9%) (0.7%)
33.6 ± 30.8 ± 30 ± 30.7 ±

Lactobacillus 1 x 106 100% 100% 100% 100%
0.68 0.38 0.51 0.20
plantarum, 17-5 CFU/mL (3/3) (3/3) (3/3) (3/3)
(2.0%) (1.2%) (1.7%) (0.7%)
33.2 ± 31.1 ± 30.1 ± 30.9 ±

Legionella 1 x 106 100% 100% 100% 100%
0.40 0.71 0.20 0.23
longbeachae CFU/mL (3/3) (3/3) (3/3) (3/3)
(1.2%) (2.3%) (0.7%) (0.7%)
33.2 ± 30.7 ± 29.7 ± 30.3 ±

Legionella 1 x 106 100% 100% 100% 100%
1.14 0.74 0.61 0.31
pneumophila CFU/mL (3/3) (3/3) (3/3) (3/3)
(3.4%) (2.4%) (2.1%) (1.0%)
32.8 ± 30.9 ± 29.4 ± 29.9 ±

1 x 106 100% 100% 100% 100%
Leptospira interrogans 0.21 0.46 0.62 0.25
copies/mL (3/3) (3/3) (3/3) (3/3)
(0.6%) (1.5%) (2.1%) (0.8%)
32.6 ± 30.8 ± 29.4 ± 29.9 ±

1 x 105 100% 100% 100% 100%
Measles 0.40 1.04 0.36 0.26
TCID50/mL (3/3) (3/3) (3/3) (3/3)
(1.2%) (3.4%) (1.2%) (0.9%)
33.1 ± 30.3 ± 28.8 ± 30 ±

1 x 105 100% 100% 100% 100%
MERS-Coronavirus 0.98 0.38 0.38 0.38
TCID50/mL (3/3) (3/3) (3/3) (3/3)
(3.0%) (1.3%) (1.3%) (1.3%)
33 ± 30.9 ± 29.8 ± 30.3 ±

1 x 106 100% 100% 100% 100%
Moraxella catarrhalis 0.95 0.68 0.30 0.20
CFU/mL (3/3) (3/3) (3/3) (3/3)
(2.9%) (2.2%) (1.0%) (0.7%)
Page 117 of 250

Organism
32.8 ± 31.5 ± 30.2 ± 30.9 ±

100% 100% 100% 100%
Mumps 1 x 105 U/mL 0.36 0.95 0.76 0.44
(3/3) (3/3) (3/3) (3/3)
(1.1%) (3.0%) (2.5%) (1.4%)
Mycobacterium 33.7 ± 31.4 ± 30 ± 30.7 ±

1 x 106 100% 100% 100% 100%
tuberculosis 0.40 0.31 0.66 0.20
copies/mL (3/3) (3/3) (3/3) (3/3)
Genomic DNA (1.2%) (1.0%) (2.2%) (0.7%)
33.5 ± 31.2 ± 29.9 ± 30.8 ±

Mycoplasma 1 x 106 100% 100% 100% 100%
0.95 0.89 0.12 0.21
pneumoniae CCU/mL (3/3) (3/3) (3/3) (3/3)
(2.8%) (2.9%) (0.4%) (0.7%)
32.9 ± 30.9 ± 29.9 ± 30.8 ±

1 x 106 100% 100% 100% 100%
Neisseria elongata 0.53 0.32 0.50 0.35
CFU/mL (3/3) (3/3) (3/3) (3/3)
(1.6%) (1.0%) (1.7%) (1.1%)
34 ± 31.1 ± 30.1 ± 30.6 ±

1 x 106 100% 100% 100% 100%
Neisseria meningitidis 1.01 1.87 0.40 0.21
CFU/mL (3/3) (3/3) (3/3) (3/3)
(3.0%) (6.0%) (1.3%) (0.7%)
33.8 ± 31.3 ± 29.7 ± 30.6 ±

100% 100% 100% 100%
Parainfluenza Type 1 1 x 105 U/mL 1.21 0.15 0.40 0.23
(3/3) (3/3) (3/3) (3/3)
(3.6%) (0.5%) (1.4%) (0.8%)
32.9 ± 31.7 ± 30 ± 30.7 ±

1 x 105 100% 100% 100% 100%
Parainfluenza Type 2 0.49 0.56 0.86 0.40
TCID50/mL (3/3) (3/3) (3/3) (3/3)
(1.5%) (1.8%) (2.9%) (1.3%)
33 ± 31.9 ± 30 ± 30.4 ±
1 x 105 100% 100% 100% 100%
Parainfluenza Type 3 0.60 1.10 0.47 0.40
TCID50/mL (3/3) (3/3) (3/3) (3/3)
(1.8%) (3.5%) (1.6%) (1.3%)
32.8 ± 30.7 ± 30 ± 30.5 ±

100% 100% 100% 100%
Parainfluenza Type 4 1 x 105 U/mL 0.76 0.47 0.46 0.21
(3/3) (3/3) (3/3) (3/3)
(2.3%) (1.5%) (1.5%) (0.7%)
32.7 ± 30.3 ± 30 ± 30.4 ±

100% 100% 100% 100%
Parechovirus Type 3 1 x 105 U/mL 0.61 0.35 0.75 0.10
(3/3) (3/3) (3/3) (3/3)
(1.9%) (1.2%) (2.5%) (0.3%)
35.2 ± 30.7 ± 30.2 ± 30.8 ±

Pseudomonas 1 x 106 100% 100% 100% 100%
1.50 0.57 0.06 0.21
aeruginosa CFU/mL (3/3) (3/3) (3/3) (3/3)
(4.3%) (1.9%) (0.2%) (0.7%)
Page 118 of 250

Organism
32.9 ± 30.9 ± 29.5 ± 30.2 ±

95% 100% 100% 100%
Rhinovirus 1A* 1 x 104 U/mL 0.59 0.47 0.50 0.51
(19/20) (20/20) (20/20) (20/20)
(1.8%) (1.5%) (1.7%) (1.7%)
32.6 ± 30.8 ± 29.5 ± 30.2 ±

1 x 105 100% 100% 100% 100%
RSV-A 0.95 0.55 0.40 0.35
TCID50/mL (3/3) (3/3) (3/3) (3/3)
(2.9%) (1.8%) (1.4%) (1.2%)
33.3 ± 31.4 ± 30.3 ± 31 ±

1 x 105 100% 100% 100% 100%
RSV-B 0.53 0.50 0.12 0.06
TCID50/mL (3/3) (3/3) (3/3) (3/3)
(1.6%) (1.6%) (0.4%) (0.2%)
33.6 ± 30.9 ± 30.7 ± 30.9 ±

Staphylococcus 1 x 106 100% 100% 100% 100%
0.75 0.45 0.51 0.40
aureus CFU/mL (3/3) (3/3) (3/3) (3/3)
(2.2%) (1.5%) (1.7%) (1.3%)
34.1 ± 31.8 ± 29.8 ± 30.7 ±

Staphylococcus 1 x 106 100% 100% 100% 100%
1.01 0.53 0.31 0.56
epidermidis CFU/mL (3/3) (3/3) (3/3) (3/3)
(3.0%) (1.7%) (1.0%) (1.8%)
32.8 ± 31.7 ± 30.3 ± 30.5 ±

Streptococcus 1 x 106 100% 100% 100% 100%
0.53 0.40 0.66 0.51
pneumoniae CFU/mL (3/3) (3/3) (3/3) (3/3)
(1.6%) (1.3%) (2.2%) (1.7%)
34.2 ± 33.2 ± 32.1 ± 32 ±

Streptococcus 1 x 106 100% 100% 100% 100%
1.03 1.25 1.05 0.25
pyogenes CFU/mL (3/3) (3/3) (3/3) (3/3)
(3.0%) (3.8%) (3.3%) (0.8%)
33.3 ± 30.9 ± 29.7 ± 30.7 ±

Streptococcus 1 x 106 100% 100% 100% 100%
0.75 0.17 0.26 0.15
salivarius CFU/mL (3/3) (3/3) (3/3) (3/3)
(2.3%) (0.6%) (0.9%) (0.5%)
33.2 ± 32.3 ± 29.4 ± 30.4 ±

Human Coronavirus 100% 100% 100% 100%
1 x 105 U/mL 0.96 0.46 0.21 0.12
RNA HKU1 (3/3) (3/3) (3/3) (3/3)
(2.9%) (1.4%) (0.7%) (0.4%)
Other
33.5 ± 31.5 ± 30 ± 31 ±
Human Genomic DNA 1 x 106 100% 100% 100% 100%
0.47 0.65 0.20 0.12
(Leukocytes) cells/mL (3/3) (3/3) (3/3) (3/3)
(1.4%) (2.1%) (0.7%) (0.4%)
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Organism
33.1 ± 30.8 ± 30.5 ± 30.1 ±

Pooled Human Nasal 100% 100% 100% 100%
1:1 dilution 0.95 0.44 0.38 0.12
Wash (3/3) (3/3) (3/3) (3/3)
(2.9%) (1.4%) (1.3%) (0.4%)
33.6 ± 31.4 ± 29.9 ± 30.4 ±

SARS-COV1 Synthetic 100% 100% 100% 100%
1 x 105 U/mL 0.57 0.38 0.44 0.06
RNA (3/3) (3/3) (3/3) (3/3)
(1.7%) (1.2%) (1.5%) (0.2%)
*Unable to obtain stock material with high titer,

Det = Detected, Test = Tested, Ct = Cycle Threshold, SD = Standard Deviation, %CV = Percent Coefficient of Variation, N/A = Not
Applicable, U/mL = Units/mL, TCID50/mL = Tissue culture infectious dose, CCU/mL = Color changing units/mL, CFU/mL = Colony
forming units/mL, IFU/mL = Infectious units/mL
Summary of Control Results – Microbial Inhibition
Table 18.2.3. Results for Controls – Microbial Inhibition

Instrument % Mean % Mean % Mean % Mean

Control
SN Detection Ct ± Detection Ct ± Detection Ct ± Detection Ct ±
(#Detection/ SD (#Detection/ SD (#Detection/ SD (#Detection/ SD
#Tested) (%CV) #Tested) (%CV) #Tested) (%CV) #Tested) (%CV)
N/A ± N/A ± N/A ± 29.9 ±

100090 0.0% (0/3) N/A 0.0% (0/3) N/A 0.0% (0/3) N/A 100.0% (3/3) 0.26
(N/A%) (N/A%) (N/A%) (0.9%)
N/A ± N/A ± N/A ± 31.4 ±

100122 0.0% (0/3) N/A 0.0% (0/3) N/A 0.0% (0/3) N/A 100.0% (3/3) 1.67
(N/A%) (N/A%) (N/A%) (5.3%)
NTC
N/A ± N/A ± N/A ± 30.2 ±
100157 0.0% (0/2) N/A 0.0% (0/2) N/A 0.0% (0/2) N/A 100.0% (2/2) 0.14
(N/A%) (N/A%) (N/A%) (0.5%)
N/A ± N/A ± N/A ± 30.8 ±

100215 0.0% (0/5) N/A 0.0% (0/5) N/A 0.0% (0/5) N/A 100.0% (5/5) 0.82
(N/A%) (N/A%) (N/A%) (2.7%)
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Control
N/A ± N/A ± N/A ± 31 ±

100222 0.0% (0/1) N/A 0.0% (0/1) N/A 0.0% (0/1) N/A 100.0% (1/1) N/A
(N/A%) (N/A%) (N/A%) (N/A%)
N/A ± N/A ± N/A ± 30.9 ±

100305 0.0% (0/5) N/A 0.0% (0/5) N/A 0.0% (0/5) N/A 100.0% (5/5) 0.96
(N/A%) (N/A%) (N/A%) (3.1%)
N/A ± N/A ± N/A ± 29.3 ±

100336 0.0% (0/1) N/A 0.0% (0/1) N/A 0.0% (0/1) N/A 100.0% (1/1) N/A
(N/A%) (N/A%) (N/A%) (N/A%)
N/A ± N/A ± N/A ± 31.1 ±

100445 0.0% (0/4) N/A 0.0% (0/4) N/A 0.0% (0/4) N/A 100.0% (4/4) 0.74
(N/A%) (N/A%) (N/A%) (2.4%)
N/A ± N/A ± N/A ± 30.7 ±

100%
All 0.0% (0/24) N/A 0.0% (0/24) N/A 0.0% (0/24) N/A 0.94
(24/24)
(N/A%) N/A%) (N/A%) (3.1%)
27.2 ± 27.8 ± 27.3 ± 30.2 ±

100090 100.0% (3/3) 2.54 100.0% (3/3) 2.45 100.0% (3/3) 1.54 100.0% (3/3) 0.60
(9.3%) (8.8%) (5.6%) (2.0%)
26.3 ± 27.5 ± 27.7 ± 31.1 ±

100122 100.0% (3/3) 0.46 100.0% (3/3) 0.78 100.0% (3/3) 1.13 100.0% (3/3) 0.74
(1.8%) (2.8%) (4.1%) (2.4%)
26.3 ± 27.6 ± 27.5 ± 30 ±

100157 100.0% (2/2) 0.5 100.0% (2/2) 1.13 100.0% (2/2) 1.41 100.0% (2/2) 0.57
(1.9%) (4.1%) (5.1%) (1.9%)
PC
26.2 ± 27.1 ± 27.1 ± 30.8 ±
100215 100.0% (5/5) 0.3 100.0% (5/5) 0.68 100.0% (5/5) 0.94 100.0% (5/5) 0.64
(1.2%) (2.5%) (3.5%) (2.1%)
26.6 ± 28.5 ± 28.6 ± 30.7 ±

100222 100.0% (1/1) N/A 100.0% (1/1) N/A 100.0% (1/1) N/A 100.0% (1/1) N/A
(N/A%) (N/A%) (N/A%) (N/A%)
26.5 ± 27.6 ± 27.3 ± 30.5 ±

100305 100.0% (5/5) 0.50 100.0% (5/5) 0.94 100.0% (5/5) 1.01 100.0% (5/5) 0.39
(1.9%) (3.4%) (3.7%) (1.3%)
Page 121 of 250

Control
26.2 ± 27.7 ± 27.2 ± 29.2 ±

100336 100.0% (1/1) N/A 100.0% (1/1) N/A 100.0% (1/1) N/A 100.0% (1/1) N/A
(N/A%) (N/A%) (N/A%) (N/A%)
26.5 ± 27.6 ± 27.6 ± 31 ±

100445 100.0% (4/4) 0.30 100.0% (4/4) 0.60 100.0% (4/4) 0.77 100.0% (4/4) 0.85
(1.1%) (2.2%) (2.8%) (2.7%)
26.5 ± 27.5 ± 27.4 ± 30.6 ±

100% 100% 100% 100%
All 0.87 1.02 0.96 0.71
(24/24) (24/24) (24/24) (24/24)
(3.3%) (3.7%) (3.5%) (2.3%)
Ct = Cycle Threshold, SD = Standard Deviation %CV = Percent Coefficient of Variation, N/A = Not Applicable
Conclusions – Microbial Inhibition
For Rhinovirus 1A at a concentration of 1x104 U/mL, there was 100% positivity for Flu B and
COVID-19 targets but there was one replicate out of three (1/3) not detected for Flu A. Upon
testing an additional 17 replicates, the total number of replicates detected for Flu A was 19 out
of 20 replicates total. Therefore, there is no microbial inhibition observed with the 47 organisms
tested with the Candidate Device.
Note: Interference with rhinovirus and influenza A is described in the following reference; A
clinical data analysis and experimental infection study, Lancet (2020).
https://www.thelancet.com/action/showPdf?pii=S2666-5247%2820%2930114-2
Section 18.2 Attachments (Document) Attachment Number
Microbial Inhibition Protocol 18.2-1
Microbial Inhibition in silico BLAST Analysis Results 18.2-2
Microbial Inhibition Line Data (pdf) 18.2-3
Microbial Inhibition Line Data (xls) 18.2-4

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18.3 Analytical Reactivity
The Candidate Device was evaluated for analytical reactivity with 63 Influenza A (Flu A) strains,
21 Influenza B (Flu B) strains and five (5) strains of COVID-19.
Each set of the baseline samples [negative nasopharyngeal swab (NPS) matrix in UTM] was
tested in quintuplicate and once the baseline results were checked for validity, each of the test
samples was tested in triplicate.
The study was performed at DiaSorin Molecular, Cypress, CA for Influenza A and Influenza B
using eight (8) LIAISON MDX Instruments (100074, 100090, 100091, 100122, 100170, 100215,
100305 and 100445), one (1) lot of Candidate Device Reaction Mix (Lot V11908N), two (2) lots
of Candidate Device Positive Control (Lots V11923N and V12441N), and one (1) lot of Direct
Amplification Disc (Lot 12170N). A total of 69 experimental runs were performed by five (5)
operators across eight (8) days (2, 9, 12, 14-16, 19 and 20 July 2021).
Influenza A (California/12/2012) and Influenza A (Japan/305/1957) stocks were not available.

Therefore, analytical reactivity to these organisms was evaluated with in silico analysis using
BLAST (Basic Local Alignment Search Tool) provided by NCBI (National Center for
Biotechnology Information). All Flu A oligonucleotides (primers and probes) indicated 100%
homology when BLAST analysis was performed for both Flu A strains Therefore, Influenza A
(California/12/2012) and Influenza A (Japan/305/1957) are considered to be detectable with the
Simplexa™ COVID-19 & Flu A/B Direct assay.
Another study was performed at Diasorin Molecular, Gerenzano, IT for COVID-19 using three (3)
LIAISON® MDX Instruments (2D0005, 2D0049, 200151) one (1) lot of Candidate Device
Reaction Mix (Lot R12804N), one (1) lot of Candidate Device Positive Control (Lot R13632N),
and one (1) lot of Direct Amplification Discs Kit (Lot 13208N). A total of seven (7) experimental
runs were performed by one (1) operator on one (1) day (09 November 2021). Samples were
prepared by one (1) operator on one (1) day (09 November 2021). Samples preparation and
testing was performed by different operators of the internal site.
Results Analytical Reactivity - Flu A Strains
All baseline replicates had no detection for Flu A, Flu B or COVID-19.
Each of the Flu A strains were initially tested at 100 TCID50/mL, 100 CEID50/mL or other industry
acceptable units.
The A/Christ Church/16/2010 strain was only detected in two (2) out of three (3) replicates at 100
EID50/mL. The A/Christ Church/16/2010 strain was tested at a higher concentration (1,000
EID50/mL) in which 100% detection was observed.
The A/duck/Czechoslovakia/1956 strain was detected in zero (0) out of three (3) replicates at
100 CEID50/mL. The A/duck/Czechoslovakia/1956 strain was tested at a higher concentration
(5,000 CEID50/mL) in which 100% detection was observed.
Page 123 of 250
The A/red knot/Delaware/541/1988 strain was only detected in one (1) out of three (3) replicates
at 100 CEID50/mL. The A/red knot/Delaware/541/1988 strain was tested at a higher
concentration (1,000 CEID50/mL) in which 100% detection was observed.
The A/shorebird/Delaware Bay/211/1994 strain was only detected in two (2) out of three (3)
replicates at 100 CEID50/mL. The A/shorebird/Delaware Bay/211/1994 strain was tested at a
higher concentration (1,000 CEID50/mL) in which 100% detection was observed.
The A/shorebird/Delaware/172/2006 strain was only detected in two (2) out of three (3) replicates
at 100 CEID50/mL. The A/shorebird/Delaware/172/2006 strain was tested at a higher
The A/turkey/Massachusetts/3740/1965 strain was only detected in one (1) out of three (3)
replicates at 100 CEID50/mL. The A/turkey/Massachusetts/3740/1965 strain was tested at a
higher concentration (2,000 CEID50/mL) in which 100% detection was observed.
For all remaining Flu A strains, there was 100% detection for Flu A.
None of the Flu A strains was detected for Flu B.
None of the Flu A strains, except for the A/black-legged kittiwake/Quebec/02838-1/2009 strain,
was detected for COVID-19.
The A/black-legged kittiwake/Quebec/02838-1/2009 strain had one (1) out of three (3) replicates
detected for COVID-19 (Ct = 34.2).
BLAST analysis showed that the COVID-19 primers and probes are not expected to bind or
produce an amplification product with this strain of influenza A. Therefore, cross-reactivity is not
the cause of the false signal. Table 18.3.1 shows the results for all influenza A strains tested.
Table 18.3.1. Flu A Analytical Reactivity Summary

Flu A
Summary
Qualitative
Statistics of
Results
Sample ID Tested Concentration Flu A Ct:
% Detection
Mean ± SD
(# Detected
(%CV)
/#Tested)
Baseline
FABC-AR BL-01 N/A 0.0% (0/5) N/A
FABC-AR BL-01 N/A 0.0% (0/5) N/A
FABC-AR BL-01 N/A 0.0% (0/5) N/A
FABC-AR BL-01 N/A 0.0% (0/5) N/A

Page 124 of 250
Flu A
Summary
Qualitative
Statistics of
Results
% Detection
Mean ± SD
(# Detected
(%CV)
/#Tested)
FABC_AR_BL_002 N/A 0.0% (0/5) N/A
FABC_AR_BL_002 N/A 0.0% (0/5) N/A
FABC_AR_BL_002 N/A 0.0% (0/5) N/A
FABC_AR_BL_002 N/A 0.0% (0/5) N/A
Organism
A/American green-winged 34.2 ± 0.55

teal/Mississippi/300/2010 100 CEID50/mL 100.0% (3/3) (1.6%)
A/black-legged kittiwake/Quebec/02838- 33.3 ± 1.21

1/2009* 100 CEID50/mL 100.0% (3/3) (3.6%)
25.2 ± 0.12
A/Brisbane/02/2018 100 EID50/mL 100.0% (3/3) (0.5%)
21.6 ± 0.21
A/Brisbane/10/07 100 U/mL 100.0% (3/3) (1.0%)
21.3 ± 0.06
A/Brisbane/59/07 100 U/mL 100.0% (3/3) (0.3%)
24.7 ± 0.21
A/California/02/2014 100 TCID50/mL 100.0% (3/3) (0.9%)
28.0 ± 0.20
28.7 ± 0.00
A/chicken/Germany/N/49 100 CEID50/mL 100.0% (3/3) (0.0%)
36.9 ± 2.12
100 EID50/mL 66.7% (2/3) (5.7%)
32.1 ± 0.38
1,000 EID50/mL 100.0% (3/3) (1.2%)
33.2 ± 0.81
A/duck/Chabarovsk/1610/1972 100 CEID50/mL 100.0% (3/3) (2.4%)
Page 125 of 250
Flu A
Summary
Qualitative
Statistics of
Results
% Detection
Mean ± SD
(# Detected
(%CV)
/#Tested)
N/A ± N/A
100 CEID50/mL 0.0% (0/3) (N/A%)
A/duck/Czechoslovakia/1956
36.0 ± 0.15
5,000 CEID50/mL 100.0% (3/3) (0.4%)
29.6 ± 0.26
A/duck/Wisconsin/480/1979 100 CEID50/mL 100.0% (3/3) (0.9%)
30.1 ± 0.10
A/Guangdong-Maonan/1536/2019 100 EID50/mL 100.0% (3/3) (0.3%)
34.7 ± 0.76
A/Hawaii/15/2001 100 CEID50/mL 100.0% (3/3) (2.2%)
31.1 ± 0.81
A/Hong Kong/2671/2019 100 EID50/mL 100.0% (3/3) (2.6%)
A/Hong Kong/33982/2009(H9N2)-PR8- 34.2 ± 2.05

IDCDC_RG26 100 CEID50/mL 100.0% (3/3) (6.0%)
32.2 ± 0.40
A/Kansas/14/2017 100 EID50/mL 100.0% (3/3) (1.2%)
33.3 ± 1.11
A/mallard/Illinois/10OS4334/2010 100 CEID50/mL 100.0% (3/3) (3.3%)
30.9 ± 0.62
A/mallard/Wisconsin/4218/2009 100 CEID50/mL 100.0% (3/3) (2.0%)
32.5 ± 0.35
34.4 ± 0.44
A/Massachusetts/15/2013 100 CEID50/mL 100.0% (3/3) (1.3%)
29.1 ± 0.57
A/Mexico/4108/2009 100 CEID50/mL 100.0% (3/3) (2.0%)
30.8 ± 0.35
A/Minnesota/19/2011 100 CEID50/mL 100.0% (3/3) (1.1%)
Page 126 of 250
Flu A
Summary
Qualitative
Statistics of
Results
% Detection
Mean ± SD
(# Detected
(%CV)
/#Tested)
22.0 ± 0.12
A/New Caledonia/20/99 100 TCID50/mL 100.0% (3/3) (0.5%)
29.8 ± 0.29
A/New York/18/2009 100 CEID50/mL 100.0% (3/3) (1.0%)
29.4 ± 0.71
A/New York/55/2004 100 CEID50/mL 100.0% (3/3) (2.4%)
31.8 ± 0.72
A/Perth/16/2009 100 EID50/mL 100.0% (3/3) (2.3%)
30.5 ± 0.10
A/quail/Italy/1117/1965 100 CEID50/mL 100.0% (3/3) (0.3%)
35.9 ± 3.66
A/red knot/Delaware Bay/240/1994 100 CEID50/mL 100.0% (3/3) (10.2%)
36.1 ± N/A
100 CEID50/mL 33.3% (1/3) (N/A%)
A/red knot/Delaware/541/1988
30.9 ± 1.63
1,000 CEID50/mL 100.0% (3/3) (5.3%)
32.3 ± 0.95
A/redhead/Alberta/192/2002 100 CEID50/mL 100.0% (3/3) (2.9%)
32.3 ± 0.40
A/Rhode Island/01/2010 100 CEID50/mL 100.0% (3/3) (1.2%)
31.4 ± 0.15
A/Santiago/7981/2006 100 CEID50/mL 100.0% (3/3) (0.5%)
35.7 ± 0.64
100 CEID50/mL 66.7% (2/3) (1.8%)
A/shorebird/Delaware Bay/211/1994
31.3 ± 0.30
1,000 CEID50/mL 100.0% (3/3) (1.0%)
34.6 ± 4.67
A/shorebird/Delaware/172/2006
100 CEID50/mL 66.7% (2/3) (13.5%)
Page 127 of 250
Flu A
Summary
Qualitative
Statistics of
Results
% Detection
Mean ± SD
(# Detected
(%CV)
/#Tested)
32.8 ± 0.99
1,000 CEID50/mL 100.0% (3/3) (3.0%)
25.8 ± 0.20
A/Solomon Island/3/2006 100 TCID50/mL 100.0% (3/3) (0.8%)
24.8 ± 0.10
A/Swine/1976/31 100 U/mL 100.0% (3/3) (0.4%)
22.7 ± 0.12
A/Swine/Iowa/15/30 100 U/mL 100.0% (3/3) (0.5%)
31.7 ± 0.47
A/swine/Ohio/09SW1477/2009 100 TCID50/mL 100.0% (3/3) (1.5%)
34.6 ± 0.79
A/swine/Ohio/09SW83E/2009 100 CEID50/mL 100.0% (3/3) (2.3%)
31.5 ± 0.21
A/Switzerland/9715293/2013 100 CEID50/mL 100.0% (3/3) (0.7%)
27.6 ± 0.20
A/Taiwan/42/06 100 U/mL 100.0% (3/3) (0.7%)
36.5 ± N/A
100 CEID50/mL 33.3% (1/3) (N/A%)
A/turkey/Massachusetts/3740/1965
31.3 ± 0.47
2,000 CEID50/mL 100.0% (3/3) (1.5%)
25.8 ± 0.06
A/WS/33 100 TCID50/mL 100.0% (3/3) (0.2%)
27.4 ± 0.12
27.6 ± 0.21
A/Hong Kong/4801/2014 100 TCID50/mL 100.0% (3/3) (0.8%)
26.0 ± 0.06
A/Indiana/08/2011 100 TCID50/mL 100.0% (3/3) (0.2%)
Page 128 of 250
Flu A
Summary
Qualitative
Statistics of
Results
% Detection
Mean ± SD
(# Detected
(%CV)
/#Tested)
31.6 ± 0.15
28.8 ± 0.25
A/NY/02/09 100 TCID50/mL 100.0% (3/3) (0.9%)
33.3 ± 0.25
A/Ohio/02/2012 100 CEID50/mL 100.0% (3/3) (0.8%)
24.6 ± 0.15
A/Port Chalmers/1/1973 100 TCID50/mL 100.0% (3/3) (0.6%)
27.7 ± 0.23
A/Singapore/INFIMH-16-0019/2016 100 TCID50/mL 100.0% (3/3) (0.8%)
26.0 ± 0.10
A/Texas/50/2012 100 TCID50/mL 100.0% (3/3) (0.4%)
26.2 ± 0.06
A/Wisconsin/67/05 100 TCID50/mL 100.0% (3/3) (0.2%)
24.9 ± 0.25
A/PR/8/34 100 TCID50/mL 100.0% (3/3) (1.0%)
29.9 ± 0.30
A/Anhui/1/2013 1:100,000 Dilution 100.0% (3/3) (1.0%)
A/chicken/Vietnam/NCVD-016/2008(H5N1)- 27.0 ± 0.15

PR8-IDCDC-RG12 1:100,000 Dilution 100.0% (3/3) (0.6%)
A/Egypt/N03072/2010(H5N1)-PR8-IDCDC- 27.1 ± 0.29

RG29 1:100,000 Dilution 100.0% (3/3) (1.1%)
28.5 ± 0.10
A/Hubei/1/2010(H5N1)-PR8-IDCDC-RG30 1:100,000 Dilution 100.0% (3/3) (0.4%)
27.8 ± 0.40
A/India/NIV/2006(H5N1)-PR8-IBCDC-RG7 1:100,000 Dilution 100.0% (3/3) (1.4%)
A/mallard/Netherlands/12/2000(H7N7)/PR8- 28.1 ± 0.21

IBCDC-1 1:100,000 Dilution 100.0% (3/3) (0.7%)
Page 129 of 250
Flu A
Summary
Qualitative
Statistics of
Results
% Detection
Mean ± SD
(# Detected
(%CV)
/#Tested)
A/pheasant/New Jersey/1355/1998(H5N2)- 27.8 ± 0.29

PR8-IBCDC-4 1:100,000 Dilution 100.0% (3/3) (1.0%)
A/turkey/Virginia/4529/2002 (H7N2)xPR8- 27.6 ± 1.76

IBCDC-5 1:100,000 Dilution 100.0% (3/3) (6.4%)
* One (1) out of three (3) replicates had COVID-19 detection [33.3% (1/3): 34.2 ± N/A (N/A%)]
Ct = Cycle Threshold, SD = Standard Deviation %CV = Percent Coefficient of Variation, N/A = Not Applicable,
TCID50 = Median Tissue Culture Infectious Dose, CEID50 = Median Chicken Embryo Infectious Dose, U= Units,
EID50 = Median Embryo Infectious Dose
Results Analytical Reactivity - Flu B strains
Replicates for all baselines had no detection for Flu A, Flu B or COVID-19.
Each of the Flu B strains were initially tested at 100 TCID50/mL, 100 CEID50/mL or other industry
acceptable units.
The B/Guangdong-Liwan/1133/2014 strain was only detected in one (1) out of three (3) replicates
at 100 CEID50/mL. The B/Guangdong-Liwan/1133/2014 strain was tested at a higher
For all remaining influenza B strains, there was 100% detection of Flu B. None of the influenza
B strains were detected in either the Flu A or COVID-19 channels. Table 18.3.2 shows the results
for the Flu B strains tested.
Page 130 of 250
Table 18.3.2. Flu B Analytical Reactivity Summary

Summary
Flu B Qualitative
Statistics
Results
Tested
Sample ID % Detection
Concentration of Flu B Ct:
(# Detected
Mean ± SD
/#Tested)
(%CV)
Baseline
FABC-ARBL-101 (100170) N/A 0.0% (0/5) N/A
FABC-ARBL-101 (100090) N/A 0.0% (0/5) N/A
FABC-ARBL-101 (100091) N/A 0.0% (0/5) N/A
FABC_AR_BL_102 (100091) N/A 0.0% (0/5) N/A
Organism
28.7 ± 0.50
B/Brisbane/33/2008 100 CEID50/mL 100.0% (3/3)
(1.7%)
22.3 ± 0.17
B/Brisbane/60/2008 100 U/mL 100.0% (3/3)
(0.8%)
23.0 ± 0.51
B/Colorado/06/2017 100 TCID50/mL 100.0% (3/3)
(2.2%)
21.5 ± 0.15
B/Florida/02/2006 100 U/mL 100.0% (3/3)
(0.7%)
28.6 ± 0.36
B/Michigan/09/2011 100 EID50/mL 100.0% (3/3)
(1.3%)
28.3 ± 0.81
B/Nevada/03/2011 100 CEID50/mL 100.0% (3/3)
(2.9%)
23.5 ± 0.12
B/Texas/02/2013 100 TCID50/mL 100.0% (3/3)
(0.5%)
31.0 ± 0.64
B/Victoria/304/2006 100 CEID50/mL 100.0% (3/3)
(2.1%)
32.1 ± 0.89
B/Washington/02/2019 100 EID50/mL 100.0% (3/3)
(2.8%)
25.3 ± 1.48
B/Christchurch/33/2004 100 TCID50/mL 100.0% (3/3)
(5.8%)
Page 131 of 250
Summary
Flu B Qualitative
Statistics
Results
Tested
Sample ID % Detection
Concentration of Flu B Ct:
(# Detected
Mean ± SD
/#Tested)
(%CV)
24.7 ± 0.68
B/Florida/04/2006 100 U/mL 100.0% (3/3)
(2.8%)
20.9 ± 0.12
B/Florida/07/04 100 U/mL 100.0% (3/3)
(0.6%)
35.3 ± N/A
100 CEID50/mL 33.3% (1/3)
(N/A%)
B/Guangdong-Liwan/1133/2014
30.4 ± 0.15
1,000 CEID50/mL 100.0% (3/3)
(0.5%)
29.6 ± 1.01
B/Maryland/1/59 100 TCID50/mL 100.0% (3/3)
(3.4%)
23.4 ± 0.15
B/Massachusetts/02/2012 100 TCID50/mL 100.0% (3/3)
(0.6%)
30.3 ± 0.12
B/New Hampshire/01/2016 100 EID50/mL 100.0% (3/3)
(0.4%)
29.1 ± 1.42
B/Panama/45/90 100 U/mL 100.0% (3/3)
(4.9%)
26.2 ± 0.56
B/Texas/81/2016 100 EID50/mL 100.0% (3/3)
(2.1%)
28.7 ± 0.32
B/Utah/09/2014 100 CEID50/mL 100.0% (3/3)
(1.1%)
26.5 ± 0.06
B/Wisconsin/01/2010 100 CEID50/mL 100.0% (3/3)
(0.2%)
29.7 ± 0.25
B/Great Lakes/1739/54 100 U/mL 100.0% (3/3)
(0.8%)
Ct = Cycle Threshold, SD = Standard Deviation %CV = Percent Coefficient of Variation, N/A = Not Applicable, TCID50 = Median
Tissue Culture Infectious Dose, CEID50 = Median Chicken Embryo Infectious Dose, U= Units, EID50 = Median Embryo Infectious
Dose
Page 132 of 250
Results Analytical Reactivity - COVID-19 strains
Replicates for the baseline had no detection in the Flu A, Flu B and COVID-19 channels.
Four (4) out of the five (5) strains of SARS-CoV-2 (Hong Kong/VM200001061/2020,
England/204820464/2020, South Africa/KRISP-EC-K005325/2020, Japan/TY7-503/2021) were
detected at a concentration of 1000 copies/mL. SARS-CoV-2 strain hCoV19/USA/PHC658/2021
was detected at a concentration of 1500 copies/mL. None of the replicates was detected in either
the Flu A or Flu B channels.
Table 18.3.3 shows the results for the COVID-19 strains tested.
Table 18.3.3. COVID-19 Analytical Reactivity Summary

FluA FluB COVID-19 IC Qualitative
Qualitative Qualitative Qualitative Results: %
Tested
Results: % Results: % Results: % Detection
COVID-19 Strain
Detection Detection Detection (# Detected
Concentration
(# Detected (# Detected (# Detected /#Tested)
Baseline N/A 0% (0/15) 0% (0/15) 0% (0/15) 100.0% (15/15)
Hong Kong/VM200001061/2020 1000 copies/mL 0% (0/3) 0% (0/3) 100% (3/3) 100.0% (3/3)
England/204820464/2020 1000 copies/mL 0% (0/3) 0% (0/3) 100% (3/3) 100.0% (3/3)
South Africa/KRISP-EC-
1000 copies/mL 0% (0/3) 0% (0/3) 100% (3/3) 100.0% (3/3)
K005325/2020
Japan/TY7-503/2021 1000 copies/mL 0% (0/3) 0% (0/3) 100% (3/3) 100.0% (3/3)
hCoV19/USA/PHC658/2021 1000 copies/mL 0% (0/3) 0% (0/3) 67% (2/3) 100.0% (3/3)
hCoV19/USA/PHC658/2021 1500 copies/mL 0% (0/3) 0% (0/3) 100% (3/3) 100.0% (3/3)
Ct = Cycle Threshold, SD = Standard Deviation %CV = Percent Coefficient of Variation, N/A = Not Applicable
Page 133 of 250
Results Analytical Reactivity Internal Control (IC) Flu A, Flu B and COVID-19 all strains
All baselines and test samples had 100% detection in the Internal Control (IC) channel except
for the A/New Caledonia/20/99 strain. Only two (2) of three (3) replicates were detected.
However, all three (3) replicates were detected in the Flu A channel. Therefore, the Internal
Control (RNA IC) detection is not required for a valid result. All three (3) replicates are valid for
the detection of the A/New Caledonia/20/99 strain.
Table 18.3.4 summarizes the IC results for all baselines and test samples.
Table 18.3.4. Analytical Reactivity IC Summary

Internal
Control (IC) Summary
Qualitative Statistics of
Tested
Set Sample ID Results IC Ct:
Concentration
% Detection Mean ± SD
(# Detected/ (%CV)
#Tested)
Baseline
30.4 ± 0.37
FABC-AR BL-01 N/A 100.0% (5/5) (1.2%)
30.3 ± 0.19
FABC-AR BL-01 N/A 100.0% (5/5) (0.6%)
31.4 ± 1.36
FABC-AR BL-01 N/A 100.0% (5/5) (4.3%)
30.8 ± 0.38
FABC-AR BL-01 N/A 100.0% (5/5) (1.2%)
Flu A
30.1 ± 0.40
FABC_AR_BL_002 N/A 100.0% (5/5) (1.3%)
29.5 ± 0.29
FABC_AR_BL_002 N/A 100.0% (5/5) (1.0%)
30.0 ± 0.54
FABC_AR_BL_002 N/A 100.0% (5/5) (1.8%)
30.2 ± 0.13
FABC_AR_BL_002 N/A 100.0% (5/5) (0.4%)
30.0 ± 0.43
Flu B FABC-ARBL-101 N/A 100.0% (5/5)
(1.4%)
Page 134 of 250
Internal
Tested
Concentration
(# Detected/ (%CV)
#Tested)
30.0 ± 0.42
FABC-ARBL-101 N/A 100.0% (5/5)
(1.4%)
29.6 ± 0.07
FABC-ARBL-101 N/A 100.0% (5/5)
(0.2%)
30.2 ± 0.22
FABC_AR_BL_102 N/A 100.0% (5/5)
(0.7%)
28.9 ± 0.5
Baseline MDX 200151 N/A 100% (5/5)
(1.8%)
28.2 ± 0.3
COVID-19 Baseline MDX 2D0005 N/A 100% (5/5)
(1.0%)
28.8 ± 0.2
Baseline MDX 2D0049 N/A 100% (5/5)
(0.8%)
Organisms
A/American green-winged 31.1 ± 0.45

100 CEID50/mL 100.0% (3/3)
teal/Mississippi/300/2010 (1.4%)
A/black-legged kittiwake/Quebec/02838- 30.6 ± 0.81

1/2009 100 CEID50/mL 100.0% (3/3) (2.6%)
30.4 ± 0.31
A/Brisbane/02/2018 100 EID50/mL 100.0% (3/3) (1.0%)
34.1 ± 0.21
Flu A
A/Brisbane/10/07 100 U/mL 100.0% (3/3) (0.6%)
34.1 ± 0.98
A/Brisbane/59/07 100 U/mL 100.0% (3/3) (2.9%)
31.1 ± 0.21
30.4 ± 0.56
Page 135 of 250
Internal
Tested
Concentration
(# Detected/ (%CV)
#Tested)
30.4 ± 0.55
A/chicken/Germany/N/49 100 CEID50/mL 100.0% (3/3) (1.8%)
31.3 ± 0.23
100 EID50/mL 100.0% (3/3) (0.7%)
1,000 29.9 ± 0.25
EID50/mL 100.0% (3/3) (0.8%)
31.1 ± 0.30
A/duck/Chabarovsk/1610/1972 100 CEID50/mL 100.0% (3/3) (1.0%)
30.7 ± 0.21
100 CEID50/mL 100.0% (3/3) (0.7%)
A/duck/Czechoslovakia/1956
5,000 29.6 ± 0.25
CEID50/mL 100.0% (3/3) (0.8%)
30.4 ± 0.25
A/duck/Wisconsin/480/1979 100 CEID50/mL 100.0% (3/3) (0.8%)
31.1 ± 0.20
A/Guangdong-Maonan/1536/2019 100 EID50/mL 100.0% (3/3) (0.6%)
31.2 ± 0.15
A/Hawaii/15/2001 100 CEID50/mL 100.0% (3/3) (0.5%)
30.9 ± 0.38
A/Hong Kong/2671/2019 100 EID50/mL 100.0% (3/3) (1.2%)
A/Hong Kong/33982/2009(H9N2)-PR8- 30.9 ± 0.20

IDCDC_RG26 100 CEID50/mL 100.0% (3/3) (0.6%)
30.9 ± 0.35
A/Kansas/14/2017 100 EID50/mL 100.0% (3/3) (1.1%)
30.9 ± 0.25
A/mallard/Illinois/10OS4334/2010 100 CEID50/mL 100.0% (3/3) (0.8%)
30.8 ± 0.31
Page 136 of 250
Internal
Tested
Concentration
(# Detected/ (%CV)
#Tested)
30.6 ± 0.21
30.4 ± 0.15
A/Massachusetts/15/2013 100 CEID50/mL 100.0% (3/3) (0.5%)
30.3 ± 0.20
A/Mexico/4108/2009 100 CEID50/mL 100.0% (3/3) (0.7%)
30.6 ± 0.15
33.8 ± 1.20
A/New Caledonia/20/99 100 TCID50/mL 66.7% (2/3) (3.6%)
30.5 ± 0.17
A/New York/18/2009 100 CEID50/mL 100.0% (3/3) (0.6%)
30.4 ± 0.44
A/New York/55/2004 100 CEID50/mL 100.0% (3/3) (1.4%)
30.5 ± 0.12
A/Perth/16/2009 100 CEID50/mL 100.0% (3/3) (0.4%)
30.4 ± 0.06
A/quail/Italy/1117/1965 100 CEID50/mL 100.0% (3/3) (0.2%)
30.4 ± 0.20
A/red knot/Delaware Bay/240/1994 100 CEID50/mL 100.0% (3/3) (0.7%)
30.7 ± 0.15
100 CEID50/mL 100.0% (3/3) (0.5%)
A/red knot/Delaware/541/1988
1,000 29.7 ± 0.06
CEID50/mL 100.0% (3/3) (0.2%)
30.3 ± 0.06
A/redhead/Alberta/192/2002 100 CEID50/mL 100.0% (3/3) (0.2%)
30.4 ± 0.32
A/Rhode Island/01/2010 100 CEID50/mL 100.0% (3/3) (1.1%)
Page 137 of 250
Internal
Tested
Concentration
(# Detected/ (%CV)
#Tested)
30.2 ± 0.12
A/Santiago/7981/2006 100 CEID50/mL 100.0% (3/3) (0.4%)
30.4 ± 0.23
100 CEID50/mL 100.0% (3/3) (0.8%)
A/shorebird/Delaware Bay/211/1994
1,000 29.7 ± 0.31
CEID50/mL 100.0% (3/3) (1.0%)
30.4 ± 0.06
100 CEID50/mL 100.0% (3/3) (0.2%)
A/shorebird/Delaware/172/2006
1,000 29.4 ± 0.15
CEID50/mL 100.0% (3/3) (0.5%)
30.3 ± 0.10
A/Solomon Island/3/2006 100 TCID50/mL 100.0% (3/3) (0.3%)
30.2 ± 0.60
A/Swine/1976/31 100 U/mL 100.0% (3/3) (2.0%)
32.9 ± 0.70
A/Swine/Iowa/15/30 100 U/mL 100.0% (3/3) (2.1%)
30.6 ± 0.47
A/swine/Ohio/09SW1477/2009 100 TCID50/mL 100.0% (3/3) (1.5%)
30.1 ± 0.06
A/swine/Ohio/09SW83E/2009 100 CEID50/mL 100.0% (3/3) (0.2%)
30.1 ± 0.32
A/Switzerland/9715293/2013 100 CEID50/mL 100.0% (3/3) (1.1%)
29.8 ± 0.23
A/Taiwan/42/06 100 U/mL 100.0% (3/3) (0.8%)
30.6 ± 0.12
100 CEID50/mL 100.0% (3/3) (0.4%)
A/turkey/Massachusetts/3740/1965
2,000 29.4 ± 0.17
CEID50/mL 100.0% (3/3) (0.6%)
Page 138 of 250
Internal
Tested
Concentration
(# Detected/ (%CV)
#Tested)
30.5 ± 0.10
A/WS/33 100 TCID50/mL 100.0% (3/3) (0.3%)
30.2 ± 0.15
30.1 ± 0.35
A/Hong Kong/4801/2014 100 TCID50/mL 100.0% (3/3) (1.2%)
30.0 ± 0.29
A/Indiana/08/2011 100 TCID50/mL 100.0% (3/3) (1.0%)
30.2 ± 0.35
30.2 ± 0.21
A/NY/02/09 100 TCID50/mL 100.0% (3/3) (0.7%)
30.2 ± 0.21
A/Ohio/02/2012 100 CEID50/mL 100.0% (3/3) (0.7%)
30.8 ± 0.30
A/Port Chalmers/1/1973 100 TCID50/mL 100.0% (3/3) (1.0%)
30.4 ± 0.21
A/Singapore/INFIMH-16-0019/2016 100 TCID50/mL 100.0% (3/3) (0.7%)
30.2 ± 0.36
A/Texas/50/2012 100 TCID50/mL 100.0% (3/3) (1.2%)
30.0 ± 0.10
A/Wisconsin/67/05 100 TCID50/mL 100.0% (3/3) (0.3%)
29.9 ± 0.32
A/PR/8/34 100 TCID50/mL 100.0% (3/3) (1.1%)
1:100,000 30.2 ± 0.10

A/Anhui/1/2013 Dilution 100.0% (3/3) (0.3%)
A/chicken/Vietnam/NCVD-016/2008(H5N1)- 1:100,000 29.9 ± 0.10

PR8-IDCDC-RG12 Dilution 100.0% (3/3) (0.3%)
Page 139 of 250
Internal
Tested
Concentration
(# Detected/ (%CV)
#Tested)
A/Egypt/N03072/2010(H5N1)-PR8-IDCDC- 1:100,000 29.5 ± 0.17

RG29 Dilution 100.0% (3/3) (0.6%)
1:100,000 29.5 ± 0.35

A/Hubei/1/2010(H5N1)-PR8-IDCDC-RG30 Dilution 100.0% (3/3) (1.2%)
1:100,000 29.6 ± 0.15

A/India/NIV/2006(H5N1)-PR8-IBCDC-RG7 Dilution 100.0% (3/3) (0.5%)
A/mallard/Netherlands/12/2000(H7N7)/PR8- 1:100,000 29.6 ± 0.15

IBCDC-1 Dilution 100.0% (3/3) (0.5%)
A/pheasant/New Jersey/1355/1998(H5N2)- 1:100,000 29.6 ± 0.21

PR8-IBCDC-4 Dilution 100.0% (3/3) (0.7%)
A/turkey/Virginia/4529/2002 (H7N2)xPR8- 1:100,000 29.5 ± 0.31

IBCDC-5 Dilution 100.0% (3/3) (1.1%)
29.6 ± 0.10
B/Brisbane/33/2008 100 CEID50/mL 100.0% (3/3)
(0.3%)
29.6 ± 0.06
B/Brisbane/60/2008 100 U/mL 100.0% (3/3)
(0.2%)
29.5 ± 0.61
B/Colorado/06/2017 100 TCID50/mL 100.0% (3/3)
(2.1%)
29.7 ± 0.17
B/Florida/02/2006 100 U/mL 100.0% (3/3)
(0.6%)
Flu B
30.3 ± 0.10
B/Michigan/09/2011 100 EID50/mL 100.0% (3/3)
(0.3%)
30.1 ± 0.15
B/Nevada/03/2011 100 CEID50/mL 100.0% (3/3)
(0.5%)
29.8 ± 0.25
B/Texas/02/2013 100 TCID50/mL 100.0% (3/3)
(0.8%)
29.9 ± 0.26
B/Victoria/304/2006 100 CEID50/mL 100.0% (3/3)
(0.9%)
Page 140 of 250
Internal
Tested
Concentration
(# Detected/ (%CV)
#Tested)
30.1 ± 0.31
B/Washington/02/2019 100 EID50/mL 100.0% (3/3)
(1.0%)
29.4 ± 0.06
B/Christchurch/33/2004 100 TCID50/mL 100.0% (3/3)
(0.2%)
30.1 ± 0.15
B/Florida/04/2006 100 U/mL 100.0% (3/3)
(0.5%)
30.3 ± 0.26
B/Florida/07/04 100 U/mL 100.0% (3/3)
(0.9%)
30.2 ± 0.85
100 CEID50/mL 100.0% (3/3)
(2.8%)
B/Guangdong-Liwan/1133/2014
1,000 30.5 ± 0.35
100.0% (3/3)
CEID50/mL (1.1%)
29.9 ± 0.21
B/Maryland/1/59 100 TCID50/mL 100.0% (3/3)
(0.7%)
29.5 ± 0.20
B/Massachusetts/02/2012 100 TCID50/mL 100.0% (3/3)
(0.7%)
29.9 ± 0.17
B/New Hampshire/01/2016 100 EID50/mL 100.0% (3/3)
(0.6%)
30.0 ± 0.67
B/Panama/45/90 100 U/mL 100.0% (3/3)
(2.2%)
30.9 ± 1.42
B/Texas/81/2016 100 EID50/mL 100.0% (3/3)
(4.6%)
29.9 ± 0.23
B/Utah/09/2014 100 CEID50/mL 100.0% (3/3)
(0.8%)
29.7 ± 0.36
B/Wisconsin/01/2010 100 CEID50/mL 100.0% (3/3)
(1.2%)
29.4 ± 0.15
B/Great Lakes/1739/54 100 U/mL 100.0% (3/3)
(0.5%)
Page 141 of 250
Internal
Tested
Concentration
(# Detected/ (%CV)
#Tested)
COVID-19 Isolate 1,000 29.6 ± 0.32

100.0% (3/3)
USA/CA_CDC_5574/2020 copies/mL (1.1%)
1000 28.8 ± 0.3

Hong Kong/VM200001061/2020 100.0% (3/3)
copies/mL (1.1%)
1000 28.8 ± 0.2

England/204820464/2020 100% (3/3)
copies/mL (0.8%)
1000 27.8 ± 0.5

COVID-19 South Africa/KRISP-EC-K005325/2020 100% (3/3)
copies/mL (1.7%)
1000 28.3 ± 0.4

Japan/TY7-503/2021 100% (3/3)
copies/mL (1.3%)
1000 28.9 ± 0.4

hCoV19/USA/PHC658/2021 100% (3/3)
copies/mL (1.2%)
1500 28.9 ± 0.3

hCoV19/USA/PHC658/2021 100% (3/3)
copies/mL (1.1%)
TCID50 = Median Tissue Culture Infectious Does, CEID50 = Median Chicken Embryo Infectious Dose, U = Units, EID50 = Median
Egg Infectivity Dose, IC = Internal Control, Ct = Cycle Threshold, SD = Standard Deviation %CV = Percent Coefficient of
Summary of Control Results - Analytical Reactivity
Table 18.3.5 shows the results of the Positive Control (PC) and No Template Control (NTC). All
controls performed as expected.
Page 142 of 250
Table 18.3.5. Results for Controls – Analytical Reactivity (Flu A, Flu B and COVID-19 Isolate
USA/CA_CDC_5574/2020)

Control
(#Detected SD (#Detected SD (#Detected SD (#Detected SD
/#Tested) (%CV) /#Tested) (%CV) /#Tested) (%CV) /#Tested) (%CV)
N/A ± N/A ± N/A ± 29.8 ±

100.0%
100074 0.0% (0/2) N/A 0.0% (0/2) N/A 0.0% (0/2) N/A 0.71
(2/2)
(N/A%) (N/A%) (N/A%) (2.4%)
N/A ± N/A ± N/A ± 29.5 ±

100.0%
100090 0.0% (0/4) N/A 0.0% (0/4) N/A 0.0% (0/4) N/A 0.25
(4/4)
(N/A%) (N/A%) (N/A%) (0.8%)
N/A ± N/A ± N/A ± 30.1 ±

100.0%
100091 0.0% (0/3) N/A 0.0% (0/3) N/A 0.0% (0/3) N/A 0.06
(3/3)
(N/A%) (N/A%) (N/A%) (0.2%)
N/A ± N/A ± N/A ± 30.9 ±

100.0%
100122 0.0% (0/4) N/A 0.0% (0/4) N/A 0.0% (0/4) N/A 1.07
(4/4)
(N/A%) (N/A%) (N/A%) (3.5%)
N/A ± N/A ± N/A ± 30.0 ±

100.0%
NTC 100170 0.0% (0/4) N/A 0.0% (0/4) N/A 0.0% (0/4) N/A 0.29
(4/4)
(N/A%) (N/A%) (N/A%) (1.0%)
N/A ± N/A ± N/A ± 30.8 ±

100.0%
100215 0.0% (0/3) N/A 0.0% (0/3) N/A 0.0% (0/3) N/A 1.24
(3/3)
(N/A%) (N/A%) (N/A%) (4.0%)
N/A ± N/A ± N/A ± 30.8 ±

100.0%
100305 0.0% (0/4) N/A 0.0% (0/4) N/A 0.0% (0/4) N/A 0.91
(4/4)
(N/A%) (N/A%) (N/A%) (3.0%)
N/A ± N/A ± N/A ± 31.0 ±

100.0%
100445 0.0% (0/3) N/A 0.0% (0/3) N/A 0.0% (0/3) N/A 0.70
(3/3)
(N/A%) (N/A%) (N/A%) (2.3%)
N/A ± N/A ± N/A ± 30.4 ±

100.0%
All 0.0% (0/27) N/A 0.0% (0/27) N/A 0.0% (0/27) N/A 0.85
(27/27)
(N/A%) (N/A%) (N/A%) (2.8%)
Page 143 of 250

Control
26.0 ± 26.2 ± 25.9 ± 29.3 ±

100.0% 100.0% 100.0% 100.0%
100074 0.21 0.64 0.35 0.14
(2/2) (2/2) (2/2) (2/2)
(0.8%) (2.4%) (1.4%) (0.5%)
26.2 ± 26.3 ± 26.0 ± 29.6 ±

100.0% 100.0% 100.0% 100.0%
100090 0.10 0.24 0.40 0.39
(4/4) (4/4) (4/4) (4/4)
(0.4%) (0.9%) (1.5%) (1.3%)
26.2 ± 25.7 ± 26.0 ± 30.2 ±

100.0% 100.0% 100.0% 100.0%
100091 0.10 0.29 0.12 0.21
(3/3) (3/3) (3/3) (3/3)
(0.4%) (1.1%) (0.5%) (0.7%)
27.0 ± 28.3 ± 28.2 ± 30.6 ±

100.0% 100.0% 100.0% 100.0%
100122 0.43 1.53 1.55 0.63
(4/4) (4/4) (4/4) (4/4)
(1.6%) (5.4%) (5.5%) (2.1%)
26.0 ± 26.1 ± 25.6 ± 29.9 ±

100.0% 100.0% 100.0% 100.0%
PC 100170 0.08 0.15 0.19 0.24
(4/4) (4/4) (4/4) (4/4)
(0.3%) (0.6%) (0.7%) (0.8%)
26.8 ± 28.2 ± 28.0 ± 30.5 ±

100.0% 100.0% 100.0% 100.0%
100215 0.26 1.78 1.42 0.75
(3/3) (3/3) (3/3) (3/3)
(1.0%) (6.3%) (5.1%) (2.5%)
27.1 ± 28.7 ± 28.8 ± 30.6 ±

100.0% 100.0% 100.0% 100.0%
100305 0.24 1.18 1.42 0.73
(4/4) (4/4) (4/4) (4/4)
(0.9%) (4.1%) (4.9%) (2.4%)
27.1 ± 27.7 ± 28.1 ± 30.6 ±

100.0% 100.0% 100.0% 100.0%
100445 0.51 1.96 1.50 0.62
(3/3) (3/3) (3/3) (3/3)
(1.9%) (7.1%) (5.3%) (2.0%)
26.6 ± 27.2 ± 27.1 ± 30.2 ±

100.0% 100.0% 100.0% 100.0%
All 0.54 1.53 1.58 0.65
(27/27) (27/27) (27/27) (27/27)
(2.0%) (5.6%) (5.8%) (2.2%)
PC= Positive Control, NTC No Template Control, Ct = Cycle Threshold, SD = Standard Deviation, %CV = Percent Coefficient of
Page 144 of 250
Table 18.3.6. Results for Controls – Analytical Reactivity (All Other COVID-19 Strains)
FluA (FAM) Ct FluB (JOE) Ct COVID-19 (CFR610) IC (Q670) Ct

Ct
Instrument
Control Mean Mean Mean Mean
% Ct ± % Ct ± % Ct ± % Ct ±
SN
Detection SD Detection SD Detection SD Detection SD
(%CV) (%CV) (%CV) (%CV)
200151 0% (0/1) N/A ± 0% (0/1) N/A ± 0% (0/1) N/A ± 100% (1/1) 28.4
N/A N/A N/A
2D0005 0% (0/1) N/A ± 0% (0/1) N/A ± 0% (0/1) N/A ± 100% (1/1) 27.9
N/A N/A N/A
NTC 2D0049 0% (0/1) N/A ± 0% (0/1) N/A ± 0% (0/1) N/A ± 100% (1/1) 28.5
N/A N/A N/A
N/A ± N/A ± N/A ± 28.3±0
ALL 0% (0/3) 0% (0/3) 0% (0/3) 100% (3/3)
N/A N/A N/A .3
(N/A) (N/A) (1.1%)
200151 100% (1/1) (24.9
/ ) 100% (1/1) 27.6 100% (1/1) 26.9 100% (1/1) 28.3
2D0005 100% (1/1) 25.9 100% (1/1) 28.2 100% (1/1) 26.9 100% (1/1) 27.8
PC
2D0049 100% (1/1) 26.0 100% (1/1) 28.3 100% (1/1) 27.7 100% (1/1) 28.4
25.6±0 28.0±0 27.2±0. 28.2±0

ALL 100% (3/3) 100% (3/3) 100% (3/3) 100% (3/3)
.6 .4 5 .3
(2.4%) (1.4%) (1.7%) (1.1%)
PC= Positive Control, NTC No Template Control, Ct = Cycle Threshold, SD = Standard Deviation, %CV = Percent Coefficient of
Conclusions – Analytical Reactivity
The Analytical Reactivity study demonstrates the Candidate Device can detect additional Flu A,
Flu B and COVID-19 strains that were not tested in the Limit of Detection study. 63 Flu A strains,
21 Flu B strains and five (5) COVID-19 strains were tested. All strains had 100% detection at
the initial concentration that was tested or at a higher concentration.
All baselines and test samples had 100% detection in the internal control channel except for the
A/New Caledonia/20/99 strain. Only two (2) of three (3) replicates were detected. However, all
3 replicates were detected in the Flu A channel and because of this, detection of the internal
control is not required for a valid result. Therefore, all three (3) replicates are valid for the
detection of the A/New Caledonia/20/99 strain. Controls performed as expected.
Please see attachment list at the conclusion of section 18.3 for a listing of study protocol and the
associated line data.
Page 145 of 250
Analytical Reactivity – COVID-19 Strain Monitoring
An in-silico inclusivity analysis of the COVID-19 target primers and probes in the Candidate
Device is performed quarterly by DiaSorin Molecular.
Based on in-silico analysis, it is predicted that the Candidate Device will detect all analyzed
SARS-CoV-2 sequences in GISAID databases, including sequences of the Omicron BA.1,
Omicron BA.2 and IHU (B.1.640.2) variants.
An in-silico inclusivity analysis of the oligonucleotide (oligo) sequences for the SARS-CoV-2
ORF1ab and S gene sets were performed against all SARS-CoV-2 sequences available in the
GISAID database submitted from November 01, 2021 to January 31, 2022. The analysis included
2,170,584 sequences in the amplicon regions of the ORF1ab and S gene oligo sets. Only target
sequences with full coverage of all three oligo-binding regions (forward primer, reverse primer,
and probe) were included in the analyses for both oligo sets. Partial target sequences and
sequences with ambiguous or degenerate bases in an oligo binding region were excluded from
this inclusivity analysis.
Detection of SARS-CoV-2 by the Candidate Device relies on the binding and extension of primers
with detection by probes designed off regions in the SARS-CoV-2 ORF1ab and S genes. The
assay makes a positive call for SARS-CoV-2 when oligos for either or both genes are able to
detect nucleic acids of the virus. The in-silico biosurveillance analysis involved assessing the
detection of the virus by any one gene oligo set in the assay. In analyzing the SARS-CoV-2
inclusivity results, there were 2,170,382 sequences (~99.99%) with no mismatches in at least
one gene oligo set and were predicted to be detected by the assay based on sequence homology.
There were 202 sequences (~0.01%) with mismatches in at least one oligo-binding region in
each gene oligo set. A melting temperature (Tm) analysis was conducted for those sequences
with mismatches in the binding sites of both gene oligo sets. The impact of mismatches in these
202 sequences on oligo Tm were evaluated by using assay oligo and template sequences with
mismatches as input. Tm calculation was performed with assay-specific conditions using TM
Mismatch Tool from IDT (https://www.idtdna.com/calc/analyzer/lna), which uses the nearest
neighbor model for calculating Tm. The Tm analysis compared the calculated mismatch Tm
values against the assay’s annealing temperature for all oligos and additionally against the RT
temperature for the RT (reverse) primers. For each of the 202 sequences, analysis showed that
there was at least one oligo gene set where the primers and probes exhibit mismatch Tm values
that were above their respective annealing temperature and the mismatches were not located at
the 3’ end for the primers; as such, detection of these sequences are not affected by the
mismatches. Table 18.3-6 below summarizes the Tm analysis results.
Page 146 of 250
Table 18.3-6. Simplexa™ COVID-19 & Flu A/B Direct In-silico inclusivity
No. seq. where at least one gene oligo set meets Tm criteria 2,170,584
Analytical Reactivity Protocol 18.3-1
Analytical Reactivity in-silico BLAST Analysis Results 18.3-2
Analytical Reactivity Line Data (pdf) 18.3-3
Analytical Reactivity Line Data (xls) 18.3-4
Analytical Reactivity COVID-19 Strains Line Data (pdf) 18.3-5
Analytical Reactivity COVID-19 Strains Line Data (xls) 18.3-6
Simplexa™ COVID-19 Clinical Strain Monitoring Report 18.3-7
18.4 Competitive Interference
The Candidate Device was evaluated for competitive interference with nasopharyngeal swabs
(NPS) in Universal Transport Medium (UTM). The study was performed at DiaSorin Molecular,
Cypress, CA using three (3) LIAISON MDX Instruments (100122, 100215 and 100305), one (1)
lot of the Candidate Device reaction mix (Lot V11909N), one (1) lot of the Candidate Device
Positive Control Pack (Lot #: V12441N) and one (1) lot of Direct Amplification Discs (Lot 11855N)
during one (1) day (23 July 2021). A total of six (6) experimental runs were performed by one (1)
operator.
Baseline samples were prepared by spiking COVID-19/USA-WA1/2020, Influenza A/Hong

Kong/8/1968 or Influenza B/Phuket/3073/2013 at 4x LoD into nasopharyngeal swab (NPS) matrix
in UTM. A high level (1,000x LoD) of each of the viruses was added to each of the baseline
samples and tested.
Results - Competitive Interference
Table 18.4.1 shows the competitive interference results for baseline samples containing COVID-
19, Influenza A or Influenza B as well as COVID-19, Influenza A, or Influenza B as competitive
interferents. Table 18.4.2 shows the Internal Control results. A high level of COVID-19 does not
impact the detection of a low level of influenza A or influenza B. A high level of influenza A does
Page 147 of 250
not impact the detection of a low level of COVID-19 or influenza B. A high level of influenza B
does not impact the detection of a low level of influenza A or COVID-19.
Table 18.4.1. Competitive Interference Results for COVID-19, Flu A, and Flu B
Competitive % % %
Mean Mean Mean
Competitive Interferent Detection Detection Detection
Baseline Ct ± Ct ± Ct ±
Interferent Concentration (# (# (#
SD SD SD
(copies/mL) Detected Detected Detected
(%CV) (%CV) (%CV)
/# Tested) /# Tested) /# Tested)
32.7 ±
100.0%
Flu A N/A N/A 1.06 0.0% (0/5) N/A 0.0% (0/5) N/A
(5/5)
(3.2%)
100.0% 31.0 ±
Flu B N/A N/A 0.0% (0/5) N/A (5/5) 0.54 0.0% (0/5) N/A
(1.7%)
29.2 ±
100.0%
COVID-19 N/A N/A 0.0% (0/5) N/A 0.0% (0/5) N/A 0.27
(5/5)
(0.9%)
32.5 ± 22.5 ±
100.0% 100.0%
Flu B 7.5x105 0.38 0.17 0.0% (0/3) N/A
(3/3) (3/3)
(1.2%) (0.8%)
Flu A
32.7 ± 21.6 ±
100.0% 100.0%
COVID-19 5.0x105 0.55 0.0% (0/3) N/A 0.21
(3/3) (3/3)
(1.7%) (1.0%)
24.4 ± 32.4 ±
100.0% 100.0%
Flu A 5.0x105 0.00 0.75 0.0% (0/3) N/A
(3/3) (3/3)
(0.0%) (2.3%)
Flu B
30.7 ± 21.2 ±
100.0% 100.0%
COVID-19 5.0x105 0.0% (0/3) N/A 0.31 0.06
(3/3) (3/3)
(1.0%) (0.3%)
24.2 ± 29.4 ±
100.0% 100.0%
Flu A 5.0x105 0.00 0.0% (0/3) N/A 0.57
(3/3) (3/3)
(0.0%) (1.9%)
COVID-19
22.8 ± 28.6 ±
100.0% 100.0%
Flu B 7.5x105 0.0% (0/3) N/A 0.29 0.46
(3/3) (3/3)
(1.3%) (1.6%)
Ct = Cycle Threshold, SD = Standard Deviation %CV = Percent Coefficient of Variation
Page 148 of 250
Table 18.4.2. Competitive Interference Results for the Internal Control
Competitive Internal Control

Competitive Interferent % Detection
Baseline Mean Ct ± SD
Interferent Concentration (# Detected
(%CV)
(copies/mL) /# Tested)
30.9 ± 0.22
Flu A N/A N/A 100.0% (5/5)
(0.7%)
30.4 ± 0.20
Flu B N/A N/A 100.0% (5/5)
(0.7%)
30.4 ± 0.11
COVID-19 N/A N/A 100.0% (5/5)
(0.4%)
30.6 ± 0.40
Flu B 7.5x105 100.0% (3/3)
(1.3%)
Flu A
30.8 ± 0.15
COVID-19 5.0x105 100.0% (3/3)
(0.5%)
30.6 ± 0.12
Flu A 5.0x105 100.0% (3/3)
(0.4%)
Flu B
30.8 ± 0.40
COVID-19 5.0x105 100.0% (3/3)
(1.3%)
30.9 ± 0.71
Flu A 5.0x105 100.0% (3/3)
(2.3%)
COVID-19
30.3 ± 0.15
Flu B 7.5x105 100.0% (3/3)
(0.5%)
Ct = Cycle Threshold, SD = Standard Deviation %CV = Percent Coefficient of Variation
Summary of Control Results – Competitive Interference
The results for the controls run in the study are presented in Table 18.4.3. All controls
performed as expected.
Table 18.4.3. Results for Controls – Competitive Interference

Instrument Mean Mean Mean Mean

Control % Detection % Detection % Detection % Detection
SN Ct ± Ct ± Ct ± Ct ±
(# Detected/# (# Detected/# (# Detected (# Detected
SD SD SD SD
Tested) Tested) /# Tested) /# Tested)
(%CV) (%CV) (%CV) (%CV)
N/A ± N/A ± N/A ± 30.6 ±

100122 0.0% (0/1) N/A 0.0% (0/1) N/A 0.0% (0/1) N/A 100.0% (1/1) N/A
(N/A%) (N/A%) (N/A%) (N/A%)
NTC
N/A ± N/A ± N/A ± 30.5 ±
1003215 0.0% (0/1) N/A 0.0% (0/1) N/A 0.0% (0/1) N/A 100.0% (1/1) N/A
(N/A%) (N/A%) (N/A%) (N/A%)
Page 149 of 250

Control % Detection % Detection % Detection % Detection
(# Detected/# (# Detected/# (# Detected (# Detected
SD SD SD SD
Tested) Tested) /# Tested) /# Tested)
(%CV) (%CV) (%CV) (%CV)
N/A ± N/A ± N/A ± 30.4 ±

100305 0.0% (0/1) N/A 0.0% (0/1) N/A 0.0% (0/1) N/A 100.0% (1/1) N/A
(N/A%) (N/A%) (N/A%) (N/A%)
N/A ± N/A ± N/A ± 30.5 ±

All 0.0% (0/3) N/A 0.0% (0/3) N/A 0.0% (0/3) N/A 100.0% (3/3) 0.10
(N/A%) (N/A%) (N/A%) (0.3%)
25.8 ± 26.5 ± 26.8 ± 30.8 ±

100122 100.0% (1/1) N/A 100.0% (1/1) N/A 100.0% (1/1) N/A 100.0% (1/1) N/A
(N/A%) (N/A%) (N/A%) (N/A%)
25.7 ± 26.7 ± 26.2 ± 31.1 ±

100215 100.0% (1/1) N/A 100.0% (1/1) N/A 100.0% (1/1) N/A 100.0% (1/1) N/A
(N/A%) (N/A%) (N/A%) (N/A%)
PC
25.8 ± 27.0 ± 26.0 ± 30.6 ±
100305 100.0% (1/1) N/A 100.0% (1/1) N/A 100.0% (1/1) N/A 100.0% (1/1) N/A
(N/A%) (N/A%) (N/A%) (N/A%)
25.8 ± 26.7 ± 26.3 ± 30.8 ±

All 100.0% (3/3) 0.06 100.0% (3/3) 0.25 100.0% (3/3) 0.42 100.0% (3/3) 0.25
(0.2%) (0.9%) (1.6%) (0.8%)
Ct = Cycle Threshold, SD = Standard Deviation %CV = Percent Coefficient of Variation, IC = Internal Control, PC = Positive Control, NTC
= No Template Control
Conclusions – Competitive Interference
A high level of Influenza A (5.0x105 copies/mL), Influenza B (7.5x105 copies/mL), or COVID-19

(5.0x105 copies/mL) does not have any impact on the detection of the other viruses. All controls
Section 18.4 Attachments (Document) Attachment Number
Competitive Interference Protocol 18.4-1
Competitive Interference Line Data (pdf) 18.4-2
Competitive Interference Line Data (xls 18.4-3

Page 150 of 250
18.5 Sample Stability (Fresh vs. Frozen)
The sample stability study for the Candidate Device is comprised of two (2) studies;

Transport Medium (UTM), Puritan UniTranz-RT and Saline (0.9% sodium chloride in
water)
b) Media Equivalency Nasopharyngeal Swabs (NPS) in Universal Transport Medium

(UTM), Puritan UniTranz-RT and saline (0.9% sodium chloride in water)
The following standard was CLSI MM13 Collection, Transport, Preparation and Storage for
Molecular Methods, 2ed.

Transport Medium (UTM), Puritan UniTranz-RT and Saline (0.9% sodium chloride in water)
The Candidate Device was evaluated for the product’s ability to detect low levels of influenza A,
influenza B SARS-CoV-2 (COVID-19), in different transport media inoculated with
nasopharyngeal swabs, which were then stored refrigerated (2-8°C) or frozen at or below -70°C.
The different collection media types evaluated were Universal Transport Medium [UTM], Puritan
UniTranz-RT and saline (0.9% sodium chloride in water). Sample stability was evaluated
following refrigeration at 2-8°C for up to seven (7) days, freezing at or below -70°C until 7 days
and eventually up to four (4) sample thaws. The study was performed at DiaSorin Molecular,
Cypress, CA using four (4) LIAISON MDX Instruments (100122, 100215, 100305 and 100445),
one (1) lot of Candidate Device Reaction Mix (Lot V11909N), two (2) lots of Candidate Device
Positive Control (Lots V11923N and V12441N) and one (1) lots of Direct Amplification Discs (Lot
12170N). A total of 81 experimental runs were performed by four (4) operators during six (6) days
(12, 15, 19, 22-23 and 26 July, 2021).
Heat-inactivated Flu A (Hong Kong/8/1968), Flu B (Phuket/3073/2013) and COVID-19 (2019-

nCoV/USA-WA1/2020) strains were diluted and spiked into each of the negative NPS samples
at 4x LoD then spiked into the transport media types. Samples were tested fresh and then stored
refrigerated (2-8˚C) or frozen (≤-70˚C) prior to testing.
Figure 18.5.1 shows a schematic of the workflow indicating the storage conditions, durations and
testing points for Universal Transport Medium [UTM], Puritan UniTranz-RT and saline (0.9%
sodium chloride in water).
Page 151 of 250
Figure 18.5.1. Sample Testing Workflow
Samples were prepared, tested in the fresh state (Time Point A) and stored refrigerated (2-8˚C).
After 3 days at 2-8˚C refrigerated storage (Time Point B), samples were split for refrigerated (2-
8˚C) or frozen (≤-70˚C) storage prior to further testing (see Figure 7).
For fresh vs. frozen stability, at least 95% detection is required for the refrigerated (2-8˚C) test
points (Time Points A, B, and C) and the frozen (≤-70˚C) Time Point D. There is no acceptance
criterion for the freeze/thaw test points E, F, G as the testing was for information only.
Results - (Fresh vs Frozen) Nasopharyngeal Swabs (NPS) in Universal Transport Medium

(UTM), Puritan UniTranz-RT and Saline (0.9% sodium chloride in water)
The fresh vs. frozen stability results are summarized in Table 18.5.1 and presented in Table
18.5.2 and Figures 18.5.2 to 18.5.4. For this analysis, the results for UTM, Puritan UniTranz-RT
and saline (0.9% sodium chloride in water) were combined. For the Flu A Ct value, Flu B Ct
value, COVID-19 Ct value or internal control Ct value, each had a %CV of ≤3.5%.
Page 152 of 250
Table 18.5.1. Summary of Fresh vs. Frozen Results

Time Point
A B C D E F G
7 days, 8 days, 11 days, 11 days,

3 days, 7 days,
Fresh ≤-70°C, ≤-70°C, ≤-70°C, ≤-70°C,
Target 2-8˚C 2-8˚C
1 thaw* 2 thaws 3 thaws 4 thaws
% % % % % % %
Detection Detection Detection Detection Detection Detection Detection
(#Detected (#Detected (#Detected (#Detected (#Detected (#Detected (#Detected/
/#Tested) /#Tested) /#Tested) /#Tested) /#Tested) /#Tested) #Tested)
100.0%
100.0% 100.0% 100.0% 100.0% 100.0% 100.0%
(60/60)
(60/60) (60/60) (60/60) (60/60) (60/60) (60/60)
Flu A
95% CI:
95% CI: 95% CI: 95% CI: 95% CI: 95% CI: 95% CI:
94 - 100% 94 - 100% 94 - 100% 94 - 100% 94 - 100% 94 - 100%
94 - 100%
100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 98.3%

(60/60) (60/60) (60/60) (60/60) (60/60) (60/60) (59/60)
Flu B
95% CI: 95% CI: 95% CI: 95% CI: 95% CI: 95% CI: 95% CI:
94 - 100% 94 - 100% 94 - 100% 94 - 100% 94 - 100% 94 - 100% 91.1 - 100%
100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0%

(60/60) (60/60) (60/60) (60/60) (60/60) (60/60) (60/60)
COVID-
19
95% CI: 95% CI: 95% CI: 95% CI: 95% CI: 95% CI: 95% CI:
94 - 100% 94 - 100% 94 - 100% 94 - 100% 94 - 100% 94 - 100% 94 - 100%
100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0%

(60/60) (60/60) (60/60) (60/60) (60/60) (60/60) (60/60)
IC
95% CI: 95% CI: 95% CI: 95% CI: 95% CI: 95% CI: 95% CI:
94 - 100% 94 - 100% 94 - 100% 94 - 100% 94 - 100% 94 - 100% 94 - 100%
*Samples frozen for time point D were initially stored for 3 days at 2-8 ˚C as shown in Figure 7.
95% CI = 95% Confidence Interval, IC = Internal Control

Page 153 of 250
Table 18.5.2. Results of Fresh vs. Frozen study
A B C D E F G
Time Point
Target 7 days @ ≤- 8 days @ 11 days @ 11 days, ≤-
3 days @ 7 days @
→ Fresh 70˚C, 1 ≤-70˚C, 2 ≤-70˚C, 3 70°C, 4
2-8˚C 2-8˚C
Thaw Thaws Thaws thaws)
Mean Ct ± 32.3 ± 0.79 32.5 ± 0.73 32.5 ± 0.79 32.5 ± 0.74 32.2 ± 0.59 32.4 ± 0.81 32.4 ± 1.13
SD (%CV) (2.4%) (2.2%) (2.4%) (2.3%) (1.8%) (2.5%) (3.5%)
%
Flu A
Detection
100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0%
(60/60) (60/60) (60/60) (60/60) (60/60) (60/60) (60/60)
(#Detected
/#Tested)
Mean Ct ± 30.0 ± 0.68 30.5 ± 0.71 30.2 ± 0.69 30.2 ± 0.53 30.2 ± 0.71 30.1 ± 0.58 29.7 ± 0.78
SD (%CV) (2.3%) (2.3%) (2.3%) (1.8%) (2.4%) (1.9%) (2.6%)
%
Flu B
Detection
100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 98.3%
(60/60) (60/60) (60/60) (60/60) (60/60) (60/60) (59/60)
(#Detected
/#Tested)
Mean Ct ± 29.3 ± 0.60 29.4 ± 0.59 29.4 ± 0.61 29.5 ± 0.59 29.2 ± 0.70 29.3 ± 0.65 29.0 ± 0.71
SD (%CV) (2.0%) (2.0%) (2.1%) (2.0%) (2.4%) (2.2%) (2.4%)
COVID- %
19 Detection
100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0%
(60/60) (60/60) (60/60) (60/60) (60/60) (60/60) (60/60)
(#Detected
/#Tested)
Mean Ct ± 30.8 ± 0.38 30.8 ± 0.45 30.8 ± 0.40 30.9 ± 0.35 30.8 ± 0.86 30.9 ± 0.37 31.2 ± 0.64
SD (%CV) (1.2%) (1.5%) (1.3%) (1.1%) (2.8%) (1.2%) (2.1%)
%
IC
Detection
100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0%
(60/60) (60/60) (60/60) (60/60) (60/60) (60/60) (60/60)
(#Detected
/#Tested)
Ct = Cycle Threshold, SD = Standard Deviation %CV = Percent Coefficient of Variation, N/A = Not Applicable, IC = Internal Control
Page 154 of 250
Figure 18.5.2. Fresh vs. frozen results across all time points
Note: error bars represent the standard error of the mean.

Page 155 of 250
Figure 18.5.3. Refrigerated stability results
Refrigerated Stability
33
32
Avergae Ct Value
31
30
Flu A
29 Flu B
COVID-19
28
RNA IC
27
A B C
(Fresh) (3 days, (7 days,
2-8˚C) 2-8˚C)
Time Point
Note: error bars represent the standard error of the mean.

Page 156 of 250
Figure 18.5.4. Frozen stability results
Frozen Stability
33
32
Average Ct Value
31
30
Flu A
29 Flu B
COVID-19
28
RNA IC
27
A D E F G
(Fresh) (7 days, (8 days, (11 days, (11 days,
≤-70°C, ≤-70°C, ≤-70°C, ≤-70°C,
1 thaw) 2 thaws) 3 thaws) 4 thaws)
Time Point
Note:
error bars represent the standard error of the mean
Following preparation, the 20 individually contrived samples were tested as fresh samples. The
samples were then subjected to refrigeration or storage at ≤ -70ºC. At each time point, 20
individual contrived samples were tested in order to determine fresh vs. frozen stability.
In order to establish fresh vs. frozen stability for a given time point, at least 95% detection across
all transport media types, is required.
At each of the time points, the data for each transport media type (UTM, Puritan UniTranz-RT
and saline [0.9% sodium chloride in water]) was combined.
Page 157 of 250
There was a 100% detection (60/60 replicates) at each of the required time points (Time Points
A, B, C, and D). At each of the time points, the 95% confidence interval was 94% – 100%.
Additionally, there was 100% detection (60/60 replicates) for Time Points E, F, and G for Flu A
and COVID-19. At each of the time points, the 95% confidence interval was 94% – 100%.
There was 100% detection (60/60 replicates) for Flu B at Time Points E and F. At each of these
time points, the 95% confidence interval was 94% – 100%. For Time Point G there was 98.3%
detection of Flu B and the 95% confidence interval was 91.1% – 100%.
Summary of Control Results - Fresh vs Frozen Nasopharyngeal Swabs (NPS) in Universal

Transport Medium (UTM), Puritan UniTranz-RT and Saline (0.9% sodium chloride in water)

Flu A (FAM) Flu B (JOE) COVID-19 (CFR610) RNA IC (Q670)

Control
N/A ± N/A ± N/A ± 30.9 ±

100.0%
100122 0.0% (0/6) N/A 0.0% (0/6) N/A 0.0% (0/6) N/A 0.72
(6/6)
(N/A%) (N/A%) (N/A%) (2.3%)
N/A ± N/A ± N/A ± 30.6 ±

100.0%
100215 0.0% (0/6) N/A 0.0% (0/6) N/A 0.0% (0/6) N/A 0.36
(6/6)
(N/A%) (N/A%) (N/A%) (1.2%)
N/A ± N/A ± N/A ± 30.6 ±

100.0%
NTC 100305 0.0% (0/6) N/A 0.0% (0/6) N/A 0.0% (0/6) N/A 1.08
(6/6)
(N/A%) (N/A%) (N/A%) (3.5%)
N/A ± N/A ± N/A ± 31.1 ±

100.0%
100445 0.0% (0/6) N/A 0.0% (0/6) N/A 0.0% (0/6) N/A 0.94
(6/6)
(N/A%) (N/A%) (N/A%) (3.0%)
N/A ± N/A ± N/A ± 30.8 ±

100.0%
All 0.0% (0/24) N/A 0.0% (0/24) N/A 0.0% (0/24) N/A 0.80
(24/24)
(N/A%) (N/A%) (N/A%) (2.6%)
26.2 ± 27.1 ± 26.9 ± 31.0 ±

100.0% 100.0% 100.0% 100.0%
PC 100122 0.51 0.92 0.50 0.56
(6/6) (6/6) (6/6) (6/6)
(1.9%) (3.4%) (1.9%) (1.8%)
Page 158 of 250
Flu A (FAM) Flu B (JOE) COVID-19 (CFR610) RNA IC (Q670)

Control
26.0 ± 27.2 ± 26.8 ± 30.7 ±

100.0% 100.0% 100.0% 100.0%
100215 0.53 0.92 0.83 0.72
(6/6) (6/6) (6/6) (6/6)
(2.0%) (3.4%) (3.1%) (2.3%)
26.2 ± 27.5 ± 26.9 ± 30.7 ±

100.0% 100.0% 100.0% 100.0%
100305 0.50 1.09 1.51 0.55
(6/6) (6/6) (6/6) (6/6)
(1.9%) (4.0%) (5.6%) (1.8%)
26.6 ± 27.1 ± 27.2 ± 31.0 ±

100.0% 100.0% 100.0% 100.0%
100445 0.52 0.66 0.59 0.65
(6/6) (6/6) (6/6) (6/6)
(2.0%) (2.4%) (2.2%) (2.1%)
26.2 ± 27.2 ± 26.9 ± 30.8 ±

100.0% 100.0% 100.0% 100.0%
All 0.53 0.86 0.89 0.61
(24/24) (24/24) (24/24) (24/24)
(2.0%) (3.2%) (3.3%) (2.0%)
Det = Detected, Test = Tested, Ct = Cycle Threshold, SD = Standard Deviation, %CV = Percent Coefficient of Variation, N/A = Not
Applicable, PC = Positive Control. NTC = No Template Control
Conclusions - Sample Stability (Fresh vs Frozen) Nasopharyngeal Swabs (NPS) in

Universal Transport Medium (UTM), Puritan UniTranz-RT and Saline (0.9% sodium
chloride in water)
The sample stability for nasopharyngeal swabs used with the Candidate Device was established
in Universal Transport Medium [UTM], Puritan UniTranz-RT and saline (0.9% sodium chloride in
water) for the following conditions; storage for up to seven (7) days refrigerated (2-8°C) and up
to 11 days at -70°C. This is based upon the fact that there was 100% detection at each of the
time points except for time point G [11 days, ≤-70°C, four (4) thaws].
Please see attachment list at the conclusion of section 18.5 for a listing of study protocol and the
associated line data.
b) Media Equivalency Nasopharyngeal Swabs (NPS) in Universal Transport Medium (UTM),

Puritan UniTranz-RT and Saline (0.9% sodium chloride in water)
The Candidate Device was evaluated the effect of samples stored in different collection media
types (Universal Transport Medium [UTM] or BD UVT [same formulation as UTM; with the only
difference is to product labeling], Puritan UniTranz-RT and Saline (0.9% NaCl in water). The
study was performed at DiaSorin Molecular, Cypress, CA using 12 LIAISON MDX Instruments
(100020, 100074, 100090, 100091, 100122, 100157, 100170, 100215, 100222, 100305, 100336,
Page 159 of 250
100445), two (2) lots of Candidate Device Reaction Mix (Lots V11908N and V11909N), one (1)
lot of Candidate Device Positive Control (Lot V11923N) and one (1) lot of Direct Amplification
Discs (Lot 12170N). A total of 34 experimental runs were performed by two (2) operators during
one (1) day (16 July 2021).
Heat-inactivated COVID-19 viral particles from the 2019-nCoV/USA-WA1/2020 strain (ATCC,

Manassas, VA), Influenza A Hong Kong/8/1968 strain and Influenza B Phuket/3073/2013 were
co-spiked into negative nasopharyngeal swab (NPS) matrix and placed into in either UTM,
Puritan UniTranz-RT or saline (0.9% NaCl in water) transport media. For each matrix
[Nasopharyngeal swab (NPS) in a specific type of transport media], one (1) bulk sample was
contrived with Influenza A, Influenza B and COVID-19 and at a final concentration of
approximately 4x LoD. Each bulk sample was split into five (5) aliquots, each of which was stored
frozen at ≤-70°C for two (2) days. A total of 40 replicates of each type of transport media (eight
[8] replicates of each aliquot) were tested using Candidate Device. In addition, two (2) aliquots
of negative matrix from each type of transport media were stored frozen at ≤-70°C for two (2)
days. Eight (8) replicates of each aliquot of negative matrix were tested for a total of 16 negative
replicates.
Results - Media Equivalency Nasopharyngeal Swabs (NPS) in Universal Transport Medium

(UTM), Puritan UniTranz-RT and Saline (0.9% sodium chloride in water)
The collection media equivalency results for contrived positive test samples are summarized in
Table 18.5.1.
For UTM, Puritan UniTranz-RT and saline, there was 100% detection for all three (3) targets
(COVID-19, Influenza A and Influenza B). As expected, each of the replicates of negative matrix
had a result of Not Detected.
Page 160 of 250
Table 18.5.4. Summary of Collection Media Equivalency

% Detection % Detection % Detection

Transport Media Type Sample Type
(# Detected (# Detected (# Detected
/# Tested) /# Tested) /# Tested)
100.0% 100.0% 100.0%

Contrived Positive
(40/40) (40/40) (40/40)
UTM
Negative Matrix 0% (0/16) 0% (0/16) 0% (0/16)
100.0% 100.0% 100.0%

Contrived Positive
(40/40) (40/40) (40/40)
Puritan UniTranz-RT
Negative Matrix 0% (0/16) 0% (0/16) 0% (0/16)
100.0% 100.0% 100.0%

Contrived Positive
(40/40) (40/40) (40/40)
Saline
Negative Matrix 0% (0/16) 0% (0/16) 0% (0/16)
The collection media equivalency results are presented in Table 18.5.6.
Table 18.5.6. Results for the Collection Media Equivalency Study

Flu A Ct Flu B Ct COVID-19 Ct IC Ct
Media % % % %
Sample Mean Mean Mean Mean
Type Detection Detection Detection Detection
Ct ± SD Ct ± SD Ct ± SD Ct ± SD
(#Detected (#Detected (#Detected (#Detected
(%CV) (%CV) (%CV) (%CV)
/#Tested) /#Tested) /#Tested) /#Tested)
32.0 ± 29.7 ± 29.1 ± 30.4 ±

100.0% 100.0% 100.0% 100.0%
UTM 0.39 0.64 0.49 0.32
(40/40) (40/40) (40/40) (40/40)
(1.2%) (2.2%) (1.7%) (1.1%)
Puritan 32.9 ± 30.8 ± 29.5 ± 31.2 ±

Contrived 100.0% 100.0% 100.0% 100.0%
UniTranz- 0.67 0.48 0.44 0.44
Positives (40/40) (40/40) (40/40) (40/40)
RT (2.0%) (1.6%) (1.5%) (1.4%)
31.6 ± 29.3 ± 28.7 ± 30.8 ±

100.0% 100.0% 100.0% 100.0%
Saline 0.49 0.53 0.40 0.56
(40/40) (40/40) (40/40) (40/40)
(1.6%) (1.8%) (1.4%) (1.8%)
N/A ± N/A ± N/A ± 30.0 ±

Negative 100.0%
UTM 0% (0/16) N/A 0% (0/16) N/A 0% (0/16) N/A 0.28
Matrix (16/16)
(N/A%) (N/A%) (N/A%) (0.9%)
Page 161 of 250
Flu A Ct Flu B Ct COVID-19 Ct IC Ct
Media % % % %
Sample Mean Mean Mean Mean
Type Detection Detection Detection Detection
(%CV) (%CV) (%CV) (%CV)
Puritan N/A ± N/A ± N/A ± 32.1 ±

100.0%
UniTranz- 0% (0/16) N/A 0% (0/16) N/A 0% (0/16) N/A 0.41
(16/16)
RT (N/A%) (N/A%) (N/A%) (1.3%)
N/A ± N/A ± N/A ± 30.4 ±

100.0%
Saline 0% (0/16) N/A 0% (0/16) N/A 0% (0/16) N/A 0.43
(16/16)
(N/A%) (N/A%) (N/A%) (1.4%)
Ct = Cycle threshold, SD= Standard Deviation, %CV = Percent Coefficient of Variation, N/A = Not applicable, IC = Internal Control
The effect of different transport media on Ct values for contrived positive samples is presented
in Table 18.5.7. For Puritan UniTranz-RT and saline, the difference in Ct value is < 4%, compared
to UTM.
Table 18.5.7. Change in Ct Values for Contrived Positive Samples in Various Transport Media
UTM Puritan UniTranz-RT Saline
Change
Change from
Target Mean Ct Change Mean Ct Mean Ct from UTM
UTM
from
(n) UTM (n) (n) (%
(% change)
change)
32.0 32.9 0.9 Ct 31.6 -0.4 Ct

Flu A N/A
(n=40) (n=40) (2.80%) (n=40) (-1.30%)
29.7 30.8 1.1 Ct 29.3 -0.4 Ct

Flu B N/A
(n=40) (n=40) (3.70%) (n=40) (-1.30%)
29.1 29.5 0.4 Ct 28.7 -0.4 Ct

COVID-19 N/A
(n=40) (n=40) (1.40%) (n=40) (-1.40%)
30.4 31.2 0.8 Ct 30.8 0.4 Ct

IC
(n=40) N/A (n=40) (2.60%) (n=40) (1.30%)
Ct = Cycle threshold, n = number of replicates, N/A = not applicable, IC = Internal Control

Page 162 of 250
The effect of different transport media on Internal Control Ct values for negative matrix collected
in each transport media is presented in Table 18.5.8. For Puritan UniTranz-RT and saline, the
difference in Ct value is < 7%, compared to UTM.
Table 18.5.8. Change in Ct Values for Negative Matrix Collected in Various Transport Media
UTM Puritan UniTranz-RT Saline
Change Change
Target Mean Ct Mean Ct from UTM Mean Ct from UTM
Change
from UTM
(n) (n) (% (n) (%
change) change)
30.0 32.1 2.1 Ct 30.4 0.4 Ct

IC N/A
(n=16) (n=16) (7.0%) (n=16) (1.3%)
Ct = Cycle threshold, n = number of replicates, N/A = not applicable, IC = Internal Control
For contrived test samples, there was 100% detection for all three (3) Candidate Device assay
targets.
All of the negative samples had a result of Not Detected as expected.
Summary of Control Results - Media Equivalency Nasopharyngeal Swabs (NPS) in

Universal Transport Medium (UTM), Puritan UniTranz-RT and Saline (0.9% sodium
chloride in water)
Page 163 of 250


Control
100020 0.0% (0/1) N/A ± 0.0% (0/1) N/A ± 0.0% (0/1) N/A ± 100.0% 29.2 ±
N/A N/A N/A (1/1) N/A
N/A ± N/A ± N/A ± 29.7 ±
100.0%
100074 0.0% (0/1) N/A 0.0% (0/1) N/A 0.0% (0/1) N/A N/A
(1/1)
(N/A%) (N/A%) (N/A%) (N/A%)
N/A ± N/A ± N/A ± 29.5 ±

100.0%
100090 0.0% (0/1) N/A 0.0% (0/1) N/A 0.0% (0/1) N/A N/A
(1/1)
(N/A%) (N/A%) (N/A%) (N/A%)
N/A ± N/A ± N/A ± 30.5 ±

100.0%
100091 0.0% (0/1) N/A 0.0% (0/1) N/A 0.0% (0/1) N/A N/A
(1/1)
(N/A%) (N/A%) (N/A%) (N/A%)
N/A ± N/A ± N/A ± 30.3 ±

100.0%
100122 0.0% (0/1) N/A 0.0% (0/1) N/A 0.0% (0/1) N/A N/A
(1/1)
(N/A%) (N/A%) (N/A%) (N/A%)
N/A ± N/A ± N/A ± 29.8 ±

100.0%
100157 0.0% (0/1) N/A 0.0% (0/1) N/A 0.0% (0/1) N/A N/A
(1/1)
NTC (N/A%) (N/A%) (N/A%) (N/A%)
N/A ± N/A ± N/A ± 29.7 ±

100.0%
100170 0.0% (0/1) N/A 0.0% (0/1) N/A 0.0% (0/1) N/A N/A
(1/1)
(N/A%) (N/A%) (N/A%) (N/A%)
N/A ± N/A ± N/A ± 30.2 ±

100.0%
100215 0.0% (0/1) N/A 0.0% (0/1) N/A 0.0% (0/1) N/A N/A
(1/1)
(N/A%) (N/A%) (N/A%) (N/A%)
N/A ± N/A ± N/A ± 30.9 ±

100.0%
100222 0.0% (0/1) N/A 0.0% (0/1) N/A 0.0% (0/1) N/A N/A
(1/1)
(N/A%) (N/A%) (N/A%) (N/A%)
N/A ± N/A ± N/A ± 30.4 ±

100.0%
100305 0.0% (0/1) N/A 0.0% (0/1) N/A 0.0% (0/1) N/A N/A
(1/1)
(N/A%) (N/A%) (N/A%) (N/A%)
N/A ± N/A ± N/A ± 29.4 ±

100.0%
100336 0.0% (0/1) N/A 0.0% (0/1) N/A 0.0% (0/1) N/A N/A
(1/1)
(N/A%) (N/A%) (N/A%) (N/A%)
Page 164 of 250

Control
N/A ± N/A ± N/A ± 31.0 ±

100.0%
100445 0.0% (0/1) N/A 0.0% (0/1) N/A 0.0% (0/1) N/A N/A
(1/1)
(N/A%) (N/A%) (N/A%) (N/A%)
N/A ± N/A ± N/A ± 30.1 ±

100.0%
All 0.0% (0/12) N/A 0.0% (0/12) N/A 0.0% (0/12) N/A 0.59
(12/12)
(N/A%) (N/A%) (N/A%) (2.0%)
100020 100.0% 26.9 ± 100.0% 27.4 ± 100.0% 27.9 ± 100.0% 29.1 ±

(1/1) N/A (1/1) N/A (1/1) N/A (1/1) N/A
26.2 ± 28.1 ± 28.8 ± 29.9 ±
100.0% 100.0% 100.0% 100.0%
100074 N/A N/A N/A N/A
(1/1) (1/1) (1/1) (1/1)
(N/A%) (N/A%) (N/A%) (N/A%)
26.2 ± 27.7 ± 28.4 ± 29.5 ±

100.0% 100.0% 100.0% 100.0%
100090 N/A N/A N/A N/A
(1/1) (1/1) (1/1) (1/1)
(N/A%) (N/A%) (N/A%) (N/A%)
26.4 ± 28.0 ± 28.6 ± 30.1 ±

100.0% 100.0% 100.0% 100.0%
100091 N/A N/A N/A N/A
(1/1) (1/1) (1/1) (1/1)
(N/A%) (N/A%) (N/A%) (N/A%)
27.2 ± 28.9 ± 28.6 ± 30.0 ±

100.0% 100.0% 100.0% 100.0%
100122 N/A N/A N/A N/A
(1/1) (1/1) (1/1) (1/1)
PC (N/A%) (N/A%) (N/A%) (N/A%)
26.7 ± 28.4 ± 28.2 ± 29.6 ±

100.0% 100.0% 100.0% 100.0%
100157 N/A N/A N/A N/A
(1/1) (1/1) (1/1) (1/1)
(N/A%) (N/A%) (N/A%) (N/A%)
26.1 ± 27.7 ± 27.9 ± 29.7 ±

100.0% 100.0% 100.0% 100.0%
100170 N/A N/A N/A N/A
(1/1) (1/1) (1/1) (1/1)
(N/A%) (N/A%) (N/A%) (N/A%)
26.5 ± 28.5 ± 28.3 ± 29.9 ±

100.0% 100.0% 100.0% 100.0%
100215 N/A N/A N/A N/A
(1/1) (1/1) (1/1) (1/1)
(N/A%) (N/A%) (N/A%) (N/A%)
26.4 ± 28.3 ± 28.4 ± 30.3 ±

100.0% 100.0% 100.0% 100.0%
100222 N/A N/A N/A N/A
(1/1) (1/1) (1/1) (1/1)
(N/A%) (N/A%) (N/A%) (N/A%)
Page 165 of 250

Control
27.1 ± 28.4 ± 28.1 ± 29.6 ±

100.0% 100.0% 100.0% 100.0%
100305 N/A N/A N/A N/A
(1/1) (1/1) (1/1) (1/1)
(N/A%) (N/A%) (N/A%) (N/A%)
26.2 ± 27.5 ± 28.2 ± 29.2 ±

100.0% 100.0% 100.0% 100.0%
100336 N/A N/A N/A N/A
(1/1) (1/1) (1/1) (1/1)
(N/A%) (N/A%) (N/A%) (N/A%)
27.0 ± 27.9 ± 28.5 ± 30.5 ±

100.0% 100.0% 100.0% 100.0%
100445 N/A N/A N/A N/A
(1/1) (1/1) (1/1) (1/1)
(N/A%) (N/A%) (N/A%) (N/A%)
26.6 ± 28.1 ± 28.3 ± 29.8 ±

100.0% 100.0% 100.0% 100.0%
All 0.39 0.45 0.28 0.42
(12/12) (12/12) (12/12) (12/12)
(1.5%) (1.6%) (1.0%) (1.4%)
Ct = Cycle threshold, SD= Standard Deviation, %CV = Percent Coefficient of Variation, N/A = Not applicable,
SN=Serial number, NTC = No Template Control, PC= Positive Control
Conclusions- Media Equivalency Nasopharyngeal Swabs (NPS) in Universal Transport

Medium (UTM), Puritan UniTranz-RT and Saline (0.9% sodium chloride in water)
For samples contrived in matrix collected in UTM, Puritan UniTranz-RT and Saline, there was
100% detection. Therefore, collection media equivalency is established for these transport media
types used with the Candidate Device.
Attachment
Number
Sample Stability Fresh Vs Frozen Protocol 18.5-1
Sample Stability Fresh Vs Frozen Report 18.5-2
Sample Stability Fresh Vs Frozen Report (Line Data) (xls) 18.5-3
Sample Stability - Media Equivalency Protocol (Other Media) 18.5-4
Sample Stability - Media Equivalency Line Data (pdf) 18.5-5
Sample Stability - Media Equivalency Line Data (xls) 18.5-6

Page 166 of 250
18.6 Reproducibility
Reproducibility of the Candidate Device assay was evaluated. The study’s objective was to
determine the effects of site-to-site, operator to operator, instrument to instrument, day to day
and within run variation on the performance of the Candidate Device assay using contrived
samples.
The reproducibility study consisted of eight (8) panel members, including six (6) contrived
samples, one (1) UTM sample used as the No Template Control (NTC), and one (1) positive
sample which was the Candidate Device Positive Control. The panel members were prepared
by diluting viral stocks of SARS-CoV-2, Flu A, and Flu B strains into native negative
nasopharyngeal swab (NPS) matrix in Universal Transport Media (UTM).
One (1) SARS-CoV-2, Flu A or Flu B strain was contrived in negative NPS in UTM. For each
analyte, the contrived samples included one (1) low positive (LP) and one (1) medium positive
(MP). Low positive samples were prepared at approximately 2x LoD and were expected to be
positive ≥ 95% of the time. Medium positive samples were prepared at approximately 4x LoD
and were expected to be positive 100% of the time.
Table 18.6.1 shows the reproducibility panel members and their approximate concentrations.
Table 18.6.1. Reproducibility Sample Panel Member Details

Panel Approximate Concentration
Panel Member or Strain Level
Member # LoD Level (copies/mL)
1 2019-nCoV/USA-WA1/2020 LP 2x 1000
2 2019-nCoV/USA-WA1/2020 MP 4x 2000
3 Influenza A/Hong Kong/8/68 LP 2x 1000
4 Influenza A/Hong Kong/8/68 MP 4x 2000
5 Influenza B/Malaysia/2506/04 LP 2x 500
6 Influenza B/Malaysia/2506/04 MP 4x 1000
7 UTM as NTC Neg N/A N/A
8 PC as is Pos N/A N/A
LP = Low Positive, MP = Medium Positive, LoD = Limit of Detection, NTC = No Template Control, PC =
Positive Control
Each sample panel member was tested by two (2) different operators at each testing site. Each
operator tested each panel member in triplicate each day. A total of ninety (90) replicates [three
(3) replicates X two (2) operators X three (3) sites X five (5) days] were tested for each panel
member. A total of six (6) LIAISON MDX Instruments [two (2) per site] were used to evaluate
the reproducibility study. (Site 1: 100089 and 100348, Site 2: 100349 and 100136, Site 3: 100122
and 100305).
At least one (1) set of controls (PC and NTC) were tested on each instrument being used in the
study per day. A total of sixty (60) evaluable controls sets were tested among the sites.
Page 167 of 250
One (1) lot of Candidate Device Reaction Mix (Lot V11906N) and three (3) lots of Candidate
Device Positive Control (Lots V11808N, V11923N, and V11849N) were used in the study. All
runs were performed on LIAISON MDX Studio Software, version 2.1.
Panel members 1 to 4 were performed on “Instrument 1” (Site 1: 100089, Site 2: 100349, Site 3:
100122) and panel members 5 to 7 and PC were performed on “Instrument 2” (Site 1: 100348,
site 2: 100136, Site 3: 100305) at each of the respective testing sites.
The reproducibility study was performed at two (2) external clinical sites and one (1) internal
clinical site. Table 18.6.2 is a list of sites that performed the Reproducibility study.
Table 18.6.2. Sample Testing Sites
Site ID Site Name Site Address Testing Dates

11633 San Vicente Blvd. 16, 17, 18, 21 and 22
1 UCLA
Los Angeles, CA 90049 June 2021
801 7th Ave. 22, 23, 24, 25, 28, 29*
2 Cook Children’s Hospital
Fort Worth, TX 76104 June 2021
11331 Valley View St. 14, 15. 17, 18, 21 June
3 DiaSorin Molecular
Cypress, CA 90630 2021
*Note: A 6 day of testing was performed due failed controls from Day 1. Additional specimen aliquots were provided to
th
repeat testing.
The reproducibility study was evaluated using restricted maximum likelihood analysis of variance.
The results for the above parameters were calculated with the overall mean and overall variability
utilizing cycle threshold (Ct) values, standard deviation (SD) and percent coefficient of variance
(%CV). Qualitative reproducibility for each panel member was also calculated.
Summary of Results – Precision (Reproducibility)
For each strain, the low positive (LP) samples were expected to be detected approximately 95%
of the time. The medium positive (MP) samples were expected to be detected 100% of the time.
The negative samples (UTM) were expected to be not detected 100% of the time. Conversely,
positive samples [Positive Control (PC)] were expected to be detected 100% of the time. All panel
members performed as expected per designated target for detection.
Table 18.6.3 below shows the expected and observed qualitative results according to the target
for each panel member.
Page 168 of 250
Table 18.6.3. Qualitative Summary of Reproducibility

Qualitative Summary of Reproducibility
Sample Panel Member Expected Qualitative Result Observed Result
100.0% (90/90) Detected

Influenza A/Hong Kong/8/68 ~95% of the tested replicates
(H3N2)_LP yield a Flu A detected result
95% CI: 95.9 to 100.0%
100.0% (90/90) Detected

Influenza A/Hong Kong/8/68 100% of the tested replicates
(H3N2)_MP yield a Flu A detected result
95% CI: 95.9 to 100.0%
100.0% (90/90) Detected

~95% of the tested replicates
Influenza B/ Malaysia/2506/04_LP
yield a Flu B detected result
95% CI: 95.9 to 100.0%
100.0% (90/90) Detected

Influenza B/ 100% of the tested replicates
Malaysia/2506/04_MP yield a Flu B detected result
95% CI: 95.9 to 100.0%
~95% of the tested replicates 100.0% (90/90) Detected

2019-nCoV/USA-WA1-2020_LP yield a COVID-19 detected
result 95% CI: 95.9 to 100.0%
100% of the tested replicates 100.0% (90/90) Detected

2019-nCoV/USA-WA1-2020_MP yield a COVID-19 detected
result 95% CI: 95.9 to 100.0%
100% of the tested replicates 100.0% (90/90) Detected

Positive (PC, as is) yield a Flu A, Flu B, and
COVID-19 detected result 95% CI: 95.9 to 100.0%
100.0% (90/90) Not Detected

100% of the tested replicates
Negative (UTM as NTC)
yield a not detected result
95% CI: 95.9 to 100.0%
LP = Low Positive, MP = Medium Positive, PC = Positive Control, NTC = No Template Control
At Site 1 on testing day 5, operator 1 performed testing for panel member Repro-4 and
unexpectedly observed the dual detection of COVID-19 and Flu A in all three (3) replicates.
Repro-4 was a contrived COVID-19 medium positive. The Cts for the unexpected Flu A detection
ranged from 33.1 to 35.7. For purposes of troubleshooting, Repro-4 was repeated by both
operators at Site 1. Upon repeat, Flu A was not observed in the re-testing; these results were for
internal use only and were not included in the analysis. Across all sites, no other replicates
observed dual detection.
Page 169 of 250
Table 18.6.4 shows the qualitative summary of results for Flu A, Flu B and COVID-19. For each
target within their respective channel(s), the coefficient of variance (CV) was ≤ 4.2%.
Table 18.6.4. Quantitative Summary of Flu A, Flu B and COVID-19 Reproducibility Results
Agreement Agreement Agreement Agreement Avg. Ct

Sample Avg. Ct
with Avg. Ct ± with Avg. Ct ± with with ± SD
± SD 95% CI
expected SD (%CV) expected SD (%CV) expected expected (%CV)
(%CV)
100.0% 100.0% 32.9 ± 100.0% 32.8 ± 100.0% 32.7 ± 95.9%
Flu A Hong Kong - 32.3 ± 0.54
1.22 0.55 0.86 to
LP (1.7%)
(30/30) (30/30) (3.7%) (30/30) (1.7%) (90/90) (2.6%) 100.0%
100.0% 100.0% 31.5 ± 100.0% 31.9 ± 100.0% 31.7 ± 95.9%

Flu A Hong Kong – 31.7 ± 0.49
0.43 0.44 0.47 to
MP (1.5%)
(30/30) (30/30) (1.4%) (30/30) (1.4%) (90/90) (1.5%) 100.0%
Flu B Malaysia - 100.0% 100.0% 30.9 ± 100.0% 31.5 ± 100.0% 31.1 ± 95.9%
30.8 ± 0.69
0.60 0.62 0.70 to
(2.2%)
LP (30/30) (30/30) (1.9%) (30/30) (2.0%) (90/90) (2.2%) 100.0%
Flu B Malaysia - 100.0% 100.0% 29.9 ± 100.0% 30.4 ± 100.0% 30.1 ± 95.9%
30.0 ± 0.35
0.44 0.48 0.48 to
(1.2%)
MP (30/30) (30/30) (1.5%) (30/30) (1.6%) (90/90) (1.6%) 100.0%
100.0% 100.0% 29.5 ± 100.0% 30.2 ± 100.0% 30.0 ± 95.9%

COVID-19 USA- 30.2 ± 0.41
0.40 0.67 0.59 to
WA1 - LP (1.4%)
(30/30) (30/30) (1.4%) (30/30) (2.2%) (90/90) (2.0%) 100.0%
100.0% 100.0% 28.8 ± 100.0% 29.4 ± 100.0% 29.1 ± 95.9%

COVID-19 USA- 29.0 ± 0.46
0.45 0.61 0.56 to
WA1 - MPa (1.6%)
(30/30) (30/30) (1.6%) (30/30) (2.1%) (90/90) (1.9%) 100.0%
(Flu A) 26.1 ± 26.0 ± 26.1 ±

26.2 ± 0.32
0.21 0.20 0.26
(1.2%)
(0.8%) (0.8%) (1.0%)
100.0% 100.0% 28.2 ± 100.0% 27.1 ± 100.0% 27.6 ± 95.9%

Positive 27.6 ± 0.87
(Flu B) 0.39 0.22 0.71 to
(PC) (3.2%)
(30/30) (30/30) (1.4%) (30/30) (0.8%) (90/90) (2.6%) 100.0%
27.2 ± 26.8 ± 27.3 ±

(COVID- 27.8 ± 1.18
0.23 0.22 0.80
19) (4.2%)
(0.9%) (0.8%) (2.9%)
Page 170 of 250
Agreement Agreement Agreement Agreement Avg. Ct

Sample Avg. Ct
with Avg. Ct ± with Avg. Ct ± with with ± SD
± SD 95% CI
expected SD (%CV) expected SD (%CV) expected expected (%CV)
(%CV)
(Flu A) 0.0 ± 0.0 ±
0.0 ± 0.00 0.0 ± 0.00
0.00 0.00
(N/A%) (N/A%)
(N/A%) (N/A%)
100.0% 100.0% 100.0% 0.0 ± 100.0% 0.0 ± 95.9%

Negative 0.0 ± 0.00 0.0 ± 0.00
(Flu B) 0.00 0.00 to
(NTC) (N/A%) (N/A%)
(30/30) (30/30) (30/30) (N/A%) (90/90) (N/A%) 100.0%
0.0 ± 0.0 ±
(COVID- 0.0 ± 0.00 0.0 ± 0.00
0.00 0.00
19) (N/A%) (N/A%)
(N/A%) (N/A%)
100.0% 100.0% 100.0% 100.0% 99.5%

Total to
(240/240) (240/240) (240/240) (720/720) 100.0%
a – Three (3) COVID medium positive replicates had an unexpected false positive Flu A result. All replicates were from one site from one operator
on one testing day.
Ct = Cycle threshold, SD= Standard Deviation, %CV = Percent Coefficient of Variation, LP = Low Positive, MP = Medium Positive, NTC = No
Template Control, PC = Positive Control
Table 18.6.5 shows the qualitative summary for the RNA IC (Q670) channel. For each target,
CV was ≤2.7%.
Page 171 of 250
Table 18.6.5. Qualitative Summary of Internal Control Reproducibility Results

with with with with
Sample expected expected expected expected Avg. Ct ±
results Avg. Ct ± results Avg. Ct ± results Avg. Ct ± results SD (%CV) 95% CI
SD (%CV) SD (%CV) SD (%CV)
(# (# (# (#
Detected/ Detected/ Detected/ Detected/
# Tested) # Tested) # Tested) # Tested)
100.0% 100.0% 100.0% 100.0%
Flu A Hong 30.4 ± 0.25 30.4 ± 0.31 31.0 ± 0.23 30.6 ± 0.39 95.9% to
Kong - LP (0.8%) (1.0%) (0.8%) (1.3%) 100.0%
(30/30) (30/30) (30/30) (90/90)
100.0% 100.0% 100.0% 100.0%

Flu A Hong 30.5 ± 0.27 30.0 ±0.25 30.7 ± 0.25 30.4 ± 0.41 95.9% to
Kong - MP (0.9%) (0.8%) (0.8%) (1.3%) 100.0%
(30/30) (30/30) (30/30) (90/90)
Flu B Malaysia - 100.0% 100.0% 100.0% 100.0%

30.4 ± 0.25 30.0 ± 0.22 30.6 ± 0.28 30.3 ± 0.37 95.9% to
(0.8%) (0.7%) (0.9%) (1.2%) 100.0%
LP (30/30) (30/30) (30/30) (90/90)
Flu B Malaysia - 100.0% 100.0% 100.0% 100.0%

30.5 ± 0.33 30.4 ± 0.26 30.9 ± 0.36 30.6 ± 0.39 95.9% to
(1.1%) (0.9%) (1.2%) (1.3%) 100.0%
MP (30/30) (30/30) (30/30) (90/90)
100.0% 100.0% 100.0% 100.0%

COVID-19 USA- 30.5 ± 0.24 30.0 ±0.28 30.7 ± 0.27 30.4 ± 0.41 95.9% to
WA1 - LP (0.8%) (0.9%) (0.9%) (1.4%) 100.0%
(30/30) (30/30) (30/30) (90/90)
100.0% 100.0% 100.0% 100.0%

COVID-19 USA- 30.4 ± 0.32 30.4 ± 0.30 30.9 ± 0.21 30.6 ± 0.37 95.9% to
WA1 - MP (1.0%) (1.0%) (0.7%) (1.2%) 100.0%
(30/30) (30/30) (30/30) (90/90)
100.0% 100.0% 100.0% 100.0%

30.8 ± 0.41 30.5 ± 0.53 31.8 ± 0.86 31.1 ± 0.84 95.9% to
Positive (PC)
(1.3%) (1.7%) (2.7%) (2.7%) 100.0%
(30/30) (30/30) (30/30) (90/90)
100.0% 100.0% 100.0% 100.0%

31.7 ± 0.53 31.2 ± 0.32 32.4 ± 0.51 31.8 ± 0.65 95.9% to
Negative (NTC)
(1.7%) (1.0%) (1.6%) (2.0%) 100.0%
(30/30) (30/30) (30/30) (90/90)
100.0% 100.0% 100.0% 100.0%

99.5% to
Total
100.0%
(240/240) (240/240) (240/240) (720/720)
Ct = Cycle threshold, SD= Standard Deviation, %CV = Percent Coefficient of Variation, LP = Low Positive, MP = Medium Positive, NTC = No
Template Control, PC = Positive Control
Page 172 of 250
Table 18.6.6 shows the variance components of between day, within operator, between operator
within site, between sites, within day (repeatability), and total reproducibility for each panel. The
results show reproducibility across all targets for each of the variance components, the coefficient
of variance (CV) was ≤ 3.1%.
Table 18.6.6. Quantitative Summary Flu A, Flu B and COVID-19 Between Day Within Operator, Between
Operator Within Site, Between Sites, Within Day (Repeatability), and Total Reproducibility
Between Total
Mean Between Day Between Site Repeatability
Panel Operator Reproducibility
N
Member
Ct
SD CV (%) SD CV (%) SD CV (%) SD CV (%) SD CV (%)
Flu A Hong
90 32.7 0.24 0.7 0.00 0.0 0.00 0.0 0.83 2.5 0.87 2.7
Kong - LP
Flu A Hong
90 31.7 0.13 0.4 0.13 0.4 0.00 0.0 0.44 1.4 0.48 1.5
Kong - MP
Flu B
Malaysia – 90 31.1 0.31 1.0 0.21 0.7 0.29 0.9 0.55 1.8 0.73 2.3
LP
Flu B
Malaysia – 90 30.1 0.26 0.9 0.00 0.0 0.17 0.6 0.40 1.3 0.50 1.7
MP
COVID-19
USA-WA1 – 90 30.0 0.37 1.2 0.00 0.0 0.13 0.4 0.49 1.6 0.63 2.1
LP
COVID-19
USA-WA1 – 90 29.1 0.28 1.0 0.00 0.0 0.05 0.2 0.51 1.7 0.58 2.0
MP
Positive
(PC)
90 26.1 0.06 0.2 0.00 0.0 0.15 0.6 0.21 0.8 0.26 1.0
(Flu A)
Positive
(PC)
90 27.6 0.50 1.8 0.00 0.0 0.48 1.7 0.32 1.2 0.76 2.8
(Flu B)
Positive
(PC)
90 27.3 0.43 1.6 0.00 0.0 0.68 2.5 0.26 1.0 0.85 3.1
COVID-19
Page 173 of 250
Between Total
N
Member
Ct
Negative
(NTC)
90 0.0 0.0 N/A 0.0 N/A 0.0 N/A 0.0 N/A 0.0 N/A
(Flu A)
Negative
(NTC)
90 0.0 0.0 N/A 0.0 N/A 0.0 N/A 0.0 N/A 0.0 N/A
(Flu B)
Negative
(NTC)
90 0.0 0.0 N/A 0.0 N/A 0.0 N/A 0.0 N/A 0.0 N/A
(COVID-19)
n = Number of replicates, Ct = Cycle threshold, SD= Standard Deviation, %CV = Percent Coefficient of Variation, LP = Low Positive, MP =
Medium Positive, NTC = No Template Control, PC = Positive Control
Table 18.6.7 shows the variance components for each panel member between days, within
operators, between operators, between sites, repeatability and total reproducibility.
Table 18.6.7. Quantitative Summary of Internal Control (IC) Between Day, Between Operator, Within
Operator, Within Site, Between Sites, Within Day (Repeatability), and Total Reproducibility
Between Total
N
Member
Ct
Flu A Hong
90 30.6 0.34 1.1 0.00 0.0 0.12 0.4 0.24 0.8 0.43 1.4
Kong - LP
Flu A Hong
90 30.4 0.38 1.3 0.00 0.0 0.15 0.5 0.21 0.7 0.46 1.5
Kong - MP
Flu B
Malaysia - 90 30.3 0.32 1.0 0.06 0.2 0.09 0.3 0.23 0.8 0.41 1.3
LP
Flu B
Malaysia - 90 30.6 0.27 0.9 0.00 0.0 0.21 0.7 0.25 0.8 0.42 1.4
MP
Page 174 of 250
Between Total
N
Member
Ct
COVID-19
USA-WA1 90 30.4 0.38 1.2 0.07 0.2 0.09 0.3 0.24 0.8 0.46 1.5
- LP
COVID-19
USA-WA1 90 30.6 0.29 1.0 0.00 0.0 0.14 0.5 0.25 0.8 0.41 1.3
- MP
Positive
90 31.1 0.67 2.2 0.15 0.5 0.09 0.3 0.61 2.0 0.93 3.0
(PC)
Negative
90 31.8 0.55 1.7 0.00 0.0 0.26 0.8 0.39 1.2 0.72 2.3
(NTC)
n = Number of replicates, Ct = Cycle threshold, SD= Standard Deviation, %CV = Percent Coefficient of Variation, LP = Low Positive, MP = Medium
Positive, NTC = No Template Control, PC = Positive Control
Summary of Controls – Precision (Reproducibility)
A total of sixty (60) evaluable controls were tested throughout the reproducibility study. Table
18.6.8 shows the summary of daily controls across all testing sites.
During the reproducibility study one positive control (PC) daily control was not detected (Ct = 0)
for the internal control target. It should be noted that for a daily positive control to be valid, the
internal control does not apply; however, for purposes of calculating averages and variance
components, the internal control was assigned a value of 40.0 Ct (the upper limit of the Ct range).
For each panel member, the mean Ct, SD, and %CV were calculated for each site.
Table 18.6.8. Summary of Daily Controls

Detected Detected Detected
Flu A Flu B COVID-19 IC Detected
% Detected % Detected % Detected % Detected IC Mean Ct
Control Site Instrument Mean Ct ± Mean Ct ± Mean Ct ±
(#Detected/ (#Detected (#Detected (#Detected ± SD
SD SD SD
#Tested /#Tested /#Tested /#Tested (%CV)
(%CV) (%CV) (%CV)
n=0 n=0 n=0 n = 10

0.0% 0.0% 0.0% 100.0%
100089 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 31.9 ± 0.35
(0/10) (0/10) (0/10) (10/10)
NTC S1 (N/A%) (N/A%) (N/A%) (1.1%)
0.0% 0.0% 0.0% 100.0% n=0 n=0 n=0 n = 10

100348
(0/10) (0/10) (0/10) (10/10) 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 32.0 ± 0.45
Page 175 of 250

SD SD SD
(%CV) (%CV) (%CV)
(N/A%) (N/A%) (N/A%) (1.4%)
n=0 n=0 n=0 n = 10

0.0% 0.0% 0.0% 100.0%
100349 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 32.0 ± 0.60
(0/10) (0/10) (0/10) (10/10)
(N/A%) (N/A%) (N/A%) (1.9%)
S2
n=0 n=0 n=0 n = 10
0.0% 0.0% 0.0% 100.0%
100136 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 31.3 ± 0.56
(0/10) (0/10) (0/10) (10/10)
(N/A%) (N/A%) (N/A%) (1.8%)
n=0 n=0 n=0 n = 10

0.0% 0.0% 0.0% 100.0%
100122 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 32.3 ± 0.28
(0/10) (0/10) (0/10) (10/10)
(N/A%) (N/A%) (N/A%) (0.9%)
S3
n=0 n=0 n=0 n = 10
0.0% 0.0% 0.0% 100.0%
100305 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 32.0 ± 0.50
(0/10) (0/10) (0/10) (10/10)
(N/A%) (N/A%) (N/A%) (1.6%)
n=0 n=0 n=0 n = 60

0.0% 0.0% 0.0%
100.0%
All 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 31.9 ± 0.55
(60/60)
(0/60) (0/60) (0/60)
(N/A%) (N/A%) (N/A%) (1.7%)
n = 10 n = 10 n = 10 n = 10
100.0% 100.0% 100.0% 100.0%
100089 25.6 ± 0.23 26.7 ± 0.50 26.7 ± 0.51 31.3 ± 0.84
(10/10) (10/10) (10/10) (10/10)
(0.9%) (1.9%) (1.9%) (2.7%)
S1
n = 10 n = 10 n = 10 n = 10
PC
100.0% 100.0% 100.0% 100.0%
100348 26.1 ± 0.25 27.4 ± 0.93 27.5 ± 1.03 30.8 ± 0.60
(10/10) (10/10) (10/10) (10/10)
(1.0%) (3.4%) (3.8%) (1.9%)
100.0% 100.0% 100.0% 100.0%

S2 100349 n = 10 n = 10 n = 10 n = 10
(10/10) (10/10) (10/10) (10/10)
Page 176 of 250

SD SD SD
(%CV) (%CV) (%CV)
26.0 ± 0.16 27.8 ± 0.33 27.4 ± 0.40 31.0 ± 0.39
(0.6%) (1.2%) (1.5%) (1.3%)
n = 10 n = 10 n = 10 n = 10a
100.0% 100.0% 100.0% 100.0%
100136 26.1 ± 0.20 28.1 ± 0.41 27.0 ± 0.42 31.6 ± 2.97
(10/10) (10/10) (10/10) (10/10)a
(0.8%) (1.5%) (1.5%) (9.4%)
n = 10 n = 10 n = 10 n = 10
100.0% 100.0% 100.0% 100.0%
100122 25.9 ± 0.16 27.0 ± 0.20 27.0 ± 0.21 32.6 ± 2.39
(10/10) (10/10) (10/10) (10/10)
(0.6%) (0.7%) (0.8%) (7.3%)
S3
n = 10 n = 10 n = 10 n = 10
100.0% 100.0% 100.0% 100.0%
100305 26.1 ± 0.13 27.3 ± 0.22 26.8 ± 0.22 31.7 ± 1.16
(10/10) (10/10) (10/10) (10/10)
(0.5%) (0.8%) (0.8%) (3.7%)
n = 60 n = 60 n = 60 n = 60
100.0% 100.0% 100.0% 100.0%
All 26.0 ± 0.24 27.4 ± 0.68 27.1 ± 0.60 31.5 ± 1.72
(60/60) (60/60) (60/60) (60/60)
(0.9%) (2.5%) (2.2%) (5.5%)
a – RNA IC for one daily control PC on instrument 100136 had a Ct of 0.0; however, the control is valid as RNA IC is not required for Positive
Controls. A Ct of 40.0 was assigned for the replicate and used in the analysis. n = number of replicates, Ct = Cycle threshold, SD= Standard
Deviation, %CV = Percent Coefficient of Variation, NTC = No Template Control, PC = Positive Control
Conclusion – Precision (Reproducibility)
Across all analyte targets (Influenza A/Hong Kong (LP and MP), Influenza B/Malaysia (LP and
MP), COVID-19 USA-WA1 (LP and MP), the negative sample (UTM as NTC), and the positive
sample), the Candidate Device assay reproducibility performance showed ≤ 3.1% CV for each
of the variance components.
The study demonstrates that the variance on a random single assay on a random site/instrument,
operator, run and day, is robust and reproducible.
Page 177 of 250
Reproducibility Protocol 18.6-1
Reproducibility Line Data (pdf) 18.6-2
Reproducibility Line Data (xls) 18.6-3
18.7 Inter-lot Precision – Reaction Mix
The Candidate Device was evaluated for potential lot-to-lot variability. The study was performed
at DiaSorin Molecular, Cypress, CA using four (4) LIAISON MDX Instruments (100122, 100215,
100305 and 100445), three (3) lots of Candidate Device kit (Lots V11906N, V11908N and
V12340N), and one (1) lot of Candidate Device Positive Control (PC) (Lot V12441N) run as a
sample. Two (2) lots of Candidate Device Positive Control (PC) (Lot V12441N and V11808N)
were run as daily controls. A total of 193 experimental runs were performed by four (4) operators
over the course of 13 non-consecutive days (22 and 30 June and 01, 07, 28, 29 and 30 July and
03, 04, 05, 06, 10, 11 August, 2021).
The runs on dates 22 and 30 June 2021 were considered Day 1 for the purpose of the study. A
portion of samples were tested per protocol (in duplicate per kit lot per run, on each of two (2)
runs per day per operator) for each day. Specifically, four (4) samples were run on 22 June and
four (4) samples were run on 30 June.
Each sample panel member other than described above was tested in duplicate per kit lot per
run, on each of two (2) runs per day for a total of 12 non-consecutive days. A total of 144
evaluable replicates (2 replicates/kit lot X 3 kit lots/run X 2 runs/day X 12 days) were tested for
each sample panel member. A total of 1,258 replicates (eight [8] panel members X 144 evaluable
replicates per panel member plus 106 controls) were tested during the study.
The panel consisted of a positive control (PC), Universal Transport Media (UTM) as a negative
sample, a low positive (LP) and a medium positive (MP) of the 2019-nCoV/USA-WA1/2020 strain,
a low positive (LP) and a medium positive (MP) of the Influenza A/Hong Kong/8/68 strain, and a
low positive (LP) and a medium positive (MP) of the Influenza B/Malaysia/2506/04 strain. Each
contrived sampled was prepared by spiking a specific concentration of inactivated viral particles
into pooled negative nasopharyngeal swab matrix in UTM.
The variance components were analyzed using the restricted maximum likelihood (REML)
method. The REML method uses a random effects model to estimate the variance components
of the individual factors, such as lot of reaction mix or testing days.
A description of each of the eight (8) panel members is shown in Table 18.7.1. The Positive
Control listed was tested as an unknown sample, in the same manner as the other panel
members.
Page 178 of 250
Table 18.7.1. Panel Composition
Panel Member Concentration

SARS-CoV-2 – LP (low positive) 1000 copies/mL (2x LoD)
SARS-CoV-2 – MP (medium positive) 2000 copies/mL (4x LoD)
Influenza A – LP (low positive) 1000 copies/mL (2x LoD)
Influenza A – MP (medium positive) 2000 copies/mL (4x LoD)
Influenza B – LP (low positive) 500 copies/mL (2x LoD)
Influenza B – MP (medium positive) 1000 copies/mL (4x LoD)
UTM as NTC (panel member) N/A
LoD = Limit of Detection
Summary of Results – Inter-Lot Precision (Reaction Mix)
Table 18.7.2 provides a summary of the qualitative performance of the assay for each of the eight
(8) panel members. There was 100% positivity (36/36 replicates) for the low positive (LP) sample,
medium positive (MP) sample and the positive control (PC) for all targets. There was no detection
for the negative sample (UTM) on all targets.
Table 18.7.2. Summary of Qualitative Reaction Mix Inter-Lot Reproducibility
Expected % Detection % Detection % Detection

Panel
Qualitative Result
Member (# Detected / # (# Detected / # (# Detected / #
for target
95% CI range% 95% CI range% 95% CI range%
99.3% (143/144)
~95% of the tested 0% (0/144)
Influenza A Positive 0.7% (1/144) *
LP Flu A replicate results are
95% CI: 96.2% to Influenza B Positive COVID-19 Positive
Flu A Positive
99.98%
100.0% (144/144)
100% of the tested
Influenza A Positive 0% (0/144) 0% (0/144)
MP Flu A replicate results are
Flu A Positive 95% CI: 97.5 to Influenza B Positive COVID-19 Positive
100.0%
100.0% (144/144)
~95% of the tested 0% (0/144) 0% (0/144)
Influenza B Positive
LP Flu B replicate results are
Flu B Positive Influenza A Positive 95% CI: 97.5 to COVID-19 Positive
100.0%
100.0% (144/144)
100% of the tested 0% (0/144) 0% (0/144)
Influenza B Positive
MP Flu B replicate results are
Flu B Positive Influenza A Positive 95% CI: 97.5 to COVID-19 Positive
100.0%
Page 179 of 250
Expected % Detection % Detection % Detection

Panel
Qualitative Result
Member (# Detected / # (# Detected / # (# Detected / #
for target
95% CI range% 95% CI range% 95% CI range%
99.3% (143/144)
~95% of the tested 0% (0/144) 0% (0/144) COVID-19 Positive
LP COVID-
replicate results are
19 Influenza A Positive Influenza B Positive 95% CI: 96.2 to
COVID-19 Positive
99.98%
100.0% (144/144)
100% of the 0% (0/144) 0% (0/144)
MP COVID- COVID-19 Positive
replicate results are
19 Influenza A Positive Influenza B Positive 95% CI: 97.5 to
COVID-19 Positive
100.0%
100% of the 100.0% (144/144) 100.0% (144/144) 100.0% (144/144)
Positive replicate results are Influenza A Positive Influenza B Positive COVID-19 Positive
Control Positive for all 95% CI: 97.5 to 95% CI: 97.5 to 95% CI: 97.5 to
targets 100.0% 100.0% 100.0%
0% of the replicate 0% (0/144) 0% (0/144) 0% (0/144)

Negative results are detected Influenza A Positive Influenza B Positive COVID-19 Positive
for targets
LP = Low Positive, MP = Medium Positive, CI = Confidence Interval

*Note one Flu A low positive sample returned COVID-19 detection
One (1) Flu A low positive sample returned a COVID-19 Ct of 34.3. The average Ct of COVID-
19 at LoD is 30.8. This detection was beyond the LoD. This occurred on the first day of testing
(22 June 2021). Additional cleaning was performed and testing continued and no further false
positives were detected. DiaSorin Molecular believes environmental contamination may have
caused the late Ct detection of COVID-19.
Results Inter-Lot Precision (Reaction Mix) - Influenza A
Table 18.7.3 shows the qualitative performance and Flu A Ct values by lot of reaction mix. For
each panel member, the mean Ct, standard deviation (SD) and %CV are shown for each lot of
reaction mix, as well as for all three (3) lots of reaction mix as combined data. When all three (3)
lots are combined, the %CV is ≤ 2.9% for the Low Positive (LP), Medium Positive (MP) and
Positive Control (PC).
Page 180 of 250
Table 18.7.3. Qualitative Performance and Flu A Ct Values by Lot of Assay Kit (Reaction Mix)
Kit lot V11906N Kit lot V11908N Kit lot V12340N All Lots Combined
% Detection % Detection % Detection % Detection Mean
Panel Mean Mean Mean Ct
Member Ct ±
(#Detected/ Ct ± SD (#Detected/ Ct ± SD (#Detected ± SD (#Detected/ SD
#Tested) (%CV) #Tested) (%CV) /#Tested) (%CV) #Tested) (%CV)
100% 32.6 +/- 97.9% 33.8 +/- 100% 32.7 +/- 99.3% 33.0
Flu A LP
(48/48) 0.65 (47/48) 0.99 (48/48) 0.65 (143/144) +/-
(2.0%) (2.9%) (2.0%) 0.96
(2.9%)
100% 32.0 +/- 100% 33.0 +/- 100% 32.0 +/- 100% 32.3
Flu A MP
(48/48) 0.53 (48/48) 0.63 (48/48) 0.56 (144/144) +/-
(1.6%) (1.9%) (1.8%) 0.74
(2.3%)
0% 0 +/- 0 0% 0 +/- 0 0% 0 +/- 0 0% 0 +/-
NTC
(0/48) (0%) (0/48) (0%) (0/48) (0%) (0/144) 0
(0%
)
100% 26.0 +/- 100% 26.2 +/- 100% 25.2 +/- 100% 25.8
PC
(48/48) 0.45 (48/48) 0.33 (48/48) 0.16 (144/144) +/-
(1.7%) (1.3%) (0.6%) 0.54
(2.1%)
Ct = Cycle threshold, SD= Standard Deviation, %CV = Percent Coefficient of Variation, LP = Low Positive, MP =
Medium Positive, NTC = No Template Control, PC = Positive Control
Table 18.7.5 shows the measurement variance components for Flu A for the panel members.
For each Flu A panel member, the standard deviation (SD) and %CV are shown for inter-lot
variability, inter-day variability, inter-run variability, residuals (remaining variability left over after
accounting for the other sources – lot, day and run) and total. When all three (3) lots of reaction
mix are combined for total variability, the %CV is ≤ 3.2% for each of the panel members.
Table 18.7.5. Measurement Variance Components of Flu A
Panel Inter-Lot Inter-Day Inter-Run Residual Total

N Mean
Member
SD %CV SD %CV SD %CV SD %CV SD %CV
Flu A LP 143 33.05 0.68 2.05 0.36 1.09 0.24 0.73 0.65 1.97 1.03 3.13
Flu A MP 144 32.34 0.57 1.78 0.25 0.77 0 0 0.52 1.60 0.81 2.51
PC 144 25.82 0.52 2.00 0.08 0.32 0.16 0.64 0.28 1.08 0.62 2.38
N = number of replicates, SD= Standard Deviation, %CV = Percent Coefficient of Variation, LP = Low Positive, MP = Medium
Positive, PC = Positive Control
Page 181 of 250
Results - Inter-Lot Precision (Reaction Mix) - Influenza B
Table 18.7.6 shows the qualitative performance and Flu B Ct values by lot of reaction mix. For
each panel member, the mean Ct, standard deviation (SD) and %CV are shown for each lot of
lots are combined, the %CV is equal to ≤ 2% for each of the panel members.
Table 18.7.6. Qualitative Performance and Flu B Ct Values by Lot of Assay Kit (Reaction Mix)
Panel Mean Mean Mean Mean
Member Ct ± SD Ct ± SD Ct ± SD Ct ± SD
100% 31.1 +/- 100% 31.2 +/- 100% 31.5 +/- 100% 31.3 +/-
Flu B LP (48/48) 0.61 (48/48) 0.61 (48/48) 0.64 (144/144) 0.64
(2.0%) (2.0%) (2.0%) (2.0%)
100% 30.6 +/- 100% 30.8 +/- 100% 31.1 +/- 100% 30.8 +/-
Flu B MP (48/48) 0.39 (48/48) 0.64 (48/48) 0.58 (144/144) 0.58
(1.3%) (2.1%) (1.9%) (1.9%)
0% 0 +/- 0 0% 0 +/- 0 0% 0 +/- 0 0% 0 +/- 0
NTC (0/48) (0%) (0/48) (0%) (0/48) (0%) (0/144) (0%)
100% 27.0 +/- 100% 26.5 +/- 100% 26.5 +/- 100% 26.7 +/-
PC (48/48) 0.44 (48/48) 0.33 (48/48) 0.34 (144/144) 0.44
(1.6%) (1.2%) (1.3%) (1.7%)
Ct = Cycle threshold, SD= Standard Deviation, %CV = Percent Coefficient of Variation, LP = Low Positive, MP = Medium
Table 18.7.7 shows the measurement variance components for Flu B for the panel members.
For each Flu B panel member, the standard deviation (SD) and %CV are shown for inter-lot
Table 18.7.7. Measurement Variance Components of Flu B

N Mean
Member
Flu B LP 144 31.30 0.19 0.59 0.24 0.76 0.30 0.95 0.49 1.57 0.65 2.07
Flu B MP 144 30.82 0.24 0.77 0.10 0.32 0.04 0.12 0.53 1.73 0.59 1.93
PC 144 26.65 0.29 1.09 0.26 0.99 0.08 0.28 0.25 0.95 0.47 1.78
Page 182 of 250
Inter-Lot Precision (Reaction Mix) - Results for COVID-19
Table 18.7.8 shows the qualitative performance and COVID-19 Ct values by lot of reaction mix.
For each panel member, mean Ct, standard deviation (SD) and %CV are shown for each lot of
lots are combined, the %CV is ≤ 2% for each of the panel members.
Table 18.7.8. Qualitative performance and COVID-19 Ct values by lot of assay kit
100% 31.1 +/- 100% 31.2 +/- 100% 31.5 +/- 100% 31.3 +/-
COVID-
(48/48) 0.61 (48/48) 0.61 (48/48) 0.64 (144/144) 0.64
19 LP (2.0%) (2.0%) (2.0%) (2.0%)
100% 30.6 +/- 100% 30.8 +/- 100% 31.1 +/- 100% 30.8 +/-
COVID-
(48/48) 0.39 (48/48) 0.64 (48/48) 0.58 (144/144) 0.58
19 MP (1.3%) (2.1%) (1.9%) (1.9%)
0% 0 +/- 0 0% 0 +/- 0 0% 0 +/- 0 0% 0 +/- 0
NTC (0/48) (0%) (0/48) (0%) (0/48) (0%) (0/144) (0%)
100% 27.0 +/- 100% 26.5 +/- 100% 26.5 +/- 100% 26.7 +/-
PC (48/48) 0.44 (48/48) 0.33 (48/48) 0.34 (144/144) 0.44
(1.6%) (1.2%) (1.3%) (1.7%)
Table 18.7.9 shows the measurement variance components for COVID-19. For each COVID-19
panel member, the standard deviation (SD) and %CV are shown for inter-lot variability, inter-day
variability, inter-run variability, residuals (remaining variability left over after accounting for the
other sources – lot, day and run) and total. When all three (3) lots of reaction mix are combined
for total variability, the %CV ≤ 2.4% for each of the panel members.
Page 183 of 250
Table 18.7.9. Measurement Variance Components of COVID-19

N Mean
Member
COVID- 30.41 0 0 0.02 0.08 0.15 0.50 0.54 1.76 0.56 1.83
143
19 LP
COVID- 29.11 0 0 0.16 0.54 0.25 0.86 0.51 1.74 0.59 2.01
144
19 MP
PC 144 26.50 0.53 1.99 0 0 0.18 0.69 0.26 0.98 0.61 2.32
Results Inter-Lot Precision (Reaction Mix) – Internal Control
Table 18.7.10 shows the qualitative performance and internal control (IC) Ct values by lot of
reaction mix. For each panel member, the mean Ct, standard deviation (SD) and %CV are shown
for each lot of reaction mix, as well as for all three (3) lots combined. When all three (3) lots are
combined, the %CV is ≤ 3% for each of the panel members.
Table 18.7.10. Qualitative Performance and Internal Control Ct Values by Lot of Assay Kit
COVID - 30.8 +/-

100% 31.0 +/- 100% 30.6 +/- 100% 30.8 +/- 100%
0.33
(48/48) 0.27 (48/48) 0.37 (48/48) 0.26 (144/144)
19 LP (1.1%)
COVID - 30.5 +/-

100% 100% 100% 100%
(0.9%) (1.2%) (0.9%) 0.42
(48/48) (48/48) (48/48) (144/144
19 MP (1.4%)
30.6 +/-
100% 30.6 +/- 100% 30.3 +/- 100% 30.4 +/- 100%
Flu A LP 0.42
(48/48) 0.38 (48/48) 0.51 (48/48) 0.28 (144/144)
(1.4%)
30.7 +/-
100% 100% 100% 100%
Flu A MP (1.2%) (1.7%) (0.9%) 0.38
(48/48) (48/48) (48/48) (144/144)
(1.2%)
30.7 +/- 30.5 +/- 30.6 +/- 30.8 +/-
100% 100% 100% 100%
Flu B LP 0.41 0.51 0.30 0.34
(48/48) (48/48) (48/48) (144/144)
(1.3%) (1.7%) (1.0%) (1.1%)
30.8 +/- 30.5 +/- 30.6 +/- 30.5 +/-
100% 100% 100% 100%
Flu B MP 0.32 0.43 0.31 0.38
(48/48) (48/48) (48/48) (144/144)
(1.0%) (1.4%) (1.0%) (1.3%)
Page 184 of 250
30.92 30.6 +/- 30.8 +/- 30.7 +/-
100% 100% 100% 100%
NTC +/- 0.31 0.38 0.25 0.92
(48/48) (48/48) (48/48) (144/144)
(1.0%) (1.2%) (0.8%) (3.0%)
30.7 +/- 30.3 +/- 30.5 +/- 30.6 +/-
100% 100% 100% 100%
PC 0.34 0.42 0.27 0.9
(48/48) (48/48) (48/48) (144/144)
(1.1%) (1.4%) (0.9%) (2.9%)
Table 18.7.11 shows the measurement variance components for internal control, for all panel
members. For each panel member, the standard deviation (SD) and %CV are shown for inter-lot
Table 18.7.11. Measurement Variance Components of Internal Control

Inter-Lot Inter-Day Inter-Run Residuals Total
Panel
N Mean
Member
COVID-
144 30.81 0.16 0.52 0.11 0.37 0.12 0.4 0.26 0.83 0.34 1.12
19 LP
COVID-
144 30.46 0.15 0.49 0.13 0.43 0.04 0.12 0.38 1.25 0.43 1.41
19 MP
Flu A LP 144 30.60 0.08 0.26 0.23 0.74 0.01 0.05 0.35 1.15 0.43 1.39
Flu A
144 30.68 0.13 0.43 0.00 0.00 0.20 0.66 0.30 0.97 0.38 1.25
MP
Flu B LP 144 30.76 0.12 0.40 0.05 0.16 0.19 0.62 0.25 0.82 0.34 1.12
Flu B
144 30.48 0.19 0.63 0.11 0.38 0.07 0.23 0.32 1.06 0.40 1.31
MP
NTC 144 30.69 0.91 2.97 0.43 1.40 0 0 0.32 1.04 1.06 3.45
PC 144 30.61 0.82 2.69 0.17 0.57 0.01 0.04 0.56 1.83 1.01 3.31
Page 185 of 250
Table 18.7.12 summarizes the reproducibility for all three (3) lots of reaction mix for the COVID-
19, Flu A, Flu B and internal control targets.
Table 18.7.12. Summary of Reaction Mix Inter-Lot

Reproducibility
Target % CV
Flu A < 4%
Flu B < 3%
COVID-19 < 3%
Internal Control < 4%

% CV = % Coefficient of Variation
Summary of Controls – Inter-Lot Precision (Reproducibility)
Table 18.7.13 shows the results of the Positive Control (PC) and the No Template Control
(NTC) for each assay run. All controls performed as expected.
Table 18.7.13. Summary of Controls
COVID-19 Flu A Flu B IC
Control % % % %
(lot #) Detection Mean Ct Detection Mean Ct Detection Mean Ct Detection Mean Ct
± SD ± SD ± SD ± SD
(# Detected (%CV) (# Detected (%CV) (# Detected (%CV) (# Detected (%CV)
/ # Tested) / # Tested) / # Tested) / # Tested)
NTC 31.6 +/-
0% 0 +/- 0 0% 0 +/- 0 0% 0 +/- 0 100%
0.83
(0/53) (0) (0/53) (0) (0/53) (0) (53/53)
(2.6%)
PC 27.1 +/- 26.1 +/- 27.3 +/- 31.4 +/-
100% 100% 100% 100%
(V11808N) 0.1 0.26 0.37 0.83
(4/4) (4/4) (4/4) (4/4)
(0.4%) (1.0%) (1.4%) (2.6%)
PC 26.5 +/- 25.5 +/- 26.8 +/- 30.9 +/-
(V12441N) 100% 100% 100% 100%
0.58 0.34 0.46 0.69
(49/49) (49/49) (49/49) (49/49)
(2.2%) (1.3%) (1.7%) (2.2%)
Ct = Cycle threshold, SD= Standard Deviation, %CV = Percent Coefficient of Variation, NTC = No Template Control, PC =
Positive Control
Page 186 of 250
Conclusions – Inter-Lot Precision (Reaction Mix)
The Candidate Device Reaction Mix overall %CV for all three (3) lots was ≤ 4% for Flu A, for Flu
B and COVID-19 it was <3%. For the Internal Control (IC) Ct, the %CV was ≤ 4%. The Candidate
Device Reaction Mix demonstrated a robust precision across the three (3) lots of Reaction Mix
and performs as intended. All of the controls performed as expected.
Inter-lot Precision – Reaction Mix Protocol 18.7-1
Inter-lot Precision – Reaction Mix Line Data (pdf) 18.7-2
Inter-lot Precision – Reaction Mix Line Data (xls) 18.7-3
18.8 Inter-lot Precision – Positive Control
The Candidate Device Positive Control (MOL4260) was evaluated for potential lot-to-lot variability
(precision). The study was performed at DiaSorin Molecular, Cypress, CA using one (1) LIAISON
MDX Instrument (100170), one (1) lot of Candidate Device kit (Lot V11906N), and three (3) lots
of Candidate Device Positive Control (PC) (Lots V12441N, V11923N, V11849N). A total of six
(6) experimental runs were performed by three (3) operators over the course of 12 non-
consecutive days (30 June, 01, 02, 06, 07, 22, 23, 26, 27, 28, 29 and 30 July 2021).
Twelve replicates of each of the three (3) lots of the Candidate Device Positive Control were
tested over the course of 12 days. All targets Flu A, Flu B, COVID-19 and Internal Control were
detected in each of the 144 total replicates.
The variance components were analyzed using the restricted maximum likelihood (REML)
method.
Results – Inter-lot Precision – Positive Control
Table 18.8.1 shows the qualitative performance of the Candidate Device Positive Control inter-
lot reproducibility.
Page 187 of 250
Table 18.8.1. Summary of Qualitative Positive Control Inter-Lot Precision

Reproducibility
Panel Member Expected Qualitative Result Observed Qualitative Result
100% of the tested replicate 100.0% (144/144) Flu A Positive

results are Flu A Positive 95% CI: 97.5 to 100.0%
100% of the tested replicate 100.0% (144/144) Flu B Positive

results are Flu B Positive 95% CI: 97.5 to 100.0%
Positive Control 100.0% (144/144) COVID-19
100% of the tested replicate Positive
results are COVID-19 Positive
95% CI: 97.5 to 100.0%
100% of the tested replicate 100.0% (144/144) IC detected

results are detected for Internal
Control (IC) 95% CI: 97.5 to 100.0%
Results – Positive Control Inter-Lot Precision Flu A
Table 18.8.2 shows the qualitative reproducibility and Flu A Ct values by lot of Positive Control.
Mean Ct, standard deviation (SD) and %CV are shown for each lot of Positive Control, as well
as for all three (3) lots combined. When all three (3) lots are combined, the %CV ≤ 2.4% for the
Positive Control.
Table 18.8.2. Qualitative Reproducibility and Flu A Ct Values for Each Lot of
Positive Control (PC)
PC Lot – V12441N PC Lot – V11923N PC Lot – V11849N All Lots Combined

% Mean % Mean % Mean % Mean
Panel Ct ±
Detection Detection Ct ± Detection Ct ± Detection Ct ±
Member SD
(#Detected (#Detected SD (#Detected SD (#Detected SD
/#Tested) (%CV /#Tested) (%CV) /#Tested) (%CV) /#Tested) (%CV)
)
25.9 26.8 26.3 26.3
100% +/- 100% +/- 100% +/- 100% +/-
PC
(48/48) 0.32 (48/48) 0.67 (48/48) 0.46 (144/144) 0.62
(1.2%) (2.5%) (1.8%) (2.4%)
Ct = Cycle threshold, SD= Standard Deviation, %CV = Percent Coefficient of Variation, PC= Positive Control
Table 18.8.3 shows the measurement variance components for Flu A, for the three (3) lots of
Positive Control. The standard deviation (SD) and %CV are shown for inter-lot variability, inter-
day variability, inter-run variability, residuals (remaining variability left over after accounting for
the other sources – lot, day and run) and total. When all three (3) lots of Positive Control are
combined for total variability, the %CV ≤ 2.6%.
Page 188 of 250
Table 18.8.3. Measurement Variance Components of Flu A Quantitative

Summary of Positive Control Inter-lot Reproducibility

Sample N Mean
144 26.32 0.443 1.68 0.289 1.1 0.109 0.41 0.404 1.535 0.674 2.56
PC
Results – Positive Control Inter-Lot Precision Flu B
Table 18.8.4 shows the qualitative reproducibility and Flu B Ct values by lot of Positive Control.
Mean Ct, standard deviation (SD) and %CV are shown for each lot of Positive Control, as well
as for all three (3) lots combined. When all three (3) lots are combined, the %CV is 3.4% for the
Positive Control.
Table 18.8.4. Qualitative Reproducibility and Flu B Ct Values for Each Lot of
Positive Control (PC)
Panel
Detection Ct ± Detection Ct ± Detection Ct ± Detection Ct ±
Member
26.8 28.8 28.1 27.9
100% +/- 100% +/- 100% +/- 100% +/-
PC
(48/48) 0.39 (48/48) 0.65 (48/48) 0.34 (144/144) 0.94
(1.4%) (2.3%) (1.2%) (3.4%)
Table 18.8.5 shows the measurement variance components for Flu B, for the three (3) lots of
Positive Control. The standard deviation (SD) and %CV are shown for inter-lot variability, inter-
day variability, inter-run variability, residuals (remaining variability left over after accounting for
the other sources – lot, day and run) and total. When all three (3) lots of Positive Control are
combined for total variability, the %CV is less than 4%.
Page 189 of 250
Table 18.8.5. Measurement Variance Components of Flu B Quantitative Summary of

Positive Control Inter-lot Reproducibility

Sample N Mean
144 27.87 0.99 3.55 0.135 0.49 0.199 0.71 0.417 1.50 1.101 3.95
PC
N = number of replicates, SD= Standard Deviation, %CV = Percent Coefficient of Variation, LP = Low Positive, MP = Medium Positive,
PC = Positive Control
Results – Positive Control Inter-Lot Precision COVID-19
Table 18.8.6 shows the qualitative reproducibility and COVID-19 Ct values by lot of Positive
Control. Mean Ct, standard deviation (SD) and %CV are shown for each lot of Positive Control,
as well as for all three (3) lots combined. When all three (3) lots are combined, the %CV is 4.0%
for the Positive Control.
Table 18.8.6. Qualitative Reproducibility and COVID-19 Ct Values for Each Lot
of Positive Control (PC)

Panel
Member
26.4 28.7 27.2 27.4
100% +/- 100% +/- 100% +/- 100% +/-
PC
(48/48) 0.44 (48/48) 0.65 (48/48) 0.26 (144/144) 1.10
(1.7%) (2.3%) (1.0%) (4.0%)
Table 18.8.7 shows the measurement variance components for COVID-19, for the three (3) lots
of Positive Control. The standard deviation (SD) and %CV are shown for inter-lot variability, inter-
day variability, inter-run variability, residuals (remaining variability left over after accounting for the
other sources – lot, day and run) and total. When all three (3) lots of Positive Control are combined
for total variability, the %CV is less than 4.8%.
Page 190 of 250
Table 18.8.7. Measurement Variance Components of COVID-19 Quantitative

Summary of Positive Control Inter-lot Reproducibility

Sample N Mean
144 27.44 1.21 4.41 0.089 0.32 0.27 0.99 0.386 1.41 1.301 4.74
PC
Results – Positive Control Inter-Lot Precision Internal Control
Table 18.8.8 shows the qualitative performance and internal control (IC) Ct values by lot of
Positive Control. Mean Ct, standard deviation (SD) and %CV are shown for each lot of Positive
Control, as well as for all three (3) lots combined. When all three (3) lots are combined, the %CV
is 2.2% for the Positive Control.
Table 18.8.8. Qualitative performance and Internal Control Ct

values for each lot of Positive Control (PC)
Panel
Member
30.4 30.5 30.5 +/-
30.6+/-
100% 100% +/- 100% +/- 100% 0.67
PC 0.80
(48/48) (48/48) 0.60 (48/48) 0.60 (144/144) (2.2%)
(2.6%)
(2.0%) (2.0%)
Table 18.8.9 shows the measurement variance components for internal control, for the three (3)
lots of Positive Control. The standard deviation (SD) and %CV are shown for inter-lot variability,
inter-day variability, inter-run variability, residuals (remaining variability left over after accounting
for the other sources – lot, day and run) and total. When all three (3) lots of Positive Control are
combined for total variability, the %CV ≤ 2.3%.
Page 191 of 250
Table 18.8.9. Measurement Variance Components of Internal Control (IC)

Quantitative Summary of Positive Control Inter-lot Reproducibility

Sample N Mean
144 30.52 0 0 0.495 1.62 0.124 0.41 0.443 1.45 0.676 2.22
PC
N = number of replicates, SD= Standard Deviation, %CV = Percent Coefficient of Variation, LP = Low Positive, MP =
Medium Positive, PC = Positive Control
Summary of Control Results – Positive Control Inter-Lot Precision
For daily controls, lot V12441N was run as the PC. On the first run of each day, one aliquot of
PC Lot V12441N and No Template Control (NTC) was run, in addition to the three (3) lots of
Positive Control. Table 18.8.10 shows the results of the PC and NTC for each assay run. All
controls performed as expected.
Table 18.8.10. Summary of Controls
Control % % % %
Detection Mean Ct Detection Mean Ct Detection Mean Ct Detection Mean Ct
± SD ± SD ± SD ± SD
(# Detected (%CV) (# Detected (%CV) (# Detected (%CV) (# Detected (%CV)
/ # Tested) / # Tested) / # Tested) / # Tested)
31.4 ±
0% 0% 0% 100% 0.71
NTC N/A N/A N/A
(0/12) (0/12) (0/12) (12/12)
(2.3%)
26.7 ± 30.5 ±
25.8 ± 0.23 26.3 ± 0.39
100% 100% 0.35 100% 100% 0.53
PC
(12/12) (12/12) (12/12) (12/12)
(0.9%) (1.5%)
(1.3%) (1.7%)
Ct = Cycle threshold, SD= Standard Deviation, %CV = Percent Coefficient of Variation, NTC = No Template Control, PC=
Positive Control
Table 18.8.11 summarizes the reproducibility for all three (3) lots of Positive Control for the Flu
A, Flu B, COVID-19 and Internal Control (IC) targets.
Table 18.8.11. Summary of Positive Control Inter-Lot Reproducibility
Target % CV
Flu A 2.56%
Flu B 3.95%
COVID-19 4.74%
Internal Control 2.22%
% CV = Percent Coefficient of Variation
Page 192 of 250
Conclusion – Inter-Lot Precision – Positive Control
The Candidate Device Positive Control overall %CV for all three (3) lots was 2.56% for Flu A,
3.95% for Flu B, and 4.74% for COVID-19. For the IC the %CV was 2.22%. The Candidate Device
Positive Control demonstrated a robust precision across the three (3) lots of Positive Control and
is performing as intended. All of the controls performed as expected.
Inter-lot Precision – Positive Control Protocol 18.8-1
Inter-lot Precision – Positive Control Line Data (pdf) 18.8-2
Inter-lot Precision – Positive Control Line Data (xls) 18.8-3
18.9 Amplification Carry-Over/Direct Amplification Disc Re-Usability
Amplification Carry Over studies/Disc Re-usability studies with nasopharyngeal swabs was
previously performed as part of K120413 Simplexa™ Flu A/B & RSV Direct. As Amplification
Carry-Over testing is not analyte specific the study can be applied to the Candidate Device.
18.10 Limit of Detection (LoD)
The Analytical Sensitivity (Limit of Detection) study for the Candidate Device is comprised of two
(2) studies:
a) Limit of Detection with two (2) influenza A, two (2) influenza B and one (1) COVID-19
strain (Influenza A (Hong Kong/8/1968 and Michigan/45/2015 strains), influenza B
(Malaysia/2506/04 and Phuket/3073/2013 strains) and heat-inactivated COVID-19 viral
particles from the 2019-nCoV/USA-WA1/2020).
b) Limit of Detection using the WHO international Standard (Std designation) for COVID-
19.
a) Influenza A (Hong Kong/8/1968 and Michigan/45/2015), influenza B (Malaysia/2506/04 and

Phuket/3073/2013) and COVID-19 2019-nCoV/USA-WA1/2020
The Limit of Detection (LoD) for the Candidate Device was performed at DiaSorin Molecular,
Cypress, CA using five (5) LIAISON MDX Instruments (100090, 100122, 100215, 100305 and
Page 193 of 250
100445), two (2) lots of Candidate Device Reaction Mix (Lots V11906N and V11908N), one (1)
lot of Candidate Device Positive Control (Lot V11808N) and three (3) lots of Direct Amplification
Discs (Lots 11147N, 11098N and 11530N). In total, 77 experimental runs were performed by
three (3) operators over the course of four (4) days (6-7 and 20-21 April, 2021).
Influenza A (Hong Kong/8/1968 and Michigan/45/2015 strains), influenza B (Malaysia/2506/04

and Phuket/3073/2013 strains) and heat-inactivated COVID-19 viral particles from the 2019-
nCoV/USA-WA1/2020 strain was diluted to various concentrations and spiked into pooled
negative nasopharyngeal (NPS) specimen matrix.
The limit of detection was determined using a two-step approach. During the screening phase, a
preliminary LoD was established by testing four (4) replicates of each concentration level of
inactivated virus, using a single lot of reaction mix. The preliminary LoD was the level yielding a
100% detection rate.
During the confirmation phase, 40 replicates of the preliminary LoD were tested using two (2)
lots of reaction mix. The Analytical Sensitivity/ LoD is defined as the concentration of the virus
that resulted in at least 95% detection during confirmation testing for each of the virus strains.
Preliminary LoD Results - Influenza A/HongKong/8/68
The results of the preliminary LoD determination for Influenza A/Hong Kong/8/68 are shown in
Table 18.10.1. The preliminary LoD result was 500 copies/mL; at this concentration, there was
100% detection (4/4) of Flu A.
Page 194 of 250
Table 18.10.1. Preliminary LoD Results for Influenza A/Hong Kong/8/68
% % % %
Copies/
Detection Mean Detection Mean Ct Detection Mean Detection Mean
mL
Ct ± SD ± SD Ct ± SD Ct ± SD
(#Detected (%CV) (#Detected (%CV) (#Detected (%CV) (#Detected (%CV)
30.9 ± N/A ± N/A ± 29.1 ±

1500 100% (4/4) 0.57 0% (0/4) N/A 0% (0/4) N/A 100% (4/4) 0.45
(1.8%) (N/A%) (N/A%) (1.5%)
32.0 ± N/A ± N/A ± 29.0 ±

1200 100% (4/4) 0.91 0% (0/4) N/A 0% (0/4) N/A 100% (4/4) 0.46
(2.8%) (N/A%) (N/A%) (1.6%)
31.8 ± N/A ± N/A ± 29.0 ±

1000 100% (4/4) 0.75 0% (0/4) N/A 0% (0/4) N/A 100% (4/4) 0.62
(2.4%) (N/A%) (N/A%) (2.1%)
33.3 ± N/A ± N/A ± 29.1 ±

500 100% (4/4) 0.51 0% (0/4) N/A 0% (0/4) N/A 100% (4/4) 0.67
(1.5%) (N/A%) (N/A%) (2.3%)
33.6 ± N/A ± N/A ± 29.1 ±

250 75% (3/4) 1.57 0% (0/4) N/A 0% (0/4) N/A 100% (4/4) 0.51
(4.7%) (N/A%) (N/A%) (1.8%)
Ct = Cycle threshold, SD= Standard Deviation, %CV = Percent Coefficient of Variation, N/A = Not applicable
Confirmation LoD Results - Influenza A/Hong Kong/8/68 at 500 copies/mL
The results of the confirmation LoD determination are shown in Table 18.10.2 and summarized
in Table 18.10.3. At a concentration of 500 copies/mL, there was 100% detection (40/40) of Flu
A.
Table 18.10.2. Confirmation LoD Results for Influenza A/Hong

Kong/8/68 at 500 copies/mL
Confirmatory COVID-19 Ct
Flu A Ct Value Flu B Ct Value IC Ct Value
Replicate Value
1 32.9 0 0 30.4
2 33.4 0 0 30.2
3 32.4 0 0 30.3
4 34.5 0 0 30.4
5 33.4 0 0 30.1
Page 195 of 250
Replicate Value
6 33.1 0 0 30.3
7 32.5 0 0 30.4
8 34.9 0 0 30.8
9 33.8 0 0 30.8
10 34.9 0 0 30.4
11 33.7 0 0 30.1
12 32.2 0 0 29.6
13 32.7 0 0 30
14 32.8 0 0 30
15 33.6 0 0 30.3
16 36.8 0 0 30.6
17 33.4 0 0 30.2
18 32.9 0 0 30.7
19 32.1 0 0 30.7
20 33.3 0 0 30.1
21 34.8 0 0 30.2
22 34.6 0 0 30
23 34.8 0 0 30.6
24 35.1 0 0 30
25 33.4 0 0 29.9
26 34.3 0 0 30.2
27 34 0 0 30.3
28 36.3 0 0 30.4
29 34.6 0 0 30
30 34 0 0 29.7
31 35 0 0 30.1
32 34.1 0 0 30
33 33.3 0 0 30.3
34 33.5 0 0 29.3
35 34.5 0 0 29.9
36 36.3 0 0 30.2
37 33.9 0 0 30.3
38 34.3 0 0 30.3
39 34.1 0 0 30.3
40 34.7 0 0 30.4
Ct = Cycle threshold, IC = Internal Control
Page 196 of 250
Table 18.10.3. Summary of Confirmation for Influenza A/Hong Kong/8/68 at 500 copies/mL
% % % %
Copies/ Mean Mean Ct Mean Mean Ct
Detection Detection Detection Detection
mL Ct ± SD ± SD Ct ± SD ± SD
34.0 ± N/A ± N/A ± 30.2 ±

100% 100%
500 1.09 0% (0/40) N/A 0% (0/40) N/A 0.31
(40/40) (40/40)
(3.2%) (N/A%) (N/A%) (1.0%)
Preliminary LoD Results - Influenza A/Michigan/45/2015
The results of the preliminary LoD determination for Influenza A/Michigan/45/2015 are shown in
Table 18.10.4. The preliminary LoD result was 500 copies/mL; at this concentration, there was
100% detection (4/4) of Flu A.
Page 197 of 250
Table 18.10.4. Preliminary LoD Results for Influenza A/Michigan/45/2015
% % % %
Copies/ Mean Mean Ct Mean
Detection Detection Detection Detection Mean Ct ±
mL Ct ± SD ± SD Ct ± SD
(#Detected (#Detected (#Detected (#Detected SD (%CV)
(%CV) (%CV) (%CV)
30.7 ± N/A ± N/A ± 29.1 ±

1200 100% (4/4) 1.09 0% (0/4) N/A 0% (0/4) N/A 100% (4/4) 0.72
(3.6%) (N/A%) (N/A%) (2.5%)
30.5 ± N/A ± N/A ± 29.0 ±

1000 100% (4/4) 0.84 0% (0/4) N/A 0% (0/4) N/A 100% (4/4) 0.35
(2.8%) (N/A%) (N/A%) (1.2%)
31.1 ± N/A ± N/A ± 29.0 ±

750 100% (4/4) 0.66 0% (0/4) N/A 0% (0/4) N/A 100% (4/4) 0.66
(2.1%) (N/A%) (N/A%) (2.3%)
32.7 ± N/A ± N/A ± 29.1 ±

500 100% (4/4) 2.03 0% (0/4) N/A 0% (0/4) N/A 100% (4/4) 0.76
(6.2%) (N/A%) (N/A%) (2.6%)
33.7 ± N/A ± N/A ± 29.2 ±

250 75% (3/4) 1.39 0% (0/4) N/A 0% (0/4) N/A 100% (4/4) 0.59
(4.1%) (N/A%) (N/A%) (2.0%)
35.1 ± N/A ± N/A ± 29.2 ±

125 75% (3/4) 1.56 0% (0/4) N/A 0% (0/4) N/A 100% (4/4) 0.55
(4.4%) (N/A%) (N/A%) (1.9%)
Confirmation LoD Results - Influenza A/Michigan/45/2015 at 500 copies/mL
The results of the confirmation LoD for Influenza A/Michigan/45/2015 at 500 copies/mL are
shown in Table 18.10.5 and summarized in Table 18.10.6. At a concentration of 500 copies/mL,
there was 97.5% detection (39/40) of Flu A.
Page 198 of 250
Table 18.10.5. Confirmation LoD Results for Influenza

A/Michigan/45/2015 at 500 copies/mL
Replicate Value
1 34 0 0 29.8
2 32.3 0 0 29.7
3 32.9 0 0 30.1
4 0 0 0 30.1
5 36.8 0 0 30.1
6 32.1 0 0 30
7 32.3 0 0 30.5
8 32.9 0 0 30.3
9 32.1 0 0 30.3
10 33.6 0 0 30.4
11 31.8 0 0 30.3
12 32.1 0 0 29.9
13 32.3 0 0 30
14 31.7 0 0 30.3
15 32.2 0 0 30.3
16 32.3 0 0 30.2
17 33.5 0 0 30.3
18 33.1 0 0 29.9
19 33.4 0 0 30.4
20 33.5 0 0 30.6
21 33.2 0 0 29.3
22 32.5 0 0 29.5
23 33.9 0 0 29.1
24 34 0 0 29.7
25 33.1 0 0 30
26 34.6 0 0 30.3
27 32.8 0 0 30.2
28 33.1 0 0 30.2
29 33.7 0 0 30.1
30 32.7 0 0 30.2
31 31.4 0 0 29.7
32 33.3 0 0 29.9
33 32.1 0 0 29.7
34 32 0 0 29.7
35 33.4 0 0 30.4
36 34.1 0 0 30.2
37 34.6 0 0 29.9
38 33.4 0 0 30
39 33.5 0 0 29.8
40 32.9 0 0 30.3
Page 199 of 250
Table 18.10.6. Summary of Confirmation for Influenza A/Michigan/45/2015 at 500 copies/mL
% % % %
Copies/ Mean Ct Mean Ct Mean Mean Ct
mL ± SD ± SD Ct ± SD ± SD
33.1 ± N/A ± N/A ± 30.0 ±

97.5% 100%
500 1.01 0% (0/40) N/A 0% (0/40) N/A 0.32
(39/40) (40/40)
(3.1%) (N/A%) (N/A%) (1.1%)
Preliminary LoD Results - Influenza B/Malaysia/2506/04
For the preliminary LoD determination for Influenza B/Malaysia/2506/04, two (2) rounds of
screening were required. The initial round of screening resulted in all levels detected 100%. The
results are shown in Table 18.10.7, this was due to the initial round having all replicates at all
levels detected 100% for Flu B.
Page 200 of 250
Table 18.10.7. First Round Preliminary LoD Results for Influenza B/Malaysia/2506/04
% % % %
Copies/ Mean Ct Mean Ct Mean Ct Mean Ct
mL ± SD ± SD ± SD ± SD
N/A ± 29.0 ± N/A ± 28.9 ±

1200 0% (0/4) N/A 100% (4/4) 0.39 0% (0/4) N/A 100% (4/4) 0.64
(N/A%) (1.3%) (N/A%) (2.2%)
N/A ± 29.6 ± N/A ± 29.0 ±

1000 0% (0/4) N/A 100% (4/4) 0.55 0% (0/4) N/A 100% (4/4) 0.24
(N/A%) (1.9%) (N/A%) (0.8%)
N/A ± 29.3 ± N/A ± 29.2 ±

750 0% (0/4) N/A 100% (4/4) 0.74 0% (0/4) N/A 100% (4/4) 0.40
(N/A%) (2.5%) (N/A%) (1.4%)
N/A ± 30.4 ± N/A ± 29.1 ±

500 0% (0/4) N/A 100% (4/4) 1.32 0% (0/4) N/A 100% (4/4) 0.54
(N/A%) (4.3%) (N/A%) (1.9%)
N/A ± 31.3 ± N/A ± 29.1 ±

250 0% (0/4) N/A 100% (4/4) 0.74 0% (0/4) N/A 100% (4/4) 0.75
(N/A%) (2.4%) (N/A%) (2.6%)
N/A ± 32.4 ± N/A ± 29.0 ±

125 0% (0/4) N/A 100% (4/4) 0.59 0% (0/4) N/A 100% (4/4) 0.39
(N/A%) (1.8%) (N/A%) (1.3%)
The results from the second round of preliminary LoD screening for Influenza B/Malaysia/2506/04
are shown in Table 10.10.7. The preliminary LoD was determined to be 250 copies/mL. This was
the lowest level to have 100% detection of Flu B.
Page 201 of 250
Table 18.10.7. Second Round Preliminary LoD Results for Influenza B/Malaysia/2506/04
Flu Flu B COVID-19 IC
% % % %
Copies/ Mean Ct Mean Ct Mean Ct Mean Ct
(#Detected (%CV) (#Detected/ (%CV) (#Detected (%CV) (#Detected (%CV)
/#Tested) #Tested) /#Tested) /#Tested)
N/A ± 31.0 ± N/A ± 30.0 ±

500 0% (0/4) N/A 100% (4/4) 0.36 0% (0/4) N/A 100% (4/4) 0.18
(N/A%) (1.2%) (N/A%) (0.6%)
N/A ± 32.1 ± N/A ± 29.9 ±

250 0% (0/4) N/A 100% (4/4) 0.69 0% (0/4) N/A 100% (4/4) 0.06
(N/A%) (2.1%) (N/A%) (0.2%)
N/A ± 33.6 ± N/A ± 30.0 ±

125 0% (0/4) N/A 75% (3/4) 0.42 0% (0/4) N/A 100% (4/4) 0.13
(N/A%) (1.3%) (N/A%) (0.4%)
N/A ± 33.6 ± N/A ± 29.9 ±

75 0% (0/4) N/A 50% (2/4) 0.64 0% (0/4) N/A 100% (4/4) 0.58
(N/A%) (1.9%) (N/A%) (1.9%)
N/A ± 33.3 ± N/A ± 30.0 ±

50 0% (0/4) N/A 50% (2/4) 0.57 0% (0/4) N/A 100% (4/4) 0.18
(N/A%) (1.7%) (N/A%) (0.6%)
Confirmation LoD Results - Influenza B/Malaysia/2506/04 at 250 copies/mL
The results of the confirmation LoD for Influenza B/Malaysia/2506/04 at 250 copies/mL are
shown in Table 18.10.8 and summarized in Table 18.10.9. At a concentration of 250 copies/mL,
there was 97.5% detection (39/40) of Flu B.

B/Malaysia/2506/04 at 250 copies/mL
Replicate Value
1 0 31.6 0 30.5
2 0 32.4 0 30.3
3 0 31.6 0 29.9
4 0 31.6 0 30.2
5 0 31.9 0 30
6 0 31.5 0 30.6
Page 202 of 250
Replicate Value
7 0 32.8 0 30.5
8 0 0 0 29.5
9 0 31.6 0 30.4
10 0 32.1 0 30
11 0 31.7 0 29.5
12 0 31.8 0 30.7
13 0 31.8 0 30.1
14 0 31.6 0 29.8
15 0 32.3 0 29.7
16 0 33 0 30.4
17 0 32.1 0 30.5
18 0 31.4 0 30.2
19 0 33.7 0 30.2
20 0 32.2 0 30.4
21 0 31.2 0 30
22 0 32.4 0 29.8
23 0 31.9 0 29.4
24 0 31.6 0 30
25 0 31.7 0 30
26 0 32.2 0 29.9
27 0 32.8 0 30.3
28 0 30.8 0 29.9
29 0 31.3 0 29.8
30 0 33.8 0 30.4
31 0 31.2 0 29.9
32 0 30.2 0 29.4
33 0 31.7 0 29.6
34 0 31.8 0 29.3
35 0 31.6 0 29.6
36 0 32.3 0 30.6
37 0 33.9 0 30.3
38 0 31.9 0 30.3
39 0 31.5 0 30.5
40 0 32.8 0 30.3
Page 203 of 250
Table 18.10.9. Summary of Confirmation for Influenza B/Malaysia/2506/04 at 250 copies/mL
% % %
Copie Mean % Detection Mean Ct Mean Ct Mean Ct
s/ mL Ct ± SD ± SD ± SD ± SD
(#Detected/
(#Detected (%CV) (%CV) (#Detected (%CV) (#Detected (%CV)
#Tested)
N/A ± 32.0 ± N/A ± 30.1 ±

97.5% 100%
250 0% (0/40) N/A 0.76 0% (0/40) N/A 0.38
(39/40) (40/40)
(N/A%) (2.4%) (N/A%) (1.3%)
Preliminary LoD determination Results - Influenza B/Phuket/3073/2013
The preliminary LoD determination results for Influenza B/Phuket/3073/2013 are shown in Table
63. The preliminary LoD result was 750 copies/mL; at this concentration, there was 100%
detection (4/4) of Flu B.
Page 204 of 250
Table 18.10.10. Preliminary LoD Results for Influenza B/Phuket/3073/2013
% % % %
Copies Mean Ct Mean Ct Mean Ct
Detection Detection Detection Detection Mean Ct ±
/ mL ± SD ± SD ± SD
(#Detected (#Detected (#Detected (#Detected SD (%CV)
(%CV) (%CV) (%CV)
N/A ± 30.1 ± N/A ± 29.1 ±

1500 0% (0/4) N/A 100% (4/4) 0.79 0% (0/4) N/A 100% (4/4) 0.69
(N/A%) (2.6%) (N/A%) (2.4%)
N/A ± 30.7 ± N/A ± 29.1 ±

1200 0% (0/4) N/A 100% (4/4) 0.34 0% (0/4) N/A 100% (4/4) 0.59
(N/A%) (1.1%) (N/A%) (2.0%)
N/A ± 30.7 ± N/A ± 29.1 ±

1000 0% (0/4) N/A 100% (4/4) 0.53 0% (0/4) N/A 100% (4/4) 0.31
(N/A%) (1.7%) (N/A%) (1.1%)
N/A ± 31.6 ± N/A ± 29.2 ±

750 0% (0/4) N/A 100% (4/4) 1.04 0% (0/4) N/A 100% (4/4) 0.35
(N/A%) (3.3%) (N/A%) (1.2%)
N/A ± 31.9 ± N/A ± 29.2 ±

500 0% (0/4) N/A 75% (3/4) 0.29 0% (0/4) N/A 100% (4/4) 0.49
(N/A%) (0.9%) (N/A%) (1.7%)
N/A ± 32.0 ± N/A ± 28.9 ±

250 0% (0/4) N/A 100% (4/4) 0.48 0% (0/4) N/A 100% (4/4) 0.38
(N/A%) (1.5%) (N/A%) (1.3%)
Confirmation LoD Results - Influenza B/Phuket/3073/2013 at 750 copies/mL
The results of the confirmation LoD for Influenza B/Phuket/3073/2013 at 750 copies/mL are
shown in Table 18.10.11 and summarized in Table 18.10.12 . At a concentration of Influenza
B/Phuket/3073/2013 at 750 copies/mL, there was 97.5% detection (39/40) of Flu B. There was
one (1) replicate with Flu A detected, with a Ct value of 36.5 (2.5% detection). An additional thirty
(30) replicates that consisted of pooled negative matrix were tested and no false-positive issues
were observed. Therefore, the Simplexa COVID-19 & Flu A/B Direct Reaction Mix performed as
expected.
Post-LoD testing, the lab areas used for Simplexa COVID-19 & Flu A/B Direct testing were tested
for contamination and certain areas resulted in detection of Flu A, Flu B and/or COVID-19.
Therefore, environmental contamination may have caused the late Flu A Ct detection observed
Page 205 of 250
for confirmatory replicate number 26 (Table 64). (Note: Average Ct value for Flu A detection at
LoD is ~ 33 to 34. This observed Flu A Ct value was 36.5, below LoD).

B/Phuket/3073/2013 at 750 copies/mL
Replicate Value
1 0 31.9 0 30.2
2 0 33.2 0 29.7
3 0 31.6 0 29.9
4 0 33.1 0 30.3
5 0 32 0 30.1
6 0 31.2 0 30.5
7 0 31.6 0 30.3
8 0 31.3 0 30.1
9 0 32.3 0 30.8
10 0 31.4 0 29.9
11 0 31.5 0 29.8
12 0 32.1 0 29.8
13 0 31.7 0 29.8
14 0 31.5 0 29.7
15 0 31.7 0 30.2
16 0 32.1 0 30.3
17 0 32.1 0 30.1
18 0 32.5 0 30.4
19 0 31.6 0 29.9
20 0 31.6 0 30.6
21 0 32 0 30
22 0 31.8 0 30
23 0 32.2 0 30.1
24 0 33.7 0 29.7
25 0 31.2 0 30.3
26 36.5* 31.5 0 30.6
27 0 31.4 0 30.2
28 0 30.7 0 30.5
29 0 31.8 0 30.3
30 0 31.4 0 30.2
31 0 32.4 0 29.8
32 0 31 0 30.3
33 0 31.2 0 29.7
34 0 32.1 0 29.9
35 0 0 0 29.8
36 0 32.2 0 30.2
Page 206 of 250
Replicate Value
37 0 31.7 0 30
38 0 31.8 0 30.3
39 0 32.4 0 31.2
40 0 32.5 0 30.4
* An additional thirty (30) replicates that consisted of pooled negative matrix (NPS to be later used for making
COVID-19 & Flu A/B panels) were tested and no false-positive issues were observed.
Table 18.10.12. Summary of Confirmation for Influenza B/Phuket/3073/2013 at 750 copies/mL

% % %
Copies/ Mean Ct % Detection Mean Ct Mean Ct Mean Ct
(#Detected/
(#Detected (%CV) (%CV) (#Detected (%CV) (#Detected (%CV)
#Tested)
36.5 ± 31.9 ± N/A ± 30.1 ±

97.5% 100%
750 2.5% (1/40) N/A 0.60 0% (0/40) N/A 0.33
(39/40) (40/40)
(N/A%) (1.9%) (N/A%) (1.1%)
Preliminary LoD Results - COVID-19, USA-WA1/2020
For the preliminary LoD determination for COVID-19, USA-WA1/2020, two (2) rounds of
screening was required. The initial round of screening resulted in all levels detected 100%. The
results are shown in Table 18.10.13 this was due to the initial round having all replicates at all
levels detected 100% for COVID-19.
Page 207 of 250
Table 18.10.13. First Round Preliminary LoD Results for COVID-19 USA-WA1/2020
% % %
Copies % Detection Mean Ct Mean Ct Mean Ct Mean Ct
/ mL ± SD ± SD ± SD ± SD
(#Detected/
(%CV) (#Detected (%CV) (#Detected (%CV) (#Detected (%CV)
#Tested)
N/A ± N/A ± 28.8 ± 29.6 ±

1000 0% (0/4) N/A 0% (0/4) N/A 100% (4/4) 0.54 100% (4/4) 0.56
(N/A%) (N/A%) (1.9%) (1.9%)
N/A ± N/A ± 29.9 ± 29.4 ±

750 0% (0/4) N/A 0% (0/4) N/A 100% (4/4) 0.79 100% (4/4) 0.58
(N/A%) (N/A%) (2.6%) (2.0%)
N/A ± N/A ± 29.9 ± 29.2 ±

500 0% (0/4) N/A 0% (0/4) N/A 100% (4/4) 0.70 100% (4/4) 0.15
(N/A%) (N/A%) (2.3%) (0.5%)
N/A ± N/A ± 31.0 ± 29.4 ±

250 0% (0/4) N/A 0% (0/4) N/A 100% (4/4) 1.45 100% (4/4) 0.39
(N/A%) (N/A%) (4.7%) (1.3%)
N/A ± N/A ± 31.7 ± 29.2 ±

125 0% (0/4) N/A 0% (0/4) N/A 100% (4/4) 0.56 100% (4/4) 0.35
(N/A%) (N/A%) (1.8%) (1.2%)
The results from the second round of screening are shown in Table 18.10.14 . The preliminary
LoD was determined to be 250 copies/mL. This was the lowest level to have 100% detection of
COVID-19.
Page 208 of 250
Table 18.10.14. Second Round Preliminary LoD Results for COVID-19 USA-WA1/2020
% % %
Copies/ % Detection Mean Ct Mean Ct Mean Ct Mean Ct
(#Detected/
(%CV) (#Detected (%CV) (#Detected (%CV) (#Detected (%CV)
#Tested)
N/A ± N/A ± 30.9 ± 29.3 ±

500 0% (0/4) N/A 0% (0/4) N/A 100% (4/4) 1.24 100% (4/4) 0.33
(N/A%) (N/A%) (4.0%) (1.1%)
N/A ± N/A ± 31.4 ± 29.3 ±

250 0% (0/4) N/A 0% (0/4) N/A 100% (4/4) 1.16 100% (4/4) 0.10
(N/A%) (N/A%) (3.7%) (0.3%)
N/A ± N/A ± 31.9 ± 29.3 ±

125 0% (0/4) N/A 0% (0/4) N/A 50% (2/4) 0.85 100% (4/4) 0.34
(N/A%) (N/A%) (2.7%) (1.2%)
N/A ± N/A ± 31.6 ± 29.2 ±

75 0% (0/4) N/A 0% (0/4) N/A 50% (2/4) 0.49 100% (4/4) 0.30
(N/A%) (N/A%) (1.6%) (1.0%)
N/A ± N/A ± 32.2 ± 29.3 ±

50 0% (0/4) N/A 0% (0/4) N/A 25% (1/4) N/A 100% (4/4) 0.33
(N/A%) (N/A%) (N/A%) (1.1%)
Confirmation LoD Results - COVID-19 USA-WA1/2020 at 250 copies/mL and 500 copies/mL
COVID-19 USA-WA1/2020 Confirmation at 250 copies/mL
The initial results of the confirmation LoD determination of COVID-19 USA-WA1/2020 for 250
copies/mL are shown in Table 18.10.15 and summarized in Table 18.10.16. At a concentration
of 250 copies/mL, there was 90% detection (36/40) of COVID-19. There was one (1) replicate
with Flu B detected, with a Ct value of 35.2 (2.5% detection). As this was an unexpected result,
an additional thirty (30) replicates that consisted of pooled negative matrix that was tested and
no false-positive results were observed. Therefore, the Simplexa™ COVID-19 & Flu A/B Direct
Reaction Mix performed as expected.
Page 209 of 250
Table 18.10.15. Confirmation LoD Results for COVID-19 at 250 copies/mL

Confirmatory
Flu A Ct Value Flu B Ct Value COVID-19 Ct Value IC Ct Value
Replicate
1 0 0 32.1 30.2
2 0 0 30.9 29.9
3 0 0 32.2 30.1
4 0 0 32.3 30.1
5 0 0 33 30.3
6 0 0 32.5 30.2
7 0 0 31.1 30.2
8 0 0 32.2 30.4
9 0 0 32.9 30.3
10 0 0 32 30.2
11 0 0 32.2 30.4
12 0 0 31.9 30.1
13 0 0 32.3 30.3
14 0 0 32.2 30.2
15 0 0 31.7 30
16 0 0 31 29.8
17 0 0 32.2 30
18 0 0 32.2 29.7
19 0 0 31.3 30.1
20 0 0 0 30.2
21 0 35.2* 31.4 29.9
22 0 0 31.3 30.1
23 0 0 34.2 30.5
24 0 0 32 30.2
25 0 0 31.8 29.8
26 0 0 32.8 30.2
27 0 0 32.1 30
28 0 0 30.7 30
29 0 0 30.3 29.9
30 0 0 0 30.2
31 0 0 31.4 30.3
32 0 0 29.3 30.7
33 0 0 30.9 30
34 0 0 31.4 30.1
35 0 0 33.1 30
36 0 0 31.3 29.6
37 0 0 32 30.2
38 0 0 0 29.6
39 0 0 30.9 29.8
40 0 0 0 29.5
Ct = Cycle threshold, IC = Internal Control *An additional thirty (30) replicates that consisted of pooled negative matrix
(NPS to be later used for making COVID-19 & Flu A/B panels) were tested and no false-positive issues were observed.
Page 210 of 250
Table 18.10.16. Summary of Confirmation for COVID-19 at 250 copies/mL

COVID-19
Copies / Mean Ct
mL Flu A Flu B COVID-19 IC Flu A Flu B COVID-19 ± SD
(%CV)
35.2 ± 31.8 ± 30.1 ±

N/A ± N/A 100%
250 0% (0/40) 2.5% (1/40) N/A 90% (36/40) 0.89 0.25
(N/A%) (40/40)
(N/A%) (2.8%) (0.8%)
COVID-19 USA-WA1/2020 Confirmation at 500 copies/mL
Due to the fact that there was less than 95% detection of COVID-19 at 250 copies/mL, a second
round of confirmation was performed. The results of the confirmation LoD determination for 500
copies/mL are shown in Table 18.10.17 and summarized in Table 18.10.18. At a concentration
of 500 copies/mL, there was 100% detection (40/40) of COVID-19.
Table 18.10.17 Confirmation LoD Results for COVID-19 at 500 copies/mL

Confirmatory
Replicate
1 0 0 30.3 29.8
2 0 0 31.6 30
3 0 0 30.3 30.1
4 0 0 30.4 29.5
5 0 0 30.3 30.1
6 0 0 30.3 30.1
7 0 0 31.1 30.4
8 0 0 30.3 30.3
9 0 0 30.9 29.8
10 0 0 32.3 30.1
11 0 0 30.1 30.1
12 0 0 31 29.7
13 0 0 30.6 29.9
14 0 0 30.4 29.8
15 0 0 30.6 29.5
16 0 0 30.6 30.8
17 0 0 30.5 30.5
18 0 0 32 30.6
19 0 0 30.9 30.2
20 0 0 31 30.4
21 0 0 30.1 29.6
22 0 0 30.7 30
Page 211 of 250
Confirmatory
Replicate
23 0 0 28.1 29.6
24 0 0 30.1 29.8
25 0 0 30.1 30
26 0 0 30.7 30.2
27 0 0 30.7 29.5
28 0 0 31.1 30.4
29 0 0 31.4 30.1
30 0 0 30.7 30.2
31 0 0 31.1 29.8
32 0 0 30.8 29.7
33 0 0 31.2 29.5
34 0 0 31 29.7
35 0 0 30.2 30.1
36 0 0 31.5 30.2
37 0 0 30.8 30.3
38 0 0 32 30.3
39 0 0 31.2 30.4
40 0 0 31.6 30.5
Table 18.10.18. Summary of Confirmation for COVID-19 at 500 copies/mL

COVID-19
% Detection Mean Ct % Detection Mean % Detection Mean % Detection Mean Ct
Copies/
mL (# Detected / ± SD (# Detected Ct ± SD (# Detected Ct ± SD (# Detected ± SD
# Tested) (%CV) / # Tested) (%CV) / # Tested) (%CV) / # Tested) (%CV)
N/A ± N/A ± 30.8 ± 30.0 ±

100% 100%
500 0% (0/40) N/A 0% (0/40) N/A 0.70 0.33
(40/40) (40/40)
(N/A%) (N/A%) (2.3%) (1.1%)
Page 212 of 250
Summary of Control Results – Limit of Detection
Table 18.10.19 shows the results of the Positive Control (PC) and No Template Control (NTC)
for each assay run. All controls performed as expected.

Control Detection Ct ± Detection Ct ± Detection Ct ± Detection Ct ±
SN SD SD SD SD
N/A ± N/A ± N/A ± 30.5 ±

100090 0% (0/2) N/A 0% (0/2) N/A 0% (0/2) N/A 100% (2/2) 1.20
(N/A%) (N/A%) (N/A%) (3.9%)
N/A ± N/A ± N/A ± 30.4 ±
100122 0% (0/4) N/A 0% (0/4) N/A 0% (0/4) N/A 100% (4/4) 0.88
(N/A%) (N/A%) (N/A%) (2.9%)
N/A ± N/A ± N/A ± 30.4 ±

100215 0% (0/4) N/A 0% (0/4) N/A 0% (0/4) N/A 100% (4/4) 0.82
(N/A%) (N/A%) (N/A%) (2.7%)
NTC N/A ± N/A ± N/A ± 30.1 ±
100305 0% (0/2) N/A 0% (0/2) N/A 0% (0/2) N/A 100% (2/2) 0.07
(N/A%) (N/A%) (N/A%) (0.2%)
N/A ± N/A ± N/A ± 30.4 ±

100445 0% (0/4) N/A 0% (0/4) N/A 0% (0/4) N/A 100% (4/4) 1.09
(N/A%) (N/A%) (N/A%) (3.6%)
N/A ± N/A ± N/A ± 30.4 ±

100%
All 0% (0/16) N/A 0% (0/16) N/A 0% (0/16) N/A 0.80
(16/16)
(N/A%) (N/A%) (N/A%) (2.6%)
26.7 ± 26.6 ± 26.5 ± 30.3 ±

100090 100% (2/2) 0.78 100% (2/2) 0.14 100% (2/2) 0.28 100% (2/2) 0.71
(2.9%) (0.5%) (1.1%) (2.3%)
26.1 ± 26.7 ± 26.8 ± 30.9 ±

PC 100122 100% (4/4) 0.71 100% (4/4) 0.44 100% (4/4) 0.47 100% (4/4) 1.12
(2.7%) (1.6%) (1.8%) (3.6%)
26.6 ± 26.9 ± 26.9 ± 30.5 ±

100215 100% (4/4) 0.42 100% (4/4) 0.17 100% (4/4) 0.10 100% (4/4) 0.34
(1.6%) (0.6%) (0.4%) (1.1%)
Page 213 of 250
25.2 ± 26.1 ± 26.2 ± 29.6 ±

100305 100% (2/2) 0.07 100% (2/2) 0.78 100% (2/2) 0.21 100% (2/2) 0.00
(0.3%) (3.0%) (0.8%) (0.0%)
25.8 ± 26.2 ± 27.0 ± 30.5 ±

100445 100% (4/4) 0.88 100% (4/4) 0.40 100% (4/4) 0.17 100% (4/4) 0.22
(3.4%) (1.5%) (0.6%) (0.7%)
26.1 ± 26.5 ± 26.7 ± 30.5 ±

100% 100% 100% 100%
All 0.75 0.47 0.36 0.69
(16/16) (16/16) (16/16) (16/16)
(1.8%) (1.8%) (1.3%) (2.3%)
Ct = Cycle threshold, SD= Standard Deviation, %CV = Percent Coefficient of Variation, N/A = Not applicable, SN=Serial number, NTC = No
Template Control, PC= Positive Control
Conclusions – Limit of Detection
The Limit of Detection was established for the Simplexa™ COVID-19 & Flu A/B Direct. Table
18.10.20 shows the LoDs were determined to be the following;
Table 18.10.20. Simplexa™ COVID-19 & Flu A/B Direct Limit of Detection
Simplexa™ COVID-19 & Strain Limit of Detection
Flu A/B Direct Target
Flu A Influenza A/Hong Kong/8/68 500 copies/mL
Influenza A/Michigan/45/2015 500 copies/mL
Flu B Influenza B/Malaysia/2506/04 250 copies/mL
Influenza B/Phuket/3073/2013 750 copies/mL
COVID-19 COVID-19 (USA-WA1/2020) 500 copies/mL
All controls performed as expected.
Please see attachment list at the conclusion of section 18.10 for a listing of study protocol and
the associated line data.
b) Limit of Detection using WHO International Standard (COVID-19)
The Candidate Device was evaluated for limit of detection (LoD) in nasopharyngeal swab (NPS)
matrix using COVID-19 viral particles (WHO International Standard) from the England/02/2020
isolate. The study was performed at DiaSorin Molecular, Gerenzano, IT using four (4) LIAISON
MDX Instruments, two (2) lots of Candidate Device Reaction Mix, one (1) lot of Candidate Device
Page 214 of 250
Positive Control and one (1) lot of Direct Amplification Discs. A total of 12 experimental runs
were performed by one (1) operator on one (1) day (24 August 2021). A total of seven (7)
experimental samples were prepared by one (1) operator on one (1) day (24 August 2021).
Sample preparation and testing was performed by different operators of the internal site.
Acid-heat inactivated COVID-19 viral particles (WHO International Standard) from

England/02/2020 isolate were diluted to various concentrations and spiked into pooled negative
nasopharyngeal swab (NPS) matrix in UTM.
The limit of detection was determined using a two-step approach. During the screening phase
(LoD determination), a preliminary LoD was established by testing four (4) replicates of each
concentration of inactivated virus, using a single lot of reaction mix. The preliminary LoD is the
lowest tested concentration with a 100% detection rate.
During the confirmation phase, a total of 40 replicates of the preliminary LoD were tested using
two (2) lots of reaction mix. The Limit of Detection is defined as the lowest concentration of virus
with a detection rate of at least 95%.
Preliminary LoD Determination Results – COVID-19 WHO International Standard -

England/02/2020 Isolate
For the preliminary LoD determination for COVID-19 WHO International Standard -
England/02/2020 Isolate, six (6) concentrations were evaluated.
The results of screening are shown in Table 18.10.21. The preliminary LoD was 651 IU/mL.
This was the lowest level to have 100% detection of COVID-19.
Page 215 of 250
Table 18.10.21. Limit of Detection Results for COVID-19 WHO International

Standard - England/02/2020 Isolate

COVID-
% Detection Mean % Detection Mean % Detection Mean % Detection Mean
19
Ct ± Ct ± Ct ± Ct ±
IU/ mL (# Detected (# Detected (# Detected (# Detected
SD SD SD SD
30.6± 29.7±
N/A ± N/A ±
1302 0% (0/4) N/A 0% (0/4) N/A 100% (4/4) 100% (4/4)
0.93 0.97
(N/A) (N/A)
(3.0%) (3.3%)
30.9± 29.8±
N/A ± N/A ±
976 0% (0/4) N/A 0% (0/4) N/A 100% (4/4) 100% (4/4)
0.92 0.75
(N/A) (N/A)
(3.0%) (2.5%)
32.5± 29.9±
N/A ± N/A ±
651 0% (0/4) N/A 0% (0/4) N/A 100% (4/4) 100% (4/4)
1.73 0.88
(N/A) (N/A)
(5.3%) (3.0%)
31.9± 29.8±
N/A ± N/A ±
488 0% (0/4) N/A 0% (0/4) N/A 75% (3/4) 100% (4/4)
1.17 0.75
(N/A) (N/A)
(3.7%) (2.5%)
32.4± 29.3±
N/A ± N/A ±
325 0% (0/4) N/A 0% (0/4) N/A 75% (3/4) 100% (4/4)
0.78 1.21
(N/A) (N/A)
(2.4%) (4.1%)
33.5± 29.5±
N/A ± N/A ±
163 0% (0/4) N/A 0% (0/4) N/A 50% (2/4) 100% (4/4)
0.71 1.05
(N/A) (N/A)
(2.1%) (3.6%)
IU = International Units, Ct = Cycle threshold, SD= Standard Deviation, %CV = Percent Coefficient of Variation, N/A = Not
applicable, IC = Internal Control
Page 216 of 250
Confirmation LoD Results - COVID-19 WHO International Standard - England/02/2020

Isolate at 651 IU/mL
The results of the LoD confirmation for 651 IU/mL are shown in Table 18.10.22 and summarized
in Table 18.10.23. At a concentration of 651 IU/mL, there was 97.5% detection (39/40) of COVID-
19 obtained with two (2) lots of reaction mix (20 replicates for each lot).
Table 18.10.22. LoD confirmation results for COVID-19 WHO

International Standard - England/02/2020 Isolate at 651 IU/mL
Confirmatory Flu A Ct Flu B Ct COVID-19 Ct IC Ct

Replicate Value Value Value Value
1 0 0 31.9 31.1
2 0 0 31.7 31.5
3 0 0 30.0 30.6
4 0 0 33.1 30.6
5 0 0 33.1 31.1
6 0 0 31.9 29.2
7 0 0 30.1 28.9
8 0 0 31.4 29.0
9 0 0 30.5 29.1
10 0 0 30.9 28.8
11 0 0 31.0 29.4
12 0 0 31.1 29.2
13 0 0 31.3 29.2
14 0 0 30.4 28.8
15 0 0 30.8 28.7
16 0 0 36.3 29.4
17 0 0 31.9 29.4
18 0 0 32.1 28.6
19 0 0 32.7 29.8
20 0 0 33.4 28.6
21 0 0 32.2 30.6
22 0 0 32.7 30.9
23 0 0 30.0 31.1
24 0 0 31.2 30.7
25 0 0 0 30.3
26 0 0 31.1 28.6
27 0 0 32.9 28.7
28 0 0 30.9 29.2
29 0 0 30.4 28.5
30 0 0 30.2 29.1
31 0 0 31.4 28.6
32 0 0 31.7 29.1
33 0 0 32.2 28.8
Page 217 of 250
Confirmatory Flu A Ct Flu B Ct COVID-19 Ct IC Ct

Replicate Value Value Value Value
34 0 0 36.6 29.1
35 0 0 32.3 28.4
36 0 0 31.5 28.6
37 0 0 31.8 28.9
38 0 0 31.1 28.6
39 0 0 31.2 28.3
40 0 0 30.7 28.8
Table 18.10.23. Summary of LoD Confirmation for COVID-19 WHO International

Standard - England/02/2020 Isolate at 651 IU/mL
%
COVID-19 % Detection % Detection % Detection
Mean Mean Mean Detection Mean
IU / mL
(# Detected (# Detected (# Detected
(%CV) (%CV) (%CV) (# Detected (%CV)
/ # Tested) / # Tested) / # Tested)
/ # Tested)
31.7± 29.4±
N/A ± N/A ±
97.5% 100%
651 0% (0/40) N/A 0% (0/40) N/A
(39/40) 1.4 (40/40) 0.9
(N/A) (N/A)
(4.5%) (3.1%)
Ct = Cycle threshold, SD= Standard Deviation, %CV = Percent Coefficient of Variation, N/A = Not applicable, IC = Internal Control
Summary of Results for Controls - LoD for COVID-19 WHO International Standard -
Table 18.10.24 shows the results of the Positive Control (PC) and No Template Control (NTC)
for each assay run. All controls performed as expected.
Page 218 of 250

Instrument % Detection Mean % Detection Mean % Detection Mean % Mean
Control Ct ± Ct ± Ct ± Detection Ct ±
SN (# Detected / SD (# Detected / SD (# Detected SD SD
# Tested) (%CV) # Tested) (%CV) / # Tested) (%CV) (# Detected (%CV)
/ # Tested)
N/A ± N/A ± N/A ± 30.8 ±
2D0055 0% (0/1) 0% (0/1) 0% (0/1) 100% (1/1)
N/A N/A N/A N/A
(N/A) (N/A) (N/A) (N/A)
N/A ± N/A ± N/A ± 100% (1/1) 28.6 ±
200151 0% (0/1) 0% (0/1) 0% (0/1)
N/A N/A N/A N/A
(N/A) (N/A) (N/A) (N/A)
NTC N/A ± N/A ± N/A ± 100% (1/1) 28.3 ±
222063 0% (0/1) 0% (0/1) 0% (0/1)
N/A N/A N/A N/A
(N/A) (N/A) (N/A) (N/A)
2D0049 0% (0/1) N/A ± 0% (0/1) N/A ± 0% (0/1) N/A ± 100% (1/1) 28.6 ±
N/A N/A N/A N/A
(N/A) (N/A) (N/A) (N/A)
ALL 0% (0/4) N/A ± 0% (0/4) N/A ± 0% (0/4) N/A ± 100% (4/4) 29.1 ±
N/A N/A N/A 1.16
(N/A) (N/A) (N/A) (4 0%)
2D0055 100% (1/1) 26.4 ± 100% (1/1) 28.0 ± 100% (1/1) 28.4 ± 100% (1/1) 30.4 ±
N/A N/A N/A N/A
(N/A) (N/A) (N/A) (N/A)
24.8 ± 28.4 ± 27.8 ± 28.7 ±
200151 100% (1/1) 100% (1/1) 100% (1/1) 100% (1/1)
N/A N/A N/A N/A
(N/A) (N/A) (N/A) (N/A)
222063 100% (1/1) 25.8 ± 100% (1/1) 28.6 ± 100% (1/1) 28.6 ± 100% (1/1) 28.1 ±
PC
N/A N/A N/A N/A
(N/A) (N/A) (N/A) (N/A)
2D0049 100% (1/1) 25.9 ± 100% (1/1) 28.2 ± 100% (1/1) 28.1 ± 100% (1/1) 28.5 ±
N/A N/A N/A N/A
(N/A) (N/A) (N/A) (N/A)
25.7±0 28.3± 28.2± 28.9±1
ALL 100% (4/4) .67 100% (4/4) 0.26 100% (4/4) 0.35 100% (4/4) .01
(2.6%) (0.9%) (1.2%) (3.5%)
Ct = Cycle threshold, SD= Standard Deviation, %CV = Percent Coefficient of Variation, N/A = Not applicable, SN=Serial number, NTC =
No Template Control, PC= Positive Control
Conclusions – Limit of Detection COVID-19 WHO International Standard -

The Limit of Detection (LoD) for the COVID-19 WHO International Standard - England/02/2020
Isolate (NIBSC code: 20/146) was established with the Simplexa™ COVID-19 & Flu A/B Direct
and was determined to be 651 IU/mL.
Page 219 of 250
Limit of Detection Study –Protocol 18.10-1
Limit of Detection Line Data – Saliva Swab (pdf) 18.10-2
Limit of Detection Line Data – Saliva Swab (xls) 18.10-3
Limit of Detection (WHO Standard) Study - Protocol 18.10-4
Limit of Detection (WHO Standard) Line Data (pdf) 18.10-5
Limit of Detection (WHO Standard) Line Data (xls) 18.10-6
WHO Documentation 18.10-7
18.11 Interference
The Candidate Device was initially evaluated for the effects of 12 potentially interfering
endogenous and exogenous substances that may be present in nasopharyngeal swabs.
The study was performed at DiaSorin Molecular, Cypress, CA using two (2) LIAISON MDX
Instruments (100222 and 100336) one (1) lot of Candidate Device Kit (Lot V11908N), one (1) lot
of Candidate Device Positive Control Pack (Lot V12441N) and two (2) lots of Direct Amplification
Discs (Lot 11747N and 11855N). A total of 11 experimental runs were performed by one (1)
operator over the course of three (3) days (01-02 and 12 July 2021).
All samples were prepared by spiking each potentially interfering substance into a baseline
sample consisting of pooled negative nasopharyngeal swabs (NPS) in UTM and COVID-19
(2019-nCoV/USA-WA1/2020 strain), Flu A (Hong Kong/8/68) and Flu B (Malaysia/2506/04)
inactivated viral particles. The test samples contained inactivated COVID-19, Flu A and Flu B at
concentrations of 1500, 1500, and 750 copies/mL, respectively (3x limit of detection); Each
interferent was spiked into the baseline sample.
A second study was conducted to test mucin at a higher concentration and the FluMist nasal
vaccine. Unfortunately, DiaSorin Molecular was unable to source FluMist, so the potential
interferent was not tested.
The second study was performed at DiaSorin Molecular, Gerenzano (VA) Italy, using two (2)
LIAISON® MDX Instruments (222063 and 222050) one (1) lot of Simplexa™ COVID-19 & Flu
A/B Direct (MOL4251, Lot 14649N), one (1) lot of Simplexa™ COVID-19 & Flu A/B Positive
Control (MOL4261, Lot 12284N) and one (1) lot of Direct Amplification Disc MOL1455 (Lot
P15176N). A total of seven (7) experimental runs were performed by one (1) operator over the
course of three (3) days (01-02 and 03 March 2022). The samples were prepared in the same
manner as the first study in pooled negative NPS matrix spiked with COVID-19 (inactivated,
Page 220 of 250
2019-nCoV/USA-WA1/2020 strain), Flu A (active, Michigan/45/2015) and Flu B (active,

Phuket/3073/2013) viral particles. The test samples contained inactivated COVID-19, Flu A and
Flu B at concentrations of 1500, 1500, and 2250 copies/mL, respectively (3x limit of detection).
Results - Interference
The results show that none of the substances tested interfere with the detection of COVID-19,
influenza A or influenza B, with the exception of Cold Eeze throat lozenges at a concentration of
2.5% (w/v). This concentration resulted in an EC500 error on the COVID-19 target in one of three
(1/3) replicates. The EC500 error in the COVID-19 target was observed on the repeated sample.
An EC500 error may be an indication of weak or late amplification. Therefore, inhibition was
observed.
At a concentration of 1.25% (w/v), Cold Eeze throat lozenges were not found to cause
interference. Table 18.11.1 presents a summary of the results of both studies.
Table 18.11.1. Summary of results for potentially interfering substances tested

Qualitative Results: % Detection
Potentially Tested
Active
Interfering (# Detected /#Tested)
Ingredient
Substance Concentration
Baseline 1 100% 100% 100% 100%

N/A N/A
(100122) Study 1 (5/5) (5/5) (5/5) (5/5)
Baseline 1 100% 100% 100% 100%

N/A N/A
(100336) Study 1 (5/5) (5/5) (5/5) (5/5)
Baseline 2 100% 100% 100% 100%

N/A N/A
(100336) Study 1 (5/5) (5/5) (5/5) (5/5)
Baseline 1_day 1 80% 100% 100% 100%

N/A N/A
(222063) Study 2 (4/5) (5/5)* (5/5) (5/5)
Baseline 2_day 1 100% 100% 100% 100%

N/A N/A
(222063) Study 2 (5/5) (5/5)* (5/5) (5/5)
Baseline 1_day 2 100% 100% 100% 100%

N/A N/A
(222063) Study 2 (4/4) (4/4)** (4/4) (4/4)
Baseline 1_day 3 100% 100% 100% 100%

N/A N/A
(222050) Study 2 (5/5) (5/5) (5/5) (5/5)
100% 100% 100% 100.0%

Afrin Nasal Spray Oxymetazoline 15% (v/v)
(3/3) (3/3) (3/3) (3/3)
Page 221 of 250

Potentially Tested
Active
Ingredient
Antibacterial, 100% 100% 100% 100.0%

Tobramycin 4 μg/mL
systemic (3/3) (3/3) (3/3) (3/3)
Antibiotic nasal 100% 100% 100% 100.0%

Mupirocin 6.6 mg/mL
ointment (3/3) (3/3) (3/3) (3/3)
100% 100% 100% 100.0%

Blood NA 2% (v/v)
(3/3) (3/3) (3/3) (3/3)
100% 100% 100% 100.0%

60 µg/mL
Bovine (3/3) (3/3) (3/3) (3/3)
Purified Mucin
submaxillary
Protein
gland, type I-S 100% 100% 100% 100%
5 mg/mL****
(3/3) (3/3) (3/3) (3/3)
100% 100% 100% 100.0%

Cold Eeze 2.5% (w/v)
(4/4) (4/4) (2/2) *** (4/4)
(Throat lozenges,
NA
Oral anesthetic
100% 100% 100% 100.0%
and analgesic) 1.25% (w/v)
(3/3) (3/3) (3/3) (3/3)
Nasal
100% 100% 100% 100.0%
corticosteroid Beclomethasone 5% (w/v)
(3/3) (3/3) (3/3) (3/3)
(Beconase AQ)
Nasal
100% 100% 100% 100.0%
corticosteroids Fluticasone 5% (v/v)
(3/3) (3/3) (3/3) (3/3)
(Fluticasone)
Relenza Antiviral 100% 100% 100% 100.0%

Zanamivir 3.3 mg/mL
Drug (3/3) (3/3) (3/3) (3/3)
Tamiflu Antiviral 100% 100% 100% 100.0%

Oseltamivir 1 μM
drug (3/3) (3/3) (3/3) (3/3)
Luffa
opperculata,
Galphimia 100% 100% 100% 100.0%
Zicam Nasal Gel 5% (w/v)
glauca, (3/3) (3/3) (3/3) (3/3)
histaminum
hydrochloricum
Page 222 of 250

Potentially Tested
Active
Ingredient
Zicam Nasal
Spray
100% 100% 100% 100.0%
(Homeopathic NA 10% (v/v)
(3/3) (3/3) (3/3) (3/3)
allergy relief
medicine)
*EC500 error on FluB target in one of five (1/5) replicates was repeated giving valid result.
** EC500 error on FluB target in one of five (1/5) replicates was repeated giving EC500 error again.
***2.5% Cold Eeze Throat lozenges resulted in one out of three replicates with an EC500, invalid, for COVID-19. An
EC500 result was obtained on the repeat. Therefore the concentration was lowered to 1.25% and retested.
**** Concentration tested in study 2.
μg/mL = micro gram per milliliter, mg/mL = milligram per milliliter, μM = micromolar, w/v = weight per volume, v/v = volume
per volume, IC = Internal Control
Table 18.11.2 shows the results from Table 18.11.1 showing the Mean Ct ± Standard Deviation
(SD) and the percent coefficient of variation for the microbial inhibition panel tested replicates for
each assay target.
Table 18.11.2. Mean Ct ± Standard Deviation (SD) and the percent coefficient of
variation (%CV) for the Interference Panel Tested Replicates for Each Assay Target
Potentially Tested % % % %
Interfering Mean Mean Mean Mean
Substance Conc. Ct ± SD Ct ± SD Ct ± SD Ct ± SD
(#Detected/ (#Detected (#Detected (#Detected
(%CV) (%CV) (%CV) (%CV)
#Tested) /#Tested) /#Tested) /#Tested)
33.78 ± 30.92 ± 30.14 ± 30.68 ±

Baseline 1
N/A 100% (5/5) 0.68 100% (5/5) 1.17 100% (5/5) 0.67 100% (5/5) 1.02
(Study 1)
(2.01%) (3.78%) (2.22%) (3.32%)
33.3 ± 30.72 ± 28.84 ± 29.12 ±

Baseline 1
N/A 100% (5/5) 0.81 100% (5/5) 0.32 100% (5/5) 0.59 100% (5/5) 0.08
(Study 1)
(2.43%) (1.04%) (2.05%) (0.27%)
32.78 ± 30.26 ± 28.78 ± 29.4 ±

Baseline 2
N/A 100% (5/5) 0.61 100% (5/5) 0.53 100% (5/5) 0.66 100% (5/5) 0.4
(Study 1)
(1.86%) (1.75%) (2.29%) (1.36%)
33.0 ± 31.2 ± 29.2 ± 26.9 ±

Baseline 1_day 80% (4/5) 100% (5/5) 100% (5/5)
N/A 1.8 0.6 0.5 100% (5/5) 0.3
1 Study 2 Positive Positive Positive
(5.4%) (1.8%) (1.9%) (1.0%)
Page 223 of 250

(%CV) (%CV) (%CV) (%CV)
31.1 ± 30.9 ± 29.0 ± 27.1 ±

Baseline 2_day 100% (5/5) 100% (5/5)* 100% (5/5)
N/A 0.6 0.6 0.9 100% (5/5) 0.2
(2.0%) (2.1%) (3.1%) (0.6%)
30.9 ± 100% 32.0 ± 29.3 ± 27.1 ±

Baseline 1_day 100% (4/4) 100% (4/4)
N/A 0.2 (4/4)** 0.4 0.1 100% (4/4) 0.3
2 Study 2 Positive Positive
(0.8%) Positive (1.3%) (0.4%) (1.2%)
32.1 ± 31.4 ± 30.4 ± 28.5 ±

Baseline 1_day 100% (5/5) 100% (5/5) 100% (5/5)
N/A 0.6 0.6 0.5 100% (5/5) 0.2
(2.0%) (2.1%) (1.7%) (0.8%)
32.53 ± 30.57 ± 28.87 ± 29.27 ±

Afrin Nasal 15% 100.0%
100% (3/3) 0.51 100% (3/3) 0.71 100% (3/3) 0.38 0.25
Spray (v/v) (3/3)
(1.57%) (2.32%) (1.32%) (0.85%)
32.43 ± 30.47 ± 28.77 ± 29.3 ±

Antibacterial, 4 100.0%
100% (3/3) 0.15 100% (3/3) 0.31 100% (3/3) 0.47 0.2
systemic μg/mL (3/3)
(0.46%) (1.02%) (1.63%) (0.68%)
32.63 ± 30.47 ± 29.87 ± 30.47 ±

Antibiotic nasal 6.6 100.0%
100% (3/3) 0.59 100% (3/3) 0.31 100% (3/3) 0.4 0.15
ointment mg/mL (3/3)
(1.81%) (1.02%) (1.34%) (0.49%)
34.5 ± 32.27 ± 29.93 ± 31.07 ±

2% 100.0%
Blood 100% (3/3) 0.56 100% (3/3) 1.26 100% (3/3) 0.47 0.15
(v/v) (3/3)
(1.62%) (3.9%) (1.57%) (0.48%)
32.93 ± 30.37 ± 29.33 ± 29.17 ±

60 100.0%
100% (3/3) 0.4 100% (3/3) 0.06 100% (3/3) 0.61 0.15
µg/mL (3/3)
Bovine (1.21%) (0.2%) (2.08%) (0.51%)
submaxillary
gland, type I-S 5 31.5 ± 30.1 ± 29.6 ± 28.5 ±
100% (3/3) 100% (3/3) 100% (3/3)
mg/mL 0.9 0.2 0.3 100% (3/3) 0.2
Positive Positive Positive
**** (2.9%) (0.7%) (1.2%) (0.6%)
34.33 ± 32.45 ± 31.75 ± 30.6 ±

2.5% 100% (2/2) 100.0%
100% (4/4) 0.53 100% (4/4) 0.54 0.21 0.32
(w/v) *** (4/4)
(1.54%) (1.66%) (0.66%) (1.05%)
Page 224 of 250

(%CV) (%CV) (%CV) (%CV)
Cold Eeze
(Throat
lozenges, Oral 34.03 ± 31.3 ± 30.4 ± 29.67 ±
1.25% 100.0%
anesthetic and 100% (3/3) 0.76 100% (3/3) 0.62 100% (3/3) 0.36 0.91
(w/v) (3/3)
analgesic) (2.23%) (1.98%) (1.18%) (3.07%)
Nasal 32.67 ± 30.93 ± 28.87 ± 29.5 ±

5% 100.0%
corticosteroid 100% (3/3) 0.57 100% (3/3) 0.32 100% (3/3) 0.47 0.2
(w/v) (3/3)
(Beconase AQ) (1.74%) (1.03%) (1.63%) (0.68%)
Nasal 33.47 ± 31.03 ± 29.07 ± 30.33 ±

5% 100.0%
corticosteroids 100% (3/3) 0.6 100% (3/3) 0.06 100% (3/3) 0.31 0.21
(v/v) (3/3)
(Fluticasone) (1.79%) (0.19%) (1.07%) (0.69%)
33.67 ± 31.13 ± 29.8 ± 30.5 ±

Relenza 3.3 100.0%
100% (3/3) 0.12 100% (3/3) 0.47 100% (3/3) 0.79 0.26
Antiviral Drug mg/mL (3/3)
(0.36%) (1.51%) (2.65%) (0.85%)
33.2 ± 30.47 ± 29.37 ± 29.3 ±

Tamiflu Antiviral 100.0%
1 μM 100% (3/3) 0.46 100% (3/3) 0.86 100% (3/3) 0.31 0.26
drug (3/3)
(1.39%) (2.82%) (1.06%) (0.89%)
33.77 ± 30.33 ± 29.47 ± 29.6 ±

Zicam Nasal 5% 100.0%
100% (3/3) 0.83 100% (3/3) 0.32 100% (3/3) 0.9 0.44
Gel (w/v) (3/3)
(2.46%) (1.06%) (3.05%) (1.49%)
Zicam Nasal
Spray 33.93 ± 31.3 ± 29.37 ± 30.03 ±
10% 100.0%
(Homeopathic 100% (3/3) 0.85 100% (3/3) 0.1 100% (3/3) 0.4 0.12
(v/v) (3/3)
allergy relief (2.51%) (0.32%) (1.36%) (0.4%)
medicine)
*EC500 error on FluB target in one of five (1/5) replicates was repeated giving valid result.
** EC500 error on FluB target in one of five (1/5) replicates was repeated giving EC500 error again.
***2.5% Cold Eeze Throat lozenges resulted in one out of three replicates with an EC500, invalid, for COVID-19. An EC500 result was
obtained on the repeat. Therefore the concentration was lowered to 1.25% and retested.
**** Concentration tested in study 2
μg/mL = micro gram per milliliter, mg/mL = milligram per milliliter, μM = micromolar, w/v = weight per volume, v/v = volume per volume, IC =
Internal Control
Page 225 of 250
Summary of Control Results - Interference
Table 18.11.3 and 18.11.4 shows the results of the Positive Control (PC) and No Template
Control (NTC) for each assay run for the two (2) Interference studies. All controls performed as
expected.
Table 18.11.3. Results for Controls – Interference Study 1


Instrument
SN
(#Detecte SD (#Detected/ SD (#Detected/ SD (#Detected/ SD
d/#Tested) (%CV) #Tested) (%CV) #Tested) (%CV) #Tested) (%CV)
N/A N/A N/A 30.0

100122 0% (0/1) 0% (0/1) 0% (0/1) 100% (1/1)
± N/A ± N/A ± N/A ± N/A
(N/A%) (N/A%) (N/A%) (N/A%)
N/A N/A N/A 30.0

100122 0% (0/1) 0% (0/1) 0% (0/1) 100% (1/1)
± N/A ± N/A ± N/A ± N/A
(N/A%) (N/A%) (N/A%) (N/A%)
N/A N/A N/A 29.6

100336 0% (0/1) 0% (0/1) 0% (0/1) 100% (1/1)
± N/A ± N/A ± N/A ± N/A
(N/A%) (N/A%) (N/A%) (N/A%)
NTC
N/A N/A N/A 28.9
100336 0% (0/1) 0% (0/1) 0% (0/1) 100% (1/1)
± N/A ± N/A ± N/A ± N/A
(N/A%) (N/A%) (N/A%) (N/A%)
N/A N/A N/A 29.5

100336 0% (0/1) 0% (0/1) 0% (0/1) 100% (1/1)
± N/A ± N/A ± N/A ± N/A
(N/A%) (N/A%) (N/A%) (N/A%)
N/A N/A N/A 29.6

All 0% (0/5) 0% (0/5) 0% (0/5) 100% (5/5)
± N/A ± N/A ± N/A ± 0.45
(N/A%) (N/A%) (N/A%) (1.5%)
26.5 26.6 26.6 30.8

PC 100122 100% (1/1) 100% (1/1) 100% (1/1) 100% (1/1)
± N/A ± N/A ± N/A ± N/A
(N/A%) (N/A%) (N/A%) (N/A%)
Page 226 of 250

Instrument
SN
(#Detecte SD (#Detected/ SD (#Detected/ SD (#Detected/ SD
d/#Tested) (%CV) #Tested) (%CV) #Tested) (%CV) #Tested) (%CV)
26.1 26.0 26.0 30.1

100122 100% (1/1) 100% (1/1) 100% (1/1) 100% (1/1)
± N/A ± N/A ± N/A ± N/A
(N/A%) (N/A%) (N/A%) (N/A%)
25.9 26.1 25.7 29.1

100336 100% (1/1) 100% (1/1) 100% (1/1) 100% (1/1)
± N/A ± N/A ± N/A ± N/A
(N/A%) (N/A%) (N/A%) (N/A%)
26.0 26.0 25.7 28.9

100336 100% (1/1) 100% (1/1) 100% (1/1) 100% (1/1)
± N/A ± N/A ± N/A ± N/A
(N/A%) (N/A%) (N/A%) (N/A%)
26.0 26.0 25.8 29.2

100336 100% (1/1) 100% (1/1) 100% (1/1) 100% (1/1)
± N/A ± N/A ± N/A ± N/A
(N/A%) (N/A%) (N/A%) (N/A%)
26.0
26.1 26.1 29.6
All 100% (5/5) 100% (5/5) 100% (5/5) ± 0.38 100% (5/5)
± 0.23 ± 0.26 ± 0.80
(0.9%) (1.0%) (2.7%)
(1.5%)
Ct = Cycle threshold, SD= Standard Deviation, %CV = Percent Coefficient of Variation, NTC = No Template Control, PC = Positive Control
Page 227 of 250
Table 18.11.4. Results for Controls – Interference Study 2

Flu A (FAM) Flu B (JOE) COVID-19 (CFR610) IC (Q670)
Control % % Ct ± % Ct ± Ct ±
SN Ct ± SD % Detection
Detection Detection SD Detection SD SD
(%CV)
(%CV) (%CV) (%CV)
N/A N/A N/A 26.0
222063 0% (0/2) 0% (0/2) 0% (0/2) 100% (2/2)
± N/A ± N/A ± N/A ± 0.7
(N/A%) (N/A%) (N/A%) (2.7%)
NTC
N/A N/A N/A 28.2
222050 0% (0/1) 0% (0/1) 0% (0/1) 100% (1/1)
± N/A ± N/A ± N/A ± N/A
(N/A%) (N/A%) (N/A%) (N/A%)
N/A N/A N/A 26.7

ALL 0% (0/3) 0% (0/3) 0% (0/3) 100% (3/3)
± N/A ± N/A ± N/A ± 1.4
(N/A%) (N/A%) (N/A%) (5.1%)
26.1 28.2 27.3 26.4

100%
222063 100% (2/2) 100% (2/2) 100% (2/2)
(2/2) ± 0.0 ± 0.1 ± 0.1 ± 0.4
(0%) (0.5%) (0.3%) (1.6%)
26.3 28.2 28.2 27.6

100%
PC 222050 100% (1/1) 100% (1/1) 100% (1/1)
(1/1) ± N/A ± N/A ± N/A ± N/A
(N/A%) (N/A%) (N/A%) (N/A%)
26.2 28.2 27.6 26.8

100%
ALL 100% (3/3) 100% (3/3) 100% (3/3)
(3/3) ± 0.1 ± 0.1 ± 0.6 ± 0.8
(0.4%) (0.4%) (2.0%) (2.8%)
Ct = Cycle threshold, SD= Standard Deviation, %CV = Percent Coefficient of Variation, NTC = No Template Control, PC = Positive Control
Conclusions - Interference
No interference was observed with any of the 13 potentially interfering endogenous and
exogenous substances that may be present in nasopharyngeal swabs (NPS) samples. Initial
testing of Cold Eeze Throat lozenges at 2.5% resulted in one out of three (1/3) replicates with an
EC500, invalid, for COVID-19. An EC500 result was obtained on the repeat. Therefore, the
concentration was lowered to 1.25% and retested. No interference was observed at 1.25%. All
Controls performed as expected.
Page 228 of 250
Attachment
Number
Interference Protocol 18.11-1
Interference Line Data (pdf) 18.11-2
Interference Line Data (xls) 18.11-3
Interference Study 2 Line Data (pdf) 18.11-4
Interference Study 2 Line Data (xls) 18.11-5
18.12 No Template Control Specificity
The Candidate Device was evaluated for analytical specificity by testing a total of ninety-six (96)
replicates of no template control (NTC) (Universal Transport Medium [UTM]). The study was
performed at DiaSorin Molecular, Cypress, CA using four (4) LIAISON MDX Instruments, two (2)
lots of Candidate Device Reaction Mix (Lots V11908N and V11909N), one (1) lot of Candidate
Device Positive Control (PC), one (1) lot of UTM and one (1) lot of Direct Amplification Discs. A
total of 20 experimental runs were performed by two (2) operators over the course of two (2)
consecutive days (19 and 20 May, 2021).
For this study, UTM was tested directly from the manufacturer’s tube as an unknown sample.
There was no additional sample preparation or manipulation performed.
Results - Analytical Specificity
The results of this study showed that the analytical specificity was 100%.
Table 18.13.1 shows the results using Reaction Mix Lot V11908N. No false positive results
were observed. All UTM replicates had valid internal control results.
Table 18.13.1. Results for Reaction Mix Lot V11908N

%
Mean Mean Mean
% Detection % Detection % Detection Detection Mean Ct
Ct ± Ct ± Ct ±
(# Detected /# (# Detected (# Detected (# ± SD
SD SD SD
Tested) /# Tested) /# Tested) Detected /# (%CV)
(%CV) (%CV) (%CV)
Tested)
N/A ± N/A ± N/A ± 30.1 ±
100%
0% (0/48) N/A 0% (0/48) N/A 0% (0/48) N/A 0.27
(48/48)
(N/A%) (N/A%) (N/A%) (0.91%)
Ct = Cycle threshold, SD= Standard Deviation, %CV = Percent Coefficient of Variation, N/A = Not
applicable
Page 229 of 250
Table 18.13.2 shows the results using Reaction Mix Lot V11909N. No false positive results were
observed. All UTM replicates had valid internal control results.
Table 18.13.2. Results for Reaction Mix Lot V11909N

Mean Mean
% Detection % Detection % Detection Mean % Detection Mean
Ct ± Ct ±
(# Detected (# Detected (# Detected Ct ± SD (# Detected Ct ± SD
SD SD
/# Tested) /# Tested) /# Tested) (%CV) /# Tested) (%CV)
(%CV) (%CV)
N/A ± N/A ± N/A ± 30.5 ±
100%
0% (0/48) N/A 0% (0/48) N/A 0% (0/48) N/A 0.26
(48/48)
(N/A%) (N/A%) (N/A%) (0.87%)
Table 18.13.3 provides a summary of the results with both lots of Reaction Mix. Overall there
was 0% detection for Flu A, Flu B, and COVID-19 targets.
Table 18.13.3. Summary of NTC results with both lots of Reaction Mix
Mean Mean Mean Mean
Ct ± Ct ± Ct ± Ct ±
(# Detected (# Detected (# Detected (# Detected
SD SD SD SD
/# Tested) /# Tested) /# Tested) /# Tested)
(%CV) (%CV) (%CV) (%CV)
N/A ± N/A ± N/A ± 30.3 ±
100%
0% (0/96) N/A 0% (0/96) N/A 0% (0/96) N/A 0.33
(96/96)
(N/A%) (N/A%) (N/A%) (1.1%)
Ct = Cycle threshold, SD= Standard Deviation, %CV = Percent Coefficient of Variation, N/A = Not
applicable
Summary of Control Results – No Template Control Specificity
Table 18.13.4 shows the results of the PC and NTC replicates for each assay run. All controls
Page 230 of 250
Table 18.13.4. Summary of Results for Controls – No Template Control Specificity

% % % %
Control Detection Detection Detection Detection
(# (# (# (#
SD SD SD SD
Detected Detected Detected Detected
(%CV) (%CV) (%CV) (%CV)
/# Tested) /# Tested) /# Tested) /# Tested)
N/A ± N/A ± N/A ± 30.1 ±

100%
100122 0% (0/2) N/A 0% (0/2) N/A 0% (0/2) N/A 0.1
(2/2)
(N/A%) (N/A%) (N/A%) (0.3%)
N/A ± N/A ± N/A ± 30.1 ±

100%
100215 0% (0/2) N/A 0% (0/2) N/A 0% (0/2) N/A 0.3
(2/2)
(N/A%) (N/A%) (N/A%) (1.0%)
N/A ± N/A ± N/A ± 29.8 ±

100%
NTC 100305 0% (0/2) N/A 0% (0/2) N/A 0% (0/2) N/A 0.0
(2/2)
(N/A%) (N/A%) (N/A%) (0.0%)
N/A ± N/A ± N/A ± 29.9 ±

100%
100445 0% (0/2) N/A 0% (0/2) N/A 0% (0/2) N/A 0.3
(2/2)
(N/A%) (N/A%) (N/A%) (1.0%)
N/A ± N/A ± N/A ± 30.0 ±

100%
All 0% (0/8) N/A 0% (0/8) N/A 0% (0/8) N/A 0.20
(8/8)
(N/A%) (N/A%) (N/A%) (0.7%)
26.9 ± 26.7 ± 26.6 ± 30.2 ±

100.0% 100.0% 100% 100.0%
100122 0.00 0.00 0.00 0.21
(2/2) (2/2) (2/2) (2/2)
(0.0%) (0.0%) (0.0%) (0.7%)
27.0 ± 26.9 ± 27.1 ± 30.1 ±

100.0% 100.0% 100% 100.0%
100215 0.07 0.07 0.00 0.35
(2/2) (2/2) (2/2) (2/2)
(0.3%) (0.3%) (0.0%) (1.2%)
26.8 ± 27.0 ± 27.0 ± 30.0 ±

100.0% 100.0% 100% 100.0%
PC 100305 0.14 0.07 0.07 0.28
(2/2) (2/2) (2/2) (2/2)
(0.5%) (0.3%) (0.3%) (0.9%)
27.1 ± 27.3 ± 26.8 ± 30.4 ±

100.0% 100.0% 100% 100.0%
100445 0.21 0.49 0.28 0.57
(2/2) (2/2) (2/2) (2/2)
(0.8%) (1.8%) (1.0%) (1.9%)
26.9 ± 26.9 ± 26.9 ± 30.2 ±

100.0% 100.0% 100% 100.0%
All 0.14 0.29 0.23 0.33
(8/8) (8/8) (8/8) (8/8)
(0.5%) (1.1%) (0.9%) (1.1%)
Ct = Cycle threshold, SD= Standard Deviation, %CV = Percent Coefficient of Variation, N/A = Not applicable, SN=Serial number,
NTC = No Template Control, PC= Positive Control
Page 231 of 250
Conclusions – No Template Control Specificity
The analytical specificity using no template control (NTC) was established for the Candidate
Device and was determined to be 100%. All 96 replicates resulted as not detected for all targets
and all controls performed as expected.
Attachment
Number
No Template Control Protocol 18.12-1
No Template Control Line Data (pdf) 18.12-2
No Template Control Line Data (xls) 18.12-3
18.13 Testing with a Moderate Complexity User
Moderate Complexity testing was conducted and assessed in K120413. The same study is
applied to the Candidate Device as the study is not analyte specific and the workflow is
equivalent.
18.14 Direct Amplification Disc (DAD) Consumable – Disc Reusability

Direct Amplification Disc (DAD) Reusability with swabs was previously performed as part of
K120413 Simplexa™ Flu A/B & RSV Direct. As the Direct Amplification Disc (DAD) Reusability
testing is not analyte specific, the study can be applied to the Candidate Device.
Page 232 of 250
19. Performance Testing – Animal

This section is not applicable to the Candidate Devices as no animal testing was required nor performed.
Page 233 of 250
20. Performance Testing – Clinical

20.1 Clinical Agreement
A clinical agreement study was conducted using the Candidate Device with prospective and
retrospectively collected Nasopharyngeal Swab (NPS) specimens. For the prospective cohort,
fresh NPS specimens from patients with signs and symptoms of respiratory infection were
prospectively collected and tested either as fresh specimens or as frozen specimens. Namely
fresh specimens were stored refrigerated at 2-8°C, if tested within 72 hours of collection, or frozen
(≤ -70°C), if not tested within 72 hours of collection. The prospective NPS specimens were
collected from four (4) geographically diverse collection sites within the United States.
Retrospective NPS specimens were preselected for targets that exhibited low prevalence rates
in the prospective study cohort. The pre-selected clinical specimens were identified as positive
or negative by the comparator reference method. Pre-selected positive and negative specimens
were tested in a randomized, blinded manner. Retrospective NPS specimens were sourced from
sample vendors.
Only one (1) specimen was obtained per patient. Patient age, gender and date of symptom(s)
onset (vaccination status, as available) for each subject; availability of collection date and
collection time (routine respiratory methodology/test name and routine result, as available) was
obtained for each sample for each specimen. Specimens were excluded if the sample had been
exposed to three (3) or more freeze thaw cycles, the sample was collected using calcium alginate
or organic swabs, the patient was without symptoms of respiratory infection, was asymptomatic
or the sample was obtained for pre-screening purposes for travel, work and/or pre-surgical
procedures.
A total of 743 Prospective fresh and/or prospective frozen NPS specimens were collected from
four (4) geographically diverse collection sites within the United States.
Table 20.1.1 identifies sites that performed collections for the clinical agreement study. The table
includes site name, site address, geographic location and collection date range.
Table 20.1.1. Clinical Specimen Collection Site Information
Collection
Site Name Address Collection Type Location
Dates
7129 Hayvenhurst Prospective Fresh

Southeast
Ave. September 27,
MT Group 2021 to October
Suite #317
Prospective 27, 2021
Midwest
Van Nuys, CA 91406 Frozen
Page 234 of 250
Collection
Site Name Address Collection Type Location
Dates
Prospective Fresh
3317 W. Beverly Blvd.
SeraCollection October 6, 2021
Research Suite 200 West to November
Services Prospective 10, 2021
Montebello, CA 90640
Frozen
Prospective Fresh
West August 30,
Cantor 9450 Cuyamaca St.
Southeast 2021 to
BioConnect,
November 4,
LLC Santee, CA 92071
Prospective Rocky Mountain 2021
Frozen
August 25,
450 Bedford St.
Prospective 2021 to
iSpecimen Southeast
Frozen September 15.
Lexington MA 02420
2021
Table 20.1.2 lists the prospective specimen cohorts collected at each site.
Table 2. Clinical Study Specimens Collected by Site
Site Name Collection Type Number of Specimens
Prospective Fresh 123 NPS (Fresh)

MT Group
Prospective Frozen 80 NPS (Frozen)

SeraCollection

Cantor BioConnect, LLC
iSpecimen Prospective Frozen 99 NPS (Frozen)
Influenza A and B targets exhibited low prevalence rates in the prospective study cohort, the
prospective specimen set was supplemented with 258 retrospective (pre-selected) positive and
negative remnant, de-identified specimens sourced from various vendors as described in Table
20.2.3. Pre-selected specimens were characterized via the comparator methods prior to
Page 235 of 250
enrollment in the study. Specimens were then aliquoted and stored frozen according to the
clinical study protocol prior to shipment and testing at clinical sites..
Table 20.2.3. Vendors Used to Source Retrospective Specimens
Vendor Number of Specimens
PC Medical Center (PCM) 26
Laboratory Alliance of Central New York (LAC) 31
SouthwestBio (SWB) 27
NorthShore University Laboratory (NSH) 92
Tampa General Hospital (TGH) 82
Comparator reference method testing was performed on the prospectively collected clinical
specimens enrolled in the study to assess clinical performance. Prospective specimens without
a valid comparator reference method result for a target were excluded from performance analysis
for that target. The results for the Flu A and Flu B targets were evaluated against a validated
FDA-cleared PCR assay; the ARIES® Flu A/B & RSV Assay.
For the SARS-CoV-2 target, the performance of the candidate device was evaluated against a
composite testing algorithm consisting of three (3) high-sensitivity EUA-authorized SARS-CoV-2
tests: NxTAG® CoV Extended Panel, CDC 2019-nCOV Real-Time RT-PCR Diagnostic Panel and
the Quidel Lyra® SARS-CoV-2 Assay.
Table 20.1.4 below shows the SARS-CoV-2 composite reference method algorithm and
interpretation rules for the prospective cohort. Specimens were tested with the NxTAG® CoV
Extended Panel and CDC 2019-nCOV Real-Time RT-PCR Diagnostic Panel. Quidel Lyra testing
was performed as discrepant resolution if results from the NxTAG and CDC methods did not
agree. Concordance for two (2) out of the three (3) was considered the final comparator result
for SARS-CoV-2. Any specimens with a final composite reference algorithm indeterminate
interpretation were excluded from the data analysis.
Page 236 of 250
Table 20.1.4. Prospective Composite Reference Method (CRM) Algorithm
NxTAG CDC Lyra Final CRM
Positive Positive Not Tested Positive
Positive Negative Positive Positive
Positive Negative Negative Negative
Negative Negative Not Tested Negative
Negative Positive Positive Positive
Negative Positive Negative Negative
Positive Inconclusive/Invalid Positive Positive
Negative Inconclusive/Invalid Negative Negative
Invalid Positive Positive Positive
Invalid Negative Negative Negative
Positive Inconclusive/Invalid Negative Indeterminate
Negative Inconclusive/Invalid Positive Indeterminate
Invalid Positive Negative Indeterminate
Invalid Negative Positive Indeterminate
Invalid Inconclusive/Invalid Not Tested Indeterminate
Note: Final CRM interpretation of Indeterminate were excluded from the data analysis.
Due to the discontinuation of the CDC 2019-nCOV Real-Time RT-PCR Diagnostic Panel on
January 1, 2022, pre-selected specimens were screened with the Quidel Lyra® SARS-CoV-2
Assay as a replacement. Specimens were then aliquoted and stored according to the clinical
study protocol for enrollment in the study.
Discordant resolution was performed by Biofire® Respiratory 2.1 (RP2.1) Panel and/or bi-
directional sequencing (BDS) assays when the Candidate Device results were different from
those of the comparator assays. The results from the discordant analysis are not included in the
performance tables but are included as footnotes in the performance tables.
Page 237 of 250
Specimens were tested at two (2) external clinical sites and one (1) internal site. A fourth site
(UCLA) was initially included in the study as a testing site but was later disqualified due to
amplicon contamination. Specimens from this site were removed from the data analysis. Table
20.1.5 lists the sites that performed candidate device testing. These sites represented the testing
environment where the device will ultimately be used and by individuals who will perform the test
in clinical practice.
Table 20.1.5. Candidate Device Testing Sites

Site Name Address Cohort Testing Dates Location
October 18,
801 Seventh Ave.
Cook Children’s 2021 to
Prospective Southwest
Hospital October 21,
Ft. Worth, TX 76104
2021
October 6,
12212 Technology Blvd. Prospective
2021 to
Luminex Austin Southwest
November 19,
Austin, TX 78727
Pre-Selected 2021
1001 Woodward Pl. NE February 3,

Tricore Reference 2021 to Rocky
Pre-Selected
Laboratory Albuquerque, NM February 18, Mountain
87102 2022
11633 San Vicente Blvd

UCLA (disqualified) Prospective Not Applicable West
Los Angeles, CA 90050
Table 20.1.6 lists the sites where reference method testing was performed.
Table 20.1.6. Reference Method Testing Sites
Site Name Address CRM Assay Method
ARIES® Flu A/B & RSV Assay
12212 Technology Blvd.

NxTAG® CoV Extended Panel
Luminex Austin
Austin, TX 78727
CDC 2019-nCOV Real-Time RT-PCR Diagnostic
Panel
Page 238 of 250
Site Name Address CRM Assay Method
1224 Deming Way

CDC 2019-nCOV Real-Time RT-PCR Diagnostic
Luminex Madison
Panel
Madison, WI 53717
1001 Woodward Pl. NE Quidel Lyra® SARS-CoV-2 Assay

TriCore Reference
Laboratory Albuquerque, NM CDC 2019-nCOV Real-Time RT-PCR Diagnostic
87102 Panel
Testing Sites were instructed to test one (1) Positive Control (PC) and one (1) No Template
Control at the beginning of each testing day on each instrument. Controls could be run by
themselves or with specimens at the beginning of each testing day. Sample results were not
considered valid unless controls were valid for that testing day for each instrument. All runs were
performed on LIAISON MDX software version 2.1 (Cooks and Luminex) and 2.2 (TriCore).
Estimates of positive percent agreement (PPA) and negative percent agreement (NPA) for the
combined prospective and pre-selected data set were calculated based on a two-by-two table
(reference method result vs. result from the Candidate Device) for each target. In addition,
respective two-sided 95% confidence intervals were calculated using the Wilson Score method.
The PPA was calculated as 100% x (TP / (TP +FN)). A true positive (TP) indicates that both the
Candidate Device and the reference method had a positive result for the specific target, and false
negative (FN) indicates that the Candidate Device had a negative target result while the reference
method was positive. The NPA was calculated as 100% x (TN / (TN +FP). A true negative (TN)
indicates that both the Candidate Device and the reference method had a negative result for the
specific target, and false positive (FP) indicates that the Candidate Device had a positive target
result while the reference method was negative. The two-sided 95% CI was calculated using the
Wilson method.
To ensure biases were not introduced, prospectively collected specimens were de-identified by
an honest broker and shipped to the testing sites for candidate device and comparator device
testing (e.g., Operators that performed candidate device testing did not test CRM and vice versa).
Pre-selected specimens were blinded, randomized, and tested along with unique negative
clinical specimens to ensure that the operators performing the candidate device testing remained
blinded to any specimen’s expected results. Results from pre-selected specimens were analyzed
separately from the prospective data set and in combination with the prospective data set.
Prospective Clinical Agreement Specimens
Of the 743 prospective specimens enrolled, 226 specimens tested at UCLA were removed from
the data set due to site disqualification (amplicon contamination) and 16 specimens were excluded
from accuracy determination and further analysis. Of the 16 specimens, 11 specimens were never
received for testing, four (4) specimens were excluded due to specimen leaking, and one (1)
Page 239 of 250
specimen was excluded due to leakage and lack of volume in several sample aliquots. The
remaining 501 clinical specimens were used for subsequent analysis of the prospective clinical
data set.
Table 20.1.9 provides a summary of the general demographic information of the 501 prospectively
collected specimens that were included in the prospective analysis. Demographic information is
also presented by clinical collection site.
Table 20.1.9. General Demographic Details of the Prospective Study Population (N = 501)
Cantor Bioconnect MT Group SeraCollection iSpecimen All Sites

Gender
182
Male 39 (37.5%) 88 (44.0%) 39 (22.7%) 16 (64.0%)
(36.3%)
319
Female 65 (62.5%) 112 (56.0%) 133 (77.3%) 9 (36.0%)
(63.7%)
25 501
Total 104 (100.0%) 200 (100.0%) 172 (100.0%)
(100.0%) (100.0%)
Age (Years)
0-1 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%)
>1-5 2 (1.9%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 2 (0.4%)
>5-21 15 (14.4%) 5 (2.5%) 22 (12.8%) 1 (4.0%) 43 (8.6%)
417
>21-65 79 (76.0%) 167 (83.5%) 147 (85.5%) 24 (96.0%)
(83.2%)
>65 8 (7.7%) 28 (14.0%) 3 (1.7%) 0 (0.0%) 39 (7.8%)
25 501
Total 104 (100.0%) 200 (100.0%) 172 (100.0%)
(100.0%) (100.0%)
Retrospective Preselected Clinical Agreement Specimens
A total of 82 positive influenza A specimens, 114 positive influenza B specimens, and 62 negative
specimens were blinded, randomized, and enrolled in the study. No pre-selected specimens were
excluded from the data analysis.
Table 20.1.10 provides a summary of subject demographic information (age and gender) from the
258 pre-selected clinical specimens that were included in the data analysis of the pre-selected
study.
Page 240 of 250
Table 20.1.10. General Demographic Details of the

Pre-selected Study Population (N=258)
# Specimens (%)
Gender
Male 111 (43.0%)
Female 147 (57.0%)
Total 258 (100.0%)
Age (Years)
0-1 21 (8.1%)
>1-5 27 (10.5%)
>5-21 32 (12.4%)
>21-65 70 (27.1%)
>65 16 (6.2%)
Unknown 92 (35.7%)
Total 258 (100.0%)
Results - Clinical Agreement - Prospective Specimens
Of the 501 clinical specimens included in the prospective study analysis, 495 (98.8%) generated
valid Candidate Device results on the first attempt. After a single retest, 501 specimens generated
valid Candidate Device results for a final success rate of 100%.
Table 20.1.11 shows a comparison of the Candidate Device and comparator assay results for
the prospective cohort.
Page 241 of 250
Table 20.1.11. Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA)
for the Prospective Data Set
Positive Percent Agreement Negative Percent Agreement

TP / TN /
PPA (%) 95% CI NPA (%) 95% CI
(TP+FN) (TN+FP)
0% - 99% -
0/2 0% 323/323 100%
Fresh 66% 100%
0% - 98% -
Influenza A 0/1 0% 175/175 100%
Frozen 79% 100%
0% - 99% -
0/3a 0% 498/498 100%
Overall 56% 100%
0% - 99% -
0/3 0% 322/322 100%
Fresh 56% 100%
0% - 98% -
Influenza B 0/2 0% 174/174 100%
Frozen 66% 100%
0% - 99% -
0/5b 0% 496/496 100%
Overall 43% 100%
86% - 97% -
24/24 100% 298/301 99%
Fresh 100% 100%
88% - 88% -
SARS-CoV-2 44/45 97.8% 123/131 93.9%
Frozen 100% 97%
92% - 95% -
68/69c 98.6% 421/432d 97.5%
Overall 100% 99%
a
All three Influenza A False Negatives were negative for Influenza A by BioFire RP 2.1.
b
All five Influenza B False Negatives were negative for Influenza B by Biofire RP 2.1.
c
d
A total of five false negatives were observed for the Influenza A target, seven false negatives
were observed for the Influenza B target and one false negative was observed for the SARS-
CoV-2 target. No false positives were observed for the Influenza A and Influenza B targets. A
total of 11 false positives were observed for the SARS-CoV-2 target. Tables 20.1.12 to 20.1.14
show the test results for these false negatives and false positives by target.
Page 242 of 250
Table 20.1.12 shows test results for the SARS-CoV-2 target discordant specimens.
Table 20.1.12. SARS-CoV-2 Target Discordant Specimen Test Results

Candidate Final NxTAG
Sample Biofire RP2.1 CDC Lyra
Device Comparator BDS result SARS-CoV-
ID Result Result Result
Result Result 2 Result
False Negative
Not
DS058 Not Detected Detected Detected Not Tested Detected Detected
Detected
False Positives
Not Not
13562025 Detected Not Detected Detected SARS-CoV-2 Detected
Detected Detected
None Not Not Not
CCC008 Detected Not Detected Detected
None Not Not
DS046 Detected Not Detected Detected Detected
None Not Not Not
DS049 Detected Not Detected Detected
Not Not Not
DS054 Detected Not Detected Detected SARS-CoV-2
None Not Not Not
DS060 Detected Not Detected Not Detected
Not Not Not
DS062 Detected Not Detected Not Detected SARS-CoV-2
Not Not
CCC061 Detected Not Detected Detected SARS-CoV-2 Detected
Detected Detected
MT1728- None Not Not Not
Detected Not Detected Detected
088 Detected Detected Detected Detected
MT1728- None Not Not
Detected Not Detected Detected Detected
107 Detected Detected Detected
MT1728- None Not Not Not
Detected Not Detected Detected
194 Detected Detected Detected Detected
Table 20.1.13 lists test results for the Influenza A target discordant specimens.
Table 20.1.13. Influenza A Target Discordant Specimen Test Results

Candidate Device Biofire RP2.1
Sample ID ARIES Result BDS result
Result Result
Influenza A H1-
DFC-026 Not Detected Flu A Flu A
2009
DFC-108 Not Detected Flu A Not Testeda Flu A

Page 243 of 250
Candidate Device Biofire RP2.1

Result Result
CCC083 Not Detected Flu A Not Detected None Detected
MT1728-036 Not Detected Flu A Not Detected None Detected
MT1728-161 Not Detected Flu A Not Detected None Detected
a
Not tested due to insufficient volume.
Table 20.1.14 lists test results for the Influenza B target discordant specimens.
Table 20.1.14. Influenza B Target Discordant Specimen Test Results

Candidate Biofire RP2.1
Device Result Result
DFC-012 Not Detected Flu B Influenza B Flu B
DFC-227 Not Detected Flu B Not Tested Not Testeda
CCC066 Not Detected Flu B Not Detected None Detected
CCC071 Not Detected Flu B Not Detected Not Testeda
MT1728-038 Not Detected Flu B Not Detected None Detected
MT1728-086 Not Detected Flu B Not Detected None Detected
SC213 Not Detected Flu B Not Detected Not Testeda
a Not tested due to insufficient volume.
Results - Clinical Agreement - Retrospective Specimens

Of the 258 clinical specimens included in the retrospective (pre-selected) study analysis, 257
(99.6%) generated valid Candidate Device results on the first attempt. After a single retest, 258
specimens generated valid Candidate Device assay results for a final success rate of 100%. Table
20.1.15 shows a comparison of the Candidate Device and comparator assay results for the
retrospective (pre-selected) cohort.
Page 244 of 250
Table 20.1.15. Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA)
for the Retrospective (Pre-Selected) Data Set

Candidate
Device
TP / TN /
Target PPA (%) 95% CI NPA (%) 95% CI
(TP+FN) (TN+FP)
Influenza A 80/82a 97.6% 92% - 99% 176/176 100% 98% - 100%
Influenza B 112/114b 98.2% 94% - 100% 144/144 100% 97% - 100%
SARS-CoV-2 0/0 N/A N/A 252/252 100% 98% - 100%
a
One the two Influenza A False Negatives was tested with Biofire RP2.1 and tested positive for Influenza A.
b
One of the two Influenza B False Negatives was tested with Biofire RP2.1 and tested positive for Influenza B.
Results Clinical Agreement - Combined Prospective and Retrospective (Pre-selected)
Of the 759 clinical specimens included in the prospective and retrospective (pre-selected) study
analysis, 752 (99.1%) generated valid Candidate Device results on the first attempt. After a single
retest, 759 specimens generated valid Candidate Device results for a final success rate of 100%.
Table 20.1.16 shows a comparison of the Candidate Device assay and comparator assay results
for the prospective and retrospective cohorts combined.
Page 245 of 250
Table 20.1.16. Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) of
Combined Prospective and Pre-Selected Data Set

TP / PPA TN / NPA
95% CI 95% CI
(TP+FN) (%) (TN+FP) (%)
0% - 99% -
Fresh 0/2 0% 323/323 100%
66% 100%
0% - 98% -
79% 100%
Influenza A 0% - 99% -
Combined 0/3 0% 498/498 100%
56% 100%
92% - 98% -
Pre-Selected 80/82 97.6% 176/176 100%
99% 100%
87% - 99% -
Overall 80/85a 94.1% 674/674 100%
97% 100%
0% - 99% -
Fresh 0/3 0% 322/322 100%
56% 100%
0% - 98% -
66% 100%
0% - 99% -
Combined 0/5 0% 496/496 100%
43% 100%
Influenza B
94% - 97% -
Pre-Selected 112/114 98.2% 144/144 100%
100% 100%
99% -
88% - 100%
Overall 112/119b 94.1% 640/640 100%
97%
Page 246 of 250

TP / PPA TN / NPA
95% CI 95% CI
(TP+FN) (%) (TN+FP) (%)
86% - 97% -
Fresh 24/24 100% 298/301 99%
100% 100%
88% - 88% -
Prospective Frozen 44/45 97.8% 123/131 93.9%
100% 97%
SARS-CoV-2 92% - 95% -

Combined 68/69 98.6% 421/432 97.5%
100% 99%
98% -
Pre-Selected 0/0 N/A N/A 252/252 100%
100%
92% - 97% -
Overall 68/69c 98.6% 673/684d 98.4%
100% 99%
a
Four of the five Influenza A False Negatives were tested with Biofire RP2.1 and three of these were negative for Influenza A.
b
Six of the seven Influenza B False Negatives were tested with Biofire RP2.1 and five of these were negative for Influenza B.
c
d
PPA = Positive Percent Agreement, NPA = Negative Percent Agreement, CI = Confidence Interval, TP = True Positive, FN
= False Negative, TN = True Negative
Clinical performance for the combined prospective and retrospective (pre-selected) cohorts
following discordant resolution with the highly sensitive Biofire® Respiratory 2.1 (RP2.1) Panel is
shown in Table 20.1.17.
Table 20.1.17 Discordant Resolution – Prospective and Pre-Selected Data Sets
Discordant Discordant
Resolution Resolution
(Positive (Negative
Specimens) Specimens)
TP / (TP+FN) TN / (TN+FP)
Prospective 0/0 501/501
Influenza A Pre-selected 80/82 176/176
Overall 80/82 677/677

Page 247 of 250
Discordant Discordant
Resolution Resolution
(Positive (Negative
Specimens) Specimens)
TP / (TP+FN) TN / (TN+FP)
Influenza B Pre-selected 112/114 144/144
Overall 112/114 645/645
SARS-CoV-2 Pre-selected 0/0 252/252
Overall 77/78 673/675
TP = True Positive, FN = False Negative, TN = True Negative
Conclusions – Clinical Agreement
For the SARS-CoV-2 target, the Candidate Device showed an overall upper respiratory positive
percent agreement of 98.6% (68/69) with a 95% lower bound confidence interval of 92% and a
negative percent agreement of 98.4% (673/684) with a 95% lower bound confidence interval of
97%.
For the Influenza A target, the Candidate Device showed an overall upper respiratory positive
percent agreement of 94.1% (80/85) with a 95% lower bound confidence interval of 87% and a
negative percent agreement of 100% (674/674) with a 95% lower bound confidence interval of
99%.
For the Influenza B target, the Candidate Device showed an overall upper respiratory positive
percent agreement of 94.1% (112/119) with a 95% lower bound confidence interval of 88% and
a negative percent agreement of 100% (640/640) with a 95% lower bound confidence interval of
99%.
Page 248 of 250
20.2 Expected Values
SimplexaTM COVID-19 & Flu A/B Direct assay positive results (expected values) after allowable
re-runs for each individual target are summarized for specimens included in the prospective study
analysis per age group and gender in Tables 20.2.1 and 20.2.2.
Table 20.2.1. Candidate Device Expected Values for Prospective Specimens by Age (N = 501)
> 21 – 65 years > 65 years
0-1 years > 1-5 years > 5-21 years Total
(N = 0) (N = 2) (N = 43) ( N= 501)
(N = 417) (N = 39)
#Po
Target #Pos (%) #Pos (%) #Pos (%) #Pos (%) (%) #Pos (%)
s
Influenza 0.0% 0.0% 0.0% 0.0% 0.0% 0.0%

0 0 0 0 0 0
A (0/0) (0/2) (0/43) (0/417) (0/39) (0/501)
Influenza 0.0% 0.0% 0.0% 0.0% 0.0% 0.0%

0 0 0 0 0 0
B (0/0) (0/2) (0/43) (0/417) (0/39) (0/501)
SARS- 0.0% 0.0% 23.3% 15.3% 12.8% 15.8%

0 0 10 64 5 79
CoV-2 (0/0) (0/2) (10/43) (64/417) (5/39) (79/501)
Table 20.2.2. Candidate Device Expected Values for Prospective Specimens by Gender
(N = 501)
Female Male Total
(N = 319) (N = 182) (N= 501)
Target #Pos (%) #Pos (%) #Pos (%)
0.0% 0.0% 0.0%

Influenza A 0 0 0
(0/319) (0/182) (0/501)
0.0% 0.0% 0.0%

Influenza B 0 0 0
(0/319) (0/182) (0/501)
12.5% 21.4% 15.8%

SARS-CoV-2 40 39 79
(40/319) (39/182) (79/501)
Page 249 of 250
Section 20 Attachments (Documents) Attachment Number
Clinical Study Protocol 20-1
Clinical Study Line Data (pdf) 20-2
Clinical Study Line Data (xls) 20-3

Page 250 of 250
21. Miscellaneous
21.1 CLIA Complexity Categorization
Scoring Recommendation;
All 510(k) cleared DiaSorin Molecular Simplexa™ have received a moderate complexity scoring.
The Candidate Device has the same workflow as these other 510(k) cleared moderate complexity
assays and the Predicate assay is also within a moderate complexity categorization. The sponsor
is requesting CLIA Moderate Categorization for the Candidate Device. DiaSorin Molecular
believes that the assay should be scored in the CLIA Categories as follows:
Category Score Justification

Knowledge 1 DiaSorin Molecular believes that minimal scientific knowledge is required to
perform the test and knowledge required to perform the test may be
obtained through on-the-job instruction. No specific technical knowledge is
required to perform any phase of the testing. The software clearly displays
the result interpretations. Moderate Complexity Studies were performed in
K120413.
Training and 1 Minimal training is required for pre-analytic, analytic and post-analytic
experience phases of the testing process; and Limited experience is required to perform
the test. There are no pre-analytic or post-analytic judgements required by
the technician. Moderate Complexity Studies were performed in K120413.
Reagents and 2 The all in one Reaction Mix contains all of the reagents required to run the
materials assay. The Reaction Mix is premeasured, once removed from the freezer
preparation the Reaction Mix needs to be used within thirty (30) minutes.
Characteristics 1 After selecting the assay and programming in the sample ID, technicians
of operational transfer, using a fixed volume pipette, fifty microliters (50 µL) of reagent to
steps the clearly labeled reaction well on the disc, and fifty microliters (50 µL) of
sample to the clearly labeled sample well and reseal the disc with the foil
seal. The disc is designed to automatically transfer the appropriate amount
of reagent (40 µL) and sample (10 µL) to the reaction well. Once the run is
started no technician intervention is required, all PCR steps proceed
automatically.
Calibration, 1 The assay does not require calibration. The assay contains an internal
quality control, control for PCR function. DiaSorin Molecular offers optional external QC
and proficiency materials that are intended for use with the assay. Controls are packaged in
testing single use aliquots and stored frozen, once thawed the controls are stable
materials for thirty (30) minutes at room temperature.
Test system 1 Limited troubleshooting and maintenance are required. The software
troubleshooting produces easy to understand error codes for failed tests. Instrument
and equipment conditions and any warnings are clearly displayed for the technician.
maintenance
Interpretation 1 The system software interprets the test results, no technician interpretation
and judgment is necessary
Total 8 Moderate Complexity

001 - Simplexa COVID-19 Flu AB Direct PreMarket Notification 30MAR22 Final

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001 - Simplexa COVID-19 Flu AB Direct PreMarket Notification 30MAR22 Final

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Simplexa™ COVID-19 & Flu A/B Direct REF MOL4250

Table of Contents (Sections)

11. Device Description ........................................................................................... 66

12. Substantial Equivalence Discussion ................................................................. 76

13. Proposed Labeling ........................................................................................... 80

13.2 Kit Component Labels 80

14. Sterilization and Shelf Life ............................................................................... 83

15. Biocompatibility ............................................................................................... 97

17. Electromagnetic Compatibility (EMC) and Electrical Safety .......................... 101

b) Limit of Detection using WHO International Standard (COVID-19) 213

19. Performance Testing – Animal ....................................................................... 232

21. Miscellaneous .............................................................................................. 250

Attachments (Documents) Attachment Number

Establishing the Cut-off Line data for NTC 11.5-1

Establishing the Cut-off Line data for LoD 11.5-2

Predicate IFU - BioFire Respiratory Panel 2.1 (RP2.1) (DEN200031) 12.1-1

Simplexa™ COVID-19 & Flu A/B Direct IFU (IFUK.US.MOL4250) 13-1

Simplexa™ COVID-19 & Flu A/B Positive Control Pack IFU

Candidate Device Stability Memo 14.2-1

Room Temperature Reaction Mix Stability Protocol 14.3-1

Room Temperature Reaction Mix Stability Line Data pdf 14.3-2

Room Temperature Reaction Mix Stability Line Data xls 14.3-3

Room Temperature Positive Control Stability Protocol 14.3-4

Room Temperature Positive Control Stability Line Data pdf 14.3-5

Room Temperature Positive Control Stability Line Data xls 14.3-6

LIAISON MDX SW 1.1 to 2.1 Technical Report 16-1

LIAISON MDX SW 1.1 to 2.1 Technical Report Data 16-2

Cross Reactivity Protocol 18.1-1

Cross Reactivity BLAST Results 18.1-2

Cross Reactivity Line Data (pdf) 18.1-3

Cross Reactivity Line Data (xls) 18.1-4

Attachments (Documents) Attachment Number

Microbial Inhibition Protocol 18.2-1

Microbial Inhibition in silico BLAST Analysis Results 18.2-2

Microbial Inhibition Line Data (pdf) 18.2-3

Microbial Inhibition Line Data (xls) 18.2-4

Analytical Reactivity Protocol 18.3-1

Analytical Reactivity in-silico BLAST Analysis Results 18.3-2

Analytical Reactivity Line Data (pdf) 18.3-3

Analytical Reactivity Line Data (xls) 18.3-4

Analytical Reactivity COVID-19 Strains Line Data (pdf) 18.3-5

Analytical Reactivity COVID-19 Strains Line Data (xls) 18.3-6

Simplexa™ COVID-19 Clinical Strain Monitoring Report 18.3-7

Competitive Interference Protocol 18.4-1

Competitive Interference Line Data (pdf) 18.4-2

Competitive Interference Line Data (xls) 18.4-3

Sample Stability Fresh Vs Frozen Protocol 18.5-1

Sample Stability Fresh Vs Frozen Report 18.5-2

Sample Stability Fresh Vs Frozen Report (Line Data) (xls) 18.5-3

Sample Stability - Media Equivalency Swab Protocol (Other Media) 18.5-4

Sample Stability - Media Equivalency Swab Line Data (pdf) 18.5-5

Sample Stability - Media Equivalency Swab Line Data (xls) 18.5-6

Reproducibility Protocol 18.6-1

Reproducibility Line Data (pdf) 18.6-2

Reproducibility Line Data (xls) 18.6-3

Inter-lot Precision – Reaction Mix Protocol 18.7-1

Attachments (Documents) Attachment Number

Inter-lot Precision – Reaction Mix Line Data (pdf) 18.7-2

Inter-lot Precision – Reaction Mix Line Data (xls) 18.7-3

Inter-lot Precision – Positive Control Protocol 18.8-1

Inter-lot Precision – Positive Control Line Data (pdf) 18.8-2

Inter-lot Precision – Positive Control Line Data (xls) 18.8-3

Limit of Detection Study –Protocol 18.10-1