DiaSorin MOL4150 Simplexa™ COVID-19 Direct Instruction Manual

: June 7, 2024
: DiaSorin

Table of Contents

DiaSorin MOL4150 Simplexa™ COVID-19 Direct Instruction Manual
INTENDED USE
SUMMARY AND EXPLANATION
PRINCIPLES OF THE PROCEDURE
MATERIALS PROVIDED
KIT DESCRIPTION
COMPONENT DESCRIPTION
MATERIALS SUPPLIED SEPARATELY
MATERIALS REQUIRED BUT NOT SUPPLIED
RECOMMENDED MATERIALS
REAGENT HANDLING AND STORAGE

DiaSorin MOL4150 Simplexa™ COVID-19 Direct Instruction Manual

INTENDED USE

The DiaSorin Molecular Simplexa™ COVID-19 Direct real-time RT-PCR assay is intended for use on the LIAISON® MDX instrument for the in vitro qualitative detection of nucleic acid from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swabs (NPS), anterior nasal swabs (NS), nasal washes/aspirates (NW) or bronchoalveolar lavage (BAL) specimens from individuals suspected of COVID-19 by their healthcare provider. The Simplexa™ COVID-19 Direct assay is an aid in the diagnosis of SARS-CoV-2 infection.
Testing is limited to laboratories certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. §263a, that meet req uirements to perform high or moderate complexity tests.
Results are for the identification of SARS-CoV-2 RNA. The SARS-CoV-2 RNA is gen erally detectable in upper respiratory and bronchoalveolar lavage (BAL) specimens during the acute phase of infection. Positive results are indicative of the presence of SARS-CoV-2 RNA; clinical correlation with patient history and other diagnostic information is necessary to determine patient infection status. Positive results do not rule out bacterial infection or co- infection with other viruses. The ag ent detected may n o t be the definite cause of disease. Laboratories within the United States and its territories are req uired to report all results to the appropriate public health authorities.
Neg ative results do not preclude SARS-CoV-2 infection and should not be used as the sole basis for patient man ag ement decisions. Neg ative results must be combined with clinical observations, patient history, and epidemiological information.
The Simplexa™ COVID-19 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 diag nostic procedures. Simplexa™ COVID-19 Direct is only for use under the Food and Drug Administration’s Emergency Use Authorization.

SUMMARY AND EXPLANATION

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 are 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- Co V (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, o r CO VID-
SARS-CoV-2 caused an outbreak beginning in December 2019 in Wuh an City, Hubei Province, China and has spread global l y, being conseq uently declared a pandemic by the World Health Organization (WHO).2,4 Patients with COVID-19 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 lun gs.5

PRINCIPLES OF THE PROCEDURE

The DiaSorin Molecular Simplexa™ COVID-19 Direct assay system is a real-time RT-PCR system that en ables the direct amplification of Coronavirus SARS-CoV-2 RNA from nasopharyngeal swabs (NPS), nasal swabs (NS), nasal wash/aspirate (NW) or bronchoalveolar lavage (BAL) specimens. The system consists of the Simplexa™ COVID-19 Direct assay, the LIAISON® MDX (wi th LIAISON® MDX Studio Software), the Direct Amplification Disc and associated accessories.
In the Simplexa™ COVID-19 Direct assay, fluorescent probes are used together with corresponding forward and reverse primers to amplify SARS-CoV-2 viral RNA and internal control RNA. The assay targets two different regions of the SARS- CoV-2 genome, ORF1ab and S gene. 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 encod es well-conserved non- structural proteins and therefo re is less susceptible to recombination. An RNA internal control is used to detect RT-PCR failure and/or inhibition.

MATERIALS PROVIDED

The Simplexa™ COVID-19 Direct assay contains sufficient reag ents for 24 reactions. Upon receipt, store at -10 to -30 ºC (do n o t use a frost-free freezer). Each vial contains sufficient material for one use. Use within thirty (30) minutes ofthawing.

KIT DESCRIPTION

Component Name| REF| EC SYMBOL ON LABEL| Abbreviated Name| Cap Color| Number of Vials| Reactions per Vial/Kit| Volume per Vial
---|---|---|---|---|---|---|---
Simplexa™ COVID-19 Direct Reaction Mix| MOL4151| REAG| C| Co19| Brown| 24| 1/24| 50 µL

COMPONENT DESCRIPTION

Kit Component	Contents

Simplexa™ COVID-19 Direct Reaction Mix (RM)

| DNA polymerase, Reverse transcriptase, RNase inhibitor, buffer, dNTPs, encapsulated RNA Template, fluorescent probes and corresponding forward and reverse primers specific for detection of SARS-CoV-2 viral

RNA and for the Internal Control

Target

| Probe Fluorophore (Dye)| ****

Excitation (nm)

| ****

Emission (nm)

| ****

Targeted Gene

S gene

| ****

FAM

| ****

495

| ****

520

| ****

S gene

ORF1ab

| ****

JOE

| ****

520

| ****

548

| ****

ORF1ab

Internal Control RNA (IC)| ****

Q670

| ****

644

| ****

670

| ****

N/A

Simplexa™ COVID-19 Direct Barcode Card| Assay specific parameters and lot information.

MATERIALS SUPPLIED SEPARATELY

Direct Amplification Disc Kit (REF MOL1455)
Direct Amplification Discs for use on the LIAISON® MDX

MATERIALS REQUIRED BUT NOT SUPPLIED

LIAISON® MDX with LIAISON® MDX Studio Software version 1 or higher.
Simplexa™ COVID-19 Positive Control Pack (REF MOL4160).
50 µL fixed volume pipette (VWR Signature™ Fixed Volume Ergonomic High-Performance Pipettor Model VWR FE50 or eq uivalent).
Sterile, nuclease-free disposable pipette tips with filters (Extra Long tips ≥ 91 mm are recommended for pipetting directly from primary collection tubes).
Freezer (man ual defrost) at -10 to -30 °C (for kit component and/or specimen frozen storag e).
Refrigerator at 2 to 8 °C (for specimens).
Disposable, powder-free
Vortex for mixing patient
Centrifuge for collecting contents to bottom of tubes

RECOMMENDED MATERIALS

Universal Transport Media (UTM, Copan) or Universal Viral Transport (UVT, BD) to be used as a No Template Control (NTC).

REAGENT HANDLING AND STORAGE

Store reag ents at -10 to -30 °C (do not use a frost-free freezer).
Allow reag ents to thaw at room temperature (approximate range 18 to 25 °C) before
Do not use kits or reag ents beyond their expiration
After removing Reaction Mix from freezer storag e, initiate the test within thirty (30)
Do not vortex the Reaction
Do not refreeze the Reaction Mix

WARNINGS AND PRECAUTIONS

For in vitro diagnostic
For professional use
The Simplexa™ COVID-19 Direct real-time RT-PCR assay has not been FDA cleared or approved but has been authorized by FDA under an Emergency Use Authorization (EUA) for use by authorized laboratories; laboratories certified un der th e Cl i n i c al Laboratory Improvement Amendments (CLIA) of 1988, 42 S.C. §263a, that meet req uirements to perform high or moderate complexity tests.
The Simplexa™ COVID-19 Direct real-time RT-PCR assay has been authorized only for the detection of nucleic acid from SARS-CoV-2, not for any other viruses or
The emergency use of the Simplexa™ COVID-19 Direct real-time RT-PCR assay is only authorized for the duration of the
declaration that circumstances exist justifying the authorization of emergency use of in vitro diag nostics fo r d etec ti o n an d / or diagnosis of COVID-19 under Section 564(b)(1) of the Act, 21 U.S.C. § 360bbb-3(b)(1), unless the declaration is terminated or authorization is revoked sooner.
Wear personal protective equipment, such as (but not limited to) gloves and lab coats when handling kit reagents and equipment. Wash hands thoroughly when finished performing the test.
Do not pipette by mouth.
Do not smoke, drink, eat, handle contact lenses or apply make-up in areas where kit reagents and/or human specimen s are being used.
Dispose of unused kit reagents and human specimens according to local, state and federal regulations.
Treat all specimens and discs as capable of transmitting infectious agents.
Contamination of patient specimens or reagents can produce erroneous results. Use good laboratory practices and control workflow.6,7
Only use the protocol described in this insert. Deviations from the protocol or the use of times or temperatures other than those specified may give erroneous results.
Assay setup should be performed at room temperature (approximate range 18 to 25 °C).
Use calibrated fixed volume pipettes or equivalent to transfer sample and Reaction Mix.
Avoid touching the underside of the foil that will be in contact with the wells and disc surface.
To prevent potentially erroneous results, make sure that the sample and reagent are added to the appropriate input wells.
Finish loading and applying adhesive foil cover to one set of Sample and Reaction wells before opening the foi l o f ad jacen t set(s) of Sample and Reaction wells.
Initiate the run within thirty (30) minutes of removing the Reaction Mix vial from the freezer.
Do not attempt to remove adhesive foil cover from wedges that have been used or attempt to re-use Sample and Reaction ports that have been used in previous runs.
Discs may be reused until all eight (8) wedges have been used. Dispose of used discs without detaching foil cover in biohazardous waste container.
After each use store Direct Amplification Disc flat with the numbered foil side up.
Store reagents away from light.
Reaction Mix contains > 1% glycerol. Upon inhalation or skin contact, first aid measures should be taken.
If kit packaging or contents appear to be broken or damaged do not use and contact DiaSorin Molecular. Contact information is on the last page of this document.
The spectral matrix must be installed in each LIAISON® MDX and should not be changed unless an updated Quick Response
(QR) code for the instrument is provided by DiaSorin Molecular. The spectral matrix is unique to each LIAISON® MDX. The spectral matrix was provided with the LIAISON® MDX instrument on the cover of the LIAISON®MDX Hardware Manual. If th e matrix label will not scan or cannot be found contact DiaSorin Molecular Technical Services. The contact information is on the last page of this document.
Not installing or changing the spectral matrix can result in false results.

INSTRUCTIONS FOR USE

SPECIMEN COLLECTION AND HANDLING

Acceptable specimen types include:

Nasopharyngeal swabs (NPS) or nasal swabs (NS) in 3mL Copan Universal Transport Media (UTM) or BD Universal Viral Transport (UVT) or eq uivalent, Remel M5, Remel M6, Copan ESwab™ (Liquid Amies), Puritan® UniTranz-RT®, or saline (0.9% sodium chloride in water). Use only swabs with a synthetic tip (e.g. Dacron, nylon, or rayon) and an aluminum or plastic Do not use calcium alginate swabs, as they may contain substances that inhibit PCR testing.
Note: NPS or NS swabs should not be collected and placed into less than 3 mL transport media. For more information, please see the Limitations section.
Bronchoalveolar lavage (BAL) undiluted or diluted 1:1 (v/v) in a mucolytic such as Remel
Nasal wash/aspirate (NW)

A.REAL-TIME PCR INSTRUMENT SETUP

Refer to the LIAISON® MDX Operator Manual for details on how to configure the LIAISON® MDX Studio Software to ad d an assay definition, set up and an alyze runs on the LIAISON® MDX.

C DIRECT AMPLIFICATION DISC LOADING AND REAL-TIME PCR AMPLIFICATION NOTE: No sample extraction is needed prior to PCR amplification

Select samples that need to be
Thaw Reaction Mix vials at room temperature (approximate range 18 to 25 °C). Thaw one (1) Reaction Mix vial fo r eac h sample or control to be
Scan the barcode on the Simplexa™ COVID-19 Direct Reaction Mix vial or barcode card .
Scan the disc barcode on the Direct Amplification Disc (DAD).
Scan or type in each sample
For one wedge at a time, peel the adhesive foil back to expose the Sample (SAMPLE) and Reaction (R) wells without completely removing the adhesive foil cover.(Figure 1 & 2) Avoid touching the underside of the foil that will be in co n tac t with the wells and disc
Ensure that the Reaction Mix is completely thawed. Briefly spin down the tubes as (Do not vortex the Reac ti o n Mix)
Use the fixed volume pipette to transfer 50 µL of the Reaction Mix into Reaction (R)
Use the fixed volume pipette to transfer 50 µL of samples or control; pipette sample or control into Sample well (SAMPLE).
Cover the wedge sealing the wells with the peeled adhesive foil, pressing down firmly near the edge of the If th e original foil is torn do not load the wells in the wedge. Instead load another wedge.
Tear off the tab portion of the foil cover along the
Rep eat steps 6 to 11 for the next sample(s).

Figure 1 – Disc with pre-use foil lifted from Reaction [R] and Sample [SAMPLE] Wells for wedge #3| Figure 2 – Reaction [R] and Sample [SAMPLE] Wells
---|---
|

NO TES (for informational purposes – no user action/interpretation required) :

DiaSorin Molecular kits may contain version numbers for Assay Definitions. If the version number exists, it will be appended to the Assay Definition i.e. ‘Sample IVD Assay.2’. When multiple versions exist, the software automatically uses the assay definition associated with the scanned lot number.

QUALITY CONTROL

Simplexa™ COVID-19 Positive Control Pack (MOL4160) may be used as an external control for Quality Control (QC) testing, training or proficiency testing. Each laboratory should establish its own QC ranges and frequency of QC testing based on applicable local laws, reg ulations and stan dard good laboratory practice. Refer to the Simplexa™ COVID-19 Positive Control Pack (IFUC.US.MOL4160) for instructions on running the positive control.

Expected Quality Control Results

Control Type| ORF1ab target| S gene target| RNA Internal Control

(RNA IC)

---|---|---|---
Simplexa™ COVID-19

Positive Control 1

| Positive| Positive| Not applicable2
No Template Control

(NTC)

| Neg ative| Neg ative| Valid

1Typical Ct values for the Positive Control range between 22 to 32.
2Detection of the Simplexa™ RNA Internal Control (RNA IC) is not required for a valid result when SARS-CoV-2 is detected

INTERPRETATION OF RESULTS

Upon completion of the run, the software automatically calculates and displays results.
For each accession ID (Sample ID) entered, the software displays a result (“Positive”, “Neg ative”, “Invalid”, “EC500, EC505 or EC515”) for SARS-CoV-2 RNA

Results|

Interpretation

---|---
SARS-Cov-2 Target
ORF1ab| S gene
Positive| Positive| Result indicates the presence of SARS-CoV-2 RNA in the patient sample.
Positive| —-| Result indicates the presence of SARS-CoV-2 RNA in the patient sample.
—-| Positive| Result indicates the presence of SARS-CoV-2 RNA in the patient sample.
Negative| Negative| Result indicates the absence of SARS-CoV-2 RNA in the patient sample.

Invalid

| Result indicates inability to conclusively determine presence or absence of SARS-CoV- 2 RNA in the patient sample. This result may be due to 1) Internal Control (IC) failure, or 2) failure to detect sufficient specimen volume. The sample needs to be retested.

See “Invalid Results” section below.

Results	Interpretation
EC500	Data processing error due to noise, weak or late amplification in

the signal. Rep eat the sample. If the problem persists, contact Technical Service.
EC505| Insufficient information to determine whether amplification was present. If the problem persists, contact Technical Service.
EC515| Internal control amplification is not within specification. Result is invalid, rep eat the sample. If the problem persists, contact Technical Service.

In the case of one SARS-CoV-2 target positive/one SARS-CoV-2 target negative, result is suggestive of: 1) a sample at concentrations near or below the limit of detection of the test, 2) a mutation in one of the target regions, or 3) other factors.
1. Print the report as need
2. Export the results as need ed

INVALID RESULTS

In case of an “Invalid” result, re-test the sample with a new Reaction Mix vial from the same kit or a new kit. If the problem is unresolved, contact the DiaSorin Molecular Technical Services dep artment. Contact information can be found on the last p ag e o f this document

LIMITATIONS

For Emergency Use Authorization Only use
For in vitro diagnostic
For professional use
Testing of nasal swabs even if collected by a healthcare provider is limited to patients suspected of COVID-19
False-neg ative results may occur if the viruses are present at a level that is below the an alytical sensitivity of th e as s ay o r i f the virus has genomic mutations, insertions, deletions, or rearrangements or if performed very early in the course of
As with other tests, false-positive results may Rep eat testing or testing with a different device may be indicated in some settings.
This test is a qualitative test and does not provide the quantitative value of detected organisms
Information on the kit barcode can only be transferred into the LIAISON® MDX Studio Software Studio through a bar-code If the scanner is not working, or if you are unable to transfer the information for any reason, contact DiaSorin Molecular Technical Services.
NPS and NS specimens should only be placed into 3 mL transport media. Use of 1 mL transport media may result in an increased rate of Internal Control (IC) failures with some patient specimens. This may be due to a higher concentration of inhibitors present when a patient’s nasopharyngeal (NPS) or nasal swab (NS) is placed into 1 mL transport media comp ared to 3 mL transport
The clinical performance has not been established in all circulating variants but is anticipated to be reflective of the prevalent variants in circulation at the time and location of the clinical Performance at the time of testing may vary dep ending on the variants circulating, including newly emerging strains of SARS-CoV-2 and their prevalence, which chan ge over time. DiaSorin Molecular actively monitors and an alyzes the SARS-CoV-2 variants deposited in the reference database (GISAID
EpiCoV™). The Simplexa™ COVID-19 Direct assay primers and probes are checked ag ainst the database to evaluate any mutations that may impact the primer and/or probe binding.

CONDITIONS OF AUTHORIZATION FOR THE LABORATORY

The Simplexa™ COVID-19 Direct Letter of Authorization, along with the authorized Fact Sheet for Healthcare Providers, the authorized Fact Sheet for Patients, and authorized labeling are available on the FDA website: https://www.fda.gov/medical- devices/coronavirus-disease-2019-covid-19 -emergency-use-authorizations-medical-devices/in-vitro-diagnostics- euas.
However, to assist clinical laboratories using the Simplexa™ COVID-19 Direct, the relevant Conditions of Authorization are listed below:

Authorized laboratories 1 using the Simplexa™ COVID-19 Direct must include with test result reports all authorized Fact Under exigent circumstances, other appropriate methods for disseminating these Fact Sheets may be us ed, wh i c h may include mass media.
Authorized laboratories using the Simplexa™ COVID-19 Direct must use the Simplexa™ COVID-19 Direct as outlined in the authorized labeling. Deviations from the authorized proced ures, including the authorized instruments, authorized ex trac ti o n methods, authorized clinical specimen types, authorized control materials, authorized other ancillary reag ents and authori zed materials req uired to use the Simplexa™ COVID-19 Direct are not
Authorized laboratories that receive the Simplexa™ COVID-19 Direct must notify the relevant public health authorities o f th ei r intent to run the Simplexa™ COVID-19 Direct prior to initiating
a Authorized laboratories using the Simplexa™ COVID-19 Direct must have a process in place for reporting test results to healthcare providers and relevant public health authorities, as
b Authorized laboratories must collect information on the performance of the Simplexa™ COVID-19 Direct and report to DMD/OHT7-OIR/OPEQ/CDRH (via email: CDRH-EUA-Reporting@fda.hhs.gov) and DiaSorin Molecular (via phone: 800-838-4548) any suspected occurrence of false positive or false negative results and significant deviations from the establ i s h ed performance characteristics of the Simplexa™ COVID-19 Direct of which they become
c All laboratory personnel using the Simplexa™ COVID-19 Direct must be appropriately trained in RT-PCR techniques an d us e appropriate laboratory and personal protective eq uipment when han dling this kit, d d and use the Simplexa™ COVID-19 Direc t in accordance with the authorized
DiaSorin Molecular, authorized distributors, and authorized laboratories using the Simplexa™ COVID-19 Direct mus t en s ure that any records associated with this EUA are maintained until otherwise notified by FDA. Such records will be made availabl e to FDA for inspection upon request

PERFORMANCE CHARACTERISTICS CLINICAL EVALUATION

The clinical performance of the Simplexa™ COVID-19 Direct assays was established in multi-site clinical evaluation. Fresh c l in ic al NPS specimens were tested with the Simplexa™ COVID-19 Direct assay at three (3) different clinical sites from February 2020 to March 2020. For each of the sites, an established comparator was used. Sites 1 and 3 used the same comparator while Site 2 us ed a different comparator. Neg ative individually collected and positive contrived NS or NW specimens were tested intern al l y wi th th e Simplexa™ COVID-19 Direct assay in April or May 2020. Contrived samples were prep ared by spiking heat-inactivated 2019- nCoV/USA-WA1/2020 strain (ATCC® VR-1986HK™) into individual negative NS or NW specimens. BAL specimens, diluted 1:1 with Sputasol, were tested with the Simplexa™ COVID-19 Direct assay at one clinical site using an established comparator.

Simplexa™ COVID-19 Direct| Comparator| **Agreement* , ****

N/N (%)

---|---|---
Positive (+)| Negative (-)

NPS

|

Site 1

| Positive (+)| 1| 0| PPA: 1/1 (100%)
Negative (-)| 0| 20| NPA: 20/20 (100%)
Site Total| 1| 20|

Site 2

| Positive (+)| 11| 0| PPA: 11/11 (100%)
Negative (-)| 0| 0| NPA: 0/0 (100%)
Site Total| 11| 0|

Site 3

| Positive (+)| 40| 0| PPA: 40/40 (100%)
Negative (-)| 0| 36| NPA : 36/36 (100%)
Site Total| 40| 36|

NS

|

Site 4

| Positive (+)| 30| 0| PPA: 30/30 (100%)
Negative (-)| 0| 30| NPA : 30/30 (100%)
Site Total| 30| 30|

NW

|

Site 4

| Positive (+)| 29| 0| PPA: 29/30 (96.7%)
Negative (-)| 1| 30| NPA : 30/30 (100%)
Site Total| 30| 30|

BAL

|

Site 1

| Positive (+)| 11| 0| PPA: 11/11 (100%)
Negative (-)| 0| 7| NPA : 7/7 (100%)
Site Total| 11| 7|

*NPA = Negative Percent Agreement, PPA = Positive Percent Agreement
**NS and NW agreement (PPA and NPA) was determined against expected results.

ANALYTICAL SENSITIVITY/LIMIT OF DETECTION

The Limit of Detection (LoD) for NPS was determined to be the lowest detectable concentration of quantitated extracted viral genomic RNA (copies/mL) at which ≥ 95% of all replicates test positive. Initially, the tentative LoD was identified with serial diluti ons of the characterized SARS-CoV-2 viral genomic RNA tested in five (5) replicates during design and development. The lowest concentration at which all replicates were positive was interpreted as the tentative LoD. The LoD was then confirmed by testing forty-eight (48) replicates with concentrations at the tentative limit of detection. The final LoD was confirmed to be the lowest concentration resulting in positive detection with a minimum 95% positivity.
The final LoD for NPS, according to the assay results interpretation, is 500 copies/

Analytical Sensitivity/Limit of Detection for Viral Genomic RNA in UTM with RNasin

COVID-19

genomic RNA Copies/mL

| ****

Interpretation

| S gene (FAM)| ORF1ab (JOE)
---|---|---|---

% Detection (# Detected / # Tested)

| ****

Mean Ct ± SD

(%CV)

| ****

% Detection (# Detected / # Tested)

| ****

Mean Ct ± SD

(%CV)

2000| 100% (48/48)

Positive

| 100% (48/48)| 31.0 ± 0.64 (2.1%)| 100% (48/48)| 31.3 ± 0.74 (2.4%)
---|---|---|---|---|---
1000| 100% (48/48)

Positive

| 95.8% (46/48)| 32.4 ± 1.01 (3.1%)| 93.8% (45/48)| 32.7 ± 1.08 (3.3%)
500| 100% (48/48)

Positive

| 95.8% (46/48)| 33.4 ± 1.31 (3.9%)| 70.8% (34/48)| 33.9 ± 0.98 (2.9%)

The Limit of Detection (LoD) for NS was determined to be the lowest detectable concentration of inactivated titered COVID- 19 v i ral particles, strain 2019-nCoV/USA-WA1/2020, at which ≥ 95% of all replicates tested positive according to the results interpretation algorithm in pooled neg ative nasal swab specimens in UTM. Initially, the tentative LoD was identified with serial dilutions of the v i ral particles tested in four (4) replicates. The lowest concentration at which all replicates were positive was interpreted as the ten tati v e LoD. The LoD was then confirmed by testing twenty (20) replicates with concentrations at the tentative limit of detecti o n . Th e fi n al LoD was confirmed to be the lowest concentration resulting in positive detection with a minimum 95% positivity.
The final LoD for NS, according to the assay results interpretation, is 242 copies/mL

Analytical Sensitivity/Limit of Detection for Inactivated Viral Particles in NS matrix in UTM

COVID-19

Genome Copies / mL

| ****

Interpretation

| S gene (FAM)| ORF1ab (JOE)
---|---|---|---

% Detection (# Detected / # Tested)

| ****

Mean Ct ± SD

(%CV)

| ****

% Detection (# Detected / # Tested)

| ****

Mean Ct ± SD

(%CV)

242| 100% (20/20)

Positive

| 80% (16/20)| 34.0 ± 0.83 (2.4%)| 80% (16/20)| 32.9 ± 0.75 (2.3%)

The Limit of Detection (LoD) for NW was determined to be the lowest detectable concentration of inactivated titered COVID-19 v i ral particles, strain 2019-nCoV/USA-WA1/2020, at which ≥ 95% of all replicates tested positive in pooled neg ative NW specimen matrix. Initially, the tentative LoD was identified with serial dilutions of the viral particles tested in four (4) replicates. The lowest concentration at which all replicates were positive was interpreted as the tentative LoD. The LoD was then confirmed by testing twenty (20) replicates with concentrations at the tentative limit of detection. The final LoD was confirmed to be the lowest concentration resulting in positive detection with a minimum 95% positivity.
The final LoD for NW, according to the assay results interpretation, is 500 copies/mL

Analytical Sensitivity/Limit of Detection for Inactivated Viral Particles in Nasal Wash/Aspirate matrix

COVID-19

Genome Copies / mL

| ****

Interpretation

| S gene (FAM)| ORF1ab (JOE)
---|---|---|---
% Detection (# Detected / # Tested)| ****

Mean Ct ± SD (%CV)

| % Detection (# Detected / # Tested)| ****

Mean Ct ± SD (%CV)

500| 100% (20/20)

Positive

| 85.0% (17/20)| 33.1 ± 1.01 (3.0%)| 95.0% (19/20)| 32.3 ± 0.89 (2.8%)

The Limit of Detection (LoD) for BAL was determined to be the lowest detectable concentration of inactivated titered COVID-19 vi ral particles, strain 2019-nCoV/USA-WA1/2020, at which ≥ 95% of all replicates tested positive in pooled negative BAL matrix. Initi al l y , the tentative LoD was identified with serial dilutions of the viral particles tested in four (4) replicates. The lowest concentration at which all replicates were positive was interpreted as the tentative LoD. The LoD was then confirmed by testing twenty (20) replicates with concentrations at the tentative limit of detection. The final LoD was confirmed to be the lowest concentration resulting in positive detection with a minimum 95% positivity.

The final LoD for BAL, according to the assay results interpretation, is 1208 copies/mL

Analytical Sensitivity/Limit of Detection for Inactivated Viral Particles in BAL matrix

COVID-19

Genome Copies

/ mL

| ****

Interpretation

| S gene (FAM)| ORF1ab (JOE)
---|---|---|---

% Detection (# Detected / # Tested)

| ****

Mean Ct ± SD (%CV)

| ****

% Detection (# Detected / # Tested)

| ****

Mean Ct ± SD (%CV)

1208| 100% (20/20)

Positive

| 90.0% (18/20)| 32.8 ± 0.99 (3.0%)| 95% (19/20)| 32.2 ± 0.96 (3.0%)

FDA SARS-CoV-2 Reference Panel Testing: The evaluation of sensitivity and MERS-CoV cross-reactivity was performed using

reference material (T1), blinded samples and a standard protocol provided by the FDA. The study includ ed a range finding stud y
and a confirmatory study for the Limit of Detection (LoD). Blinded sample testing was used to establish specificity and to confirm the LoD. The results are summarized in the following Table

Summary of LoD Confirmation Result using the FDA SARS-CoV-2 Reference Panel

Reference Materials Provided by FDA| Specimen Type| Product LoD| Cross-Reactivity
---|---|---|---
SARS-CoV-2| ****

NPS

| 6 x 103 NDU/mL| N/A
MERS-CoV| N/A| ND

NDU/mL = RNA NAAT detectable units/mL N/A: Not applicable
ND: Not detected
NPS: Nasopharyngeal Swab

REACTIVITY/INCLUSIVITY

An in silico inclusivity analysis of the Simplexa™ COVID-19 Direct primers and probes was performed. All primer sets desig n ed fo r detection of the ORF1ab and S gene were tested ag ainst the complete available SARS-CoV-2 genome seq uence. The an alysis demonstrated that the regions recog nized by the designed primers and probes have 100% homology with all available SARS- CoV- 2 sequences from the National Center for Biotechnology Information (NCBI) and Global Initiative on Sharing Avian Influenza Data (GISAID) databases/databanks

Database

| Identity to ORF1ab| Identity to S gene
---|---|---
Primers (%)| Probe (%)| Primers (%)| Probe (%)
NCBI| 52/52 (100%)| 52/52 (100%)| 53/53 (100%)| 53/53 (100%)
GISAID| 352/352 (100%)| 350/352 (99%)| 364/364 (100%)| 364/364 (100%)

CROSS REACTIVITY

Cross-reactivity of the Simplexa™ COVID-19 Direct assay was evaluated using both in silico analysis and by testing whole organisms or purified nucleic acid from other organisms. Test specimens for laboratory testing were prep ared by s p i k i n g c ul tured isolates/inactivated organisms/purified nucleic acids (whole genome) (i.e., a minimum of 106 CFU/mL or higher for bacteria and
105 TCID50 /mL or PFU/mL or higher for viruses) into negative matrix (UTM) and determining cross reactivity based on three replicates. RNasin® was added to UTM for specimens containing extracted RNA. The results from the cross- reactivity, both in s ilic o and wet testing, are summarized below.

Microorganism

| In silico Analysis for % Identity target: ORF1ab| In silico Analysis for % Identity target: S gene
---|---|---
Human coronavirus 229E| No alignment found| No alignment found
Human coronavirus OC43| No alignment found| No alignment found
Human coronavirus HKU1| No alignment found| No alignment found
Human coronavirus NL63| No alignment found| No alignment found
SARS-coronavirus*| 90%| 80%
MERS-coronavirus| No alignment found| No alignment found
Adenovirus C| No alignment found| No alignment found
Human Metapneumovirus (hMPV)| No alignment found| No alignment found
Parainfluenza virus 1| No alignment found| No alignment found
Parainfluenza virus 2| No alignment found| No alignment found
Parainfluenza virus 3| No alignment found| No alignment found
Parainfluenza virus 4| No alignment found| No alignment found
Influenza A| No alignment found| No alignment found
Influenza B| No alignment found| No alignment found

Microorganism

| In silico Analysis for % Identity target: ORF1ab| In silico Analysis for % Identity target: S gene
---|---|---
Enterovirus (e.g. EV68)| No alignment found| No alignment found
Respiratory Syncytial Virus| No alignment found| No alignment found
Rhinovirus| No alignment found| No alignment found
Chlamydia pneumonia| No alignment found| No alignment found
Haemophilus influenzae| No alignment found| No alignment found
Mycobacterium tuberculosis| No alignment found| No alignment found
Streptococcus pneumonia| No alignment found| No alignment found
Streptococcus pyogenes| No alignment found| No alignment found
Bordetella pertussis| No alignment found| No alignment found
Mycoplasma pneumoniae| No alignment found| No alignment found
Pneumocystis jirovecii ( PJP )| No alignment found| No alignment found
Influenza C| No alignment found| No alignment found
Parechovirus| No alignment found| No alignment found
Candida albicans| No alignment found| No alignment found
Corynebacterium diphtheriae| No alignment found| No alignment found
Legionella pneumophila| No alignment found| No alignment found
Legionella non-pneumophila| No alignment found| No alignment found
Bacillus anthracis ( Anthrax )| No alignment found| No alignment found
Moraxella catarrhalis| No alignment found| No alignment found
Neisseria elongate| No alignment found| No alignment found
Neisseria meningitidis| No alignment found| No alignment found
Pseudomonas aeruginosa| No alignment found| No alignment found
Staphylococcus epidermis| No alignment found| No alignment found
Streptococcus salivarius| No alignment found| No alignment found
Leptospirosis| No alignment found| No alignment found
Chlamydia psittaci| No alignment found| No alignment found
Coxiella burnetii (Q-Fever)| No alignment found| No alignment found

Laboratory Tested Cross Reactivity Analysis

Organism

| Qualitative Results: % Detection (# Detected/#Tested)
---|---
S gene (FAM)| ORF1ab (JOE)| IC(Q670)
Adenovirus 1| 0% (0/3)| 0% (0/3)| 100% (3/3)
Bordetella pertussis| 0% (0/3)| 0% (0/3)| 100% (3/3)
Chlamydophila pneumoniae| 0% (0/3)| 0% (0/3)| 100% (3/3)
Coronavirus 229E| 0% (0/3)| 0% (0/3)| 100% (3/3)
Coronavirus HKU1*| N/A| N/A| N/A
Coronavirus NL63| 0% (0/3)| 0% (0/3)| 100% (3/3)
Coronavirus OC43| 0% (0/3)| 0% (0/3)| 100% (3/3)
Enterovirus 68| 0% (0/3)| 0% (0/3)| 100% (3/3)
Haemophilus influenzae| 0% (0/3)| 0% (0/3)| 100% (3/3)
Human metapneumovirus (hMPV-9)| 0% (0/3)| 0% (0/3)| 100% (3/3)

Organism

| Qualitative Results: % Detection

(# Detected/#Tested)

---|---
S gene (FAM)| ORF1ab (JOE)| IC(Q670)
Human leukocytes

(human genomic DNA)

| 0% (0/3)| 0% (0/3)| 100% (3/3)
Influenza A H3N2 Hong Kong/8/68| 0% (0/3)| 0% (0/3)| 100% (3/3)
Influenza B/Phuket/3073/2013| 0% (0/3)| 0% (0/3)| 100% (3/3)
Legionella pneumophila| 0% (0/3)| 0% (0/3)| 100% (3/3)
MERS- Co ronavi r us

(Extracted RNA)

| 0% (0/3)| 0% (0/3)| 100% (3/3)
Mycobacterium tuberculosis (genomic

DNA)

| 0% (0/3)| 0% (0/3)| 100% (3/3)
Mycoplasma pneumoniae| 0% (0/3)| 0% (0/3)| 100% (3/3)
Parainfluenza Type 1| 0% (0/3)| 0% (0/3)| 100% (3/3)
Parainfluenza Type 2| 0% (0/3)| 0% (0/3)| 100% (3/3)
Parainfluenza Type 3| 0% (0/3)| 0% (0/3)| 100% (3/3)
Parainfluenza Type 4A| 0% (0/3)| 0% (0/3)| 100% (3/3)
Pooled Human Nasal Fluid| 0% (0/3)| 0% (0/3)| 100% (3/3)
Rhinovirus B14| 0% (0/3)| 0% (0/3)| 100% (3/3)
RSV A Long| 0% (0/3)| 0% (0/3)| 100% (3/3)
RSV B Washington| 0% (0/3)| 0% (0/3)| 100% (3/3)
SARS-Coronavirus

(Purified RNA)

| 0% (0/3)| 0% (0/3)| 100% (3/3)
SARS-Coronavirus HKU39849 (Extracted

RNA)

| 0% (0/3)| 0% (0/3)| 100% (3/3)
Streptococcus pneumoniae| 0% (0/3)| 0% (0/3)| 100% (3/3)
Streptococcus pyogenes| 0% (0/3)| 0% (0/3)| 100% (3/3)

Coronavirus HKU1 was not available for testing; however, this organism was evaluated in silico. No alignments with Simplexa™ COVID-19 Direct primers and probes were found

POTENTIAL INTERFERING SUBSTANCES

Potential interfering substances from respiratory specimens were tested for ability to gen erate false neg ative results using s amp l es containing the extracted viral RNA at 3x LoD in nuclease free water. Testing was performed with 3 replicates per substance

Potential Interfering Substance

|

Active Ingredient

|

Tested Concentration

| COVID-19

Qualitative %

Detection

(# Detected / #Tested)

| IC Qualitative

% Detection (# Detected / #Tested)

---|---|---|---|---

Systemic antibacterial

|

Tobramycin

|

4 μg/mL

|

100% (3/3)

|

100% (3/3)

Antibiotic nasal ointment

|

Mupirocin

|

6.6 mg/mL

|

100% (3/3)

|

100% (3/3)

Nasal corticosteroids

|

Fluticasone

|

5% (v/v)

|

100% (3/3)

|

100% (3/3)

Nasal gel

| Luffa Opperculata, Galphimia glauca, histaminum

hydrochloricum

|

5% (w/v)

|

100% (3/3)

|

100% (3/3)

Homeopathic allergy relief medicine|

Not Applicable

|

10% (v/v)

|

100% (3/3)

|

100% (3/3)

Nasal spray or drops

|

Oxymetazoline

|

15% (v/v)

|

100% (3/3)

|

100% (3/3)

Cold Eeze (Throat lozenges, Oral

an esthetic and

an algesic)

|

Not Applicable

|

2.5% (w/v)

|

100% (3/3)

|

100% (3/3)

Potential Interfering Substance

|

Active Ingredient

|

Tested Concentration

| COVID-19

Qualitative %

Detection

(# Detected / #Tested)

| IC Qualitative

% Detection (# Detected / #Tested)

---|---|---|---|---

Anti-viral drug

|

Oseltamivir

|

3.3 mg/mL

|

100% (3/3)

|

100% (3/3)

Bovine submaxillary gland mucin, type I-S|

Mucin

|

60 μg/mL

|

100% (3/3)

|

100% (3/3)

Whole Blood

|

Not Applicable

|

2%(v/v)

|

100% (3/3)

|

100% (3/3)

REFERENCES

Cui J, Li F, Shi ZL. Nat Rev Microbiol. 2019 Mar;17(3):181-192. doi: 10.1038/s41579-018-0118-9.
World Health Coronavirus. https://[www.who.int/health-topics/coron avirus](http://www.who.int/health-topics/coronavirus)
Centers for Disease Control and Coron avirus. https://[www.cdc.gov/coronavirus/general-information.html](https://www.cdc.gov/coronavirus/general-information.html)
Cheng, Z.J., Shan, 2019 Novel coronavirus: where we are and what we know. Infection (2020). https://doi.org/10.1007/s15010-020-01401-y
Centers for Disease Control and Coron avirus Disease 2019 (COVID-19). https://[www.cdc.gov/coronavirus/2019-](https://www.cdc.gov/coronavirus/2019-ncov/about/index.html) ncov/abo ut/index.html
US Dep artment of Health and Human Services PHS/CDC/NIH. Biosafety in microbiology and biomedical laboratories, Washington DC: US Government Printing Office,
MM3-A2 Molecular diagnostic methods for infectious disease; approved guideline, 2nd ed. Wayne, PA: Clinical Laboratory Standards Institute, 2006

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ORDERING INFORMATION (U.S.A. only)

Telephone: 800-838-4548

Fax: 714-243-4703

| IFUK.US.MOL4150

Rev.09 Date written: 07July 2021

---|---
TECHNICAL ASSISTANCE (U.S.A. only)

Telephone: 800-838-4548

Fax: 562-240-6526

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U.S.A.

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Important Information: Please read carefully

For Emergency Use Authorization Only For In Vitro Diagnostic Use

Rx Only
Dear Valued Customer:
DiaSorin Molecular is proud to provide an easy to use sample to answer COVID-19 molecular testing system. The SimplexaTM COVID-19 Direct assay (catalog number MOL4150) and Simplexa™ COVID-19 Positive Control Pack (catalog number MOL4160 ) are designed to directly test the patient sample without the need for sample extraction and with all-in-one reagents for more rapid COVID-19 testing. The DiaSorin Molecular Simplexa™ COVID-19 Direct real-time RT-PCR assay is intended for use on the LIAISON® MDX instrument for the in vitro qualitative detection of nucleic acid from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swabs (NPS), anterior nasal swabs (NS), nasal washes/aspirates (NW) or bronchoalveolar lavage (BAL) specimens from individuals suspected of COVID-19 by their healthcare provider. The Simplexa™ COVID-19 Direct assay is an aid in the diagnosis of SARS-CoV-2 infection.

This product has not been FDA cleared or approved, but has been authorized for emergency use by FDA under an EUA for use by authorized laboratories;
This product has been authorized only for the detection of nucleic acid from SARS-CoV- 2, not for any other viruses or pathogens; and
The emergency use of this product is only authorized for the duration of the declaration that circumstances exist justifying the authorization of emergency use of in vitro diagnostics for detection and/or diagnosis of COVID-19 under Section 564(b)(1) of the Federal Food, Drug, and Cosmetic Act, 21 U.S.C. § 360bbb-3(b)(1), unless the declaration is terminated or authorization is revoked sooner

For the Instructions for Use (IFU) please visit: https://molecular.diasorin.com/us/covid19/ This card is not the full IFU. A printed copy of the IFU can be obtained free of charge by
contacting DiaSorin MolecularTechnical Service at TS.molecular@diasorin.com or 1-800-838-548, option 3.