Accelerated Emergency Use Authorization (EUA) Summary, Curative SARS-Cov-2 Assay (KorvaLabs Inc. Clinical Laboratory)
Summary
Accelerated Emergency Use Authorization summary for the Curative SARS-Cov-2 Assay, performed at the KorvaLabs Inc. Clinical Laboratory in San Dimas, CA, a CLIA-certified high-complexity laboratory. The assay is a real-time RT-PCR test for qualitative detection of SARS-CoV-2 nucleic acid in oropharyngeal, nasopharyngeal and nasal swabs and oral fluid from individuals suspected of COVID-19 by their healthcare provider. Testing is limited to the single KorvaLabs site. The summary states that negative results do not preclude SARS-CoV-2 infection and that laboratories must report all positive results to public health authorities.
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Curative SARS-Cov-2 Assay
ACCELERATED EMERGENCY USE AUTHORIZATION (EUA) SUMMARY
CURATIVE SARS-COV-2 ASSAY
(KorvaLabs Inc. Clinical Laboratory)
For in vitro diagnostic use
Rx only
For use under Emergency Use Authorization (EUA) Only
(The Curative SARS-Cov-2 Assay will be performed in the KorvaLabs Inc. Clinical
Laboratory, a Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42
U.S.C. §263a certified high-complexity laboratory, per the Instructions for Use that
were reviewed by the FDA under this EUA).
INTENDED USE
The Curative SARS-Cov-2 Assay is a real-time RT-PCR test intended for the qualitative
detection of nucleic acid from the SARS-CoV-2 in oropharyngeal (throat) swab,
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nasopharyngeal swab, nasal swab, and oral fluid specimens from individuals suspected of
COVID-19 by their healthcare provider. Testing is limited to the single site of KorvaLabs,
Inc. San Dimas, CA, that is a Clinical Laboratory Improvement Amendments of 1988
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(CLIA), 42 U.S.C. §263a certified high-complexity laboratory.
Collection of nasal swabs and oral fluid specimens is limited to symptomatic individuals
within 14 days of COVID-19 symptom onset. Specimen collection must be directly
observed and directed during the sample collection process by a trained healthcare worker
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at the specimen collection site. Negative results for SARS-CoV-2 RNA from oral fluid
specimens should be confirmed by testing of another specimen type authorized for use
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with this test if clinically indicated.
Results are for the detection of SARS-CoV-2 RNA. The SARS-CoV-2 RNA is generally
detectable in respiratory specimens during the acute phase of infection. Positive results are
indicative of the presence of SARS-CoV-2 RNA; clinical correlation with patient history
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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
agent detected may not be the definite cause of disease. Laboratories within the United
States and its territories are required to report all positive results to the appropriate public
health authorities.
Negative results do not preclude SARS-CoV-2 infection and should not be used as the
sole basis for patient management decisions. Negative results must be combined with
clinical observations, patient history, and epidemiological information.
The assay is intended for use under the Food and Drug Administration’s Emergency Use
Authorization.
Curative SARS-Cov-2 Assay
DEVICE DESCRIPTION AND TEST PRINCIPLE
The Curative SARS-CoV-2 Assay is a real-time reverse transcription polymerase chain
reaction (rRT-PCR) test. The assay uses primers and probes that were developed and
validated under the Emergency Use Authorization (EUA) for the CDC 2019-Novel
Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel and are designed to
detect RNA from SARS-CoV-2 in respiratory specimens from patients as recommended
for testing by public health authority guidelines. The purpose of this EUA request is to
enable testing of additional specimen types, including oral fluid specimens and use of
alternative nucleic acid extraction and amplification systems available in KorvaLabs, Inc.
Oropharyngeal (throat) swab, nasopharyngeal swab, and nasal swabs should be collected,
transported and stored according to standard procedures. Nasal swabs and oral fluid
specimens must be collected using a swab (Curative Inc., #K00023) into the sample
collection tube (Curative Inc., #K00016) containing DNA/RNA Shield (Zymo Research,
#R1100). Nasal swabs and oral fluid specimens must be transported and stored at ambient
temperature and tested within 4 days of collection.
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RNA Extraction for all specimen types is performed using Total RNA Purification 96-
Well Kit (Norgen Biotek Corporation) manually or using Tecan Resolvex A200. The input
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sample volume is 300µl, the elution volume is 60µl.
Reverse transcriptase-PCR (RT-PCR) is performed using Applied Biosystems TaqPath™
1-Step Multiplex Master Mix with 5µl of the extracted sample.
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INSTRUMENTS USED WITH THE TEST
The Curative SARS-CoV-2 Assay is for use with the BioRad CFX 96 Touch, BioRad
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CFX Connect Real-Time PCR systems and Roche LightCycler 480 II Real-Time PCR
systems. RT-PCR processing and data analysis is being performed by BioRad CFX
Maestro V1.1 and Roche LightCycler Software V1.5.
REAGENTS AND MATERIALS
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Table 1. Reagents and materials required for use with Curative SARS-CoV-2 Assay
Material ID Vendor Catalog #
Ultrapure water RX Bioscience P01-UPW01-500
IDT 10006606
N1 Primers/Probes Biosearch Technologies KIT-NCOV-PP1-1000
IDT 10006606
N2 Primers/Probes Biosearch Technologies KIT-NCOV-PP1-1000
IDT 10006606
RP Primers/Probes Biosearch Technologies KIT-NCOV-PP1-1000
TaqPath™ 1-Step Multiplex
Master Mix Applied Biosystems™ A28527
Curative SARS-Cov-2 Assay
LIMITATIONS
The performance of the Curative SARS-CoV-2 Assay was established using oral fluid
specimen. Nasopharyngeal swabs, oropharyngeal swabs and nasal swabs are considered
acceptable specimen types for use with the Curative SARS-CoV-2 Test but performance
has not been established.
Testing of nasal swabs and oral fluid specimens (self-collected while directly observed
and directed by a trained healthcare worker at the site of sample collection or collected by
a trained healthcare worker) is limited to symptomatic individuals within 14-days of
COVID-19 symptom onset.
CONTROLS
Negative Process Control: The negative process control consists of uninfected collection
media, and two replicates are processed with every extraction batch run.
SARS-CoV-2 and RP Positive Process Control: This control monitors the success of the
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RNA extraction and confirms the performance of the RT-PCR master mix. The
positive process control consists of uninfected collection media spiked with
quantified nCoV plasmid and Hs_RPP30 gene.
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RP Positive Extraction Control: Detection of RNase P RNA in extracted nucleic acid serves
as a positive extraction control for each sample. It also confirms the absence of PCR
inhibitors in each eluted RNA sample.
Table 2. Controls performed with Curative SARS-CoV-2 Assay
Control Type Used to Monitor
O Frequency of Testing
SARS-CoV-2 N RP
Negative
extraction
control EV Cross-contamination during
extraction
Once per run of
RT-PCR
SARS-CoV-2/ Inefficient lysis of specimen, poor Once per run of
RP Positive specimen collection, improper RT-PCR
Once per run
of RT-PCR
Once per run
of RT-PCR
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Process control assay setup, extraction failure, PCR
RP Positive
inhibition, reagent failure including
primer and probe degradation
Inefficient lysis of specimen, poor Once per run of Once per run
extraction specimen collection, improper RT-PCR of RT-PCR
control assay setup, extraction failure, PCR
inhibition, reagent failure including
primer and probe degradation
If the SARS-CoV-2 N assay is positive with a negative RP result, consider the sample valid
and proceed with the next steps.
If the SARS-CoV-2 N assay is negative in conjunction with a negative RP, the specimen
results are considered invalid and should be repeated. If the residual specimen is available,
re-extract the specimen and perform testing again. If results remain invalid, a new
specimen should be collected.
Curative SARS-Cov-2 Assay
Table 3. Ct values of controls for validation of results
Control Type Used to Monitor Expected results and Ct Values
SARS-CoV-2 N RP
Negative Cross-contamination during Negative Negative
extraction extraction Ct not detected or Ct not
control Ct>40 detected or Ct
>35
SARS-CoV-2/ Inefficient lysis of specimen, poor Positive Positive
RP Positive specimen collection, improper 0 < Ct ≤ 40 0 < Ct ≤ 35
Process control assay setup, extraction failure,
PCR inhibition, reagent failure
including primer and probe
degradation
RP Positive Inefficient lysis of specimen, poor Negative Positive
extraction specimen collection, improper Ct not detected 0 < Ct ≤ 35
control assay setup, extraction failure,
PCR inhibition, reagent failure
including primer and probe
degradation
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INTERPRETATION OF RESULTS
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The results from testing of patient samples are interpreted according to the criteria
described in Table 4.
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Table 4. Interpretation of Patient Samples
SARS-CoV-2 RP
N EV
Ct detected in ±
the range
Result
Interpretation
SARS-CoV-2
detected
Report
Presumptive
Positive SARS-
Action
Report result to
sender and local or
0 < Ct ≤ 35
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No Ct or
Ct>40
0 < Ct SARS-CoV-2 not
≤ 35 detected
CoV-2
Negative SARS-
CoV-2
state department of
health.
Report results to
submitter.
Ct detected in ± Mandatory repeat Hold reporting till Repeat extraction
the range confirmatory testing from the sample
35< Ct≤ 40
No Ct or No Ct Invalid Result Invalid Repeat extraction
Ct>40 or from the sample. If
Ct>35 the repeated result
remains invalid,
report the result to the
sender and local or
state department of
health, and
recommend the
collection of a new
Curative SARS-Cov-2 Assay
specimen from the
patient.
PERFORMANCE EVALUATION
I. Analytical Sensitivity
The LoD was determined using viral genomic RNA (BEI Resources Catalog No. NR-
52285 (Lot # 70333700) that were diluted in SARS-CoV-2 negative oral fluid specimens
from volunteer donors into DNA/RNA Shield solution (Zymo Research, #R1100). An
initial estimate of the LoD with both the Bio-Rad CFX and Roche LightCycler 480 II was
obtained by testing four replicates at each of four different target levels: 1600, 800, 400
and 200 copies/mL. At all analyte levels, four out of four replicates passed. Based on these
results, two analyte concentration ranges (200 copies/mL and 100 copies/mL) were
selected for confirmation with 20 replicates each. For both automated and manual
extraction methods and both PCR systems, all 20 replicates at 200 copies/mL produced
the expected results for the SARS-CoV-2 target, and the LoD was therefore confirmed to
be 200 copies/mL.
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Table 5: Summary of Analytical Sensitivity Results for Curative SARS-CoV-2 Assay
Concentration
of Viral RNA
Manual Extraction
BioRad
CFX
Roche
LightCycler
Automated Extraction
BioRad
CFX
Roche
LightCycler
200 copies/mL 20/20
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20/20 20/20
480
20/20
100 copies/mL
II.
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Analytical specificity
20/20 19/20 17/20
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Inclusivity
The Curative SARS-CoV-2 Assay comprises only primers and probes designed by CDC
from the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic
Panel without any changes. Inclusivity of the CDC Diagnostic panel has been previously
established.
Cross-reactivity
The Curative SARS-CoV-2 Assay comprises only primers and probes designed by CDC
from the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic
Panel without any changes. Cross-reactivity of the CDC Diagnostic panel has been
previously established.
III. Clinical evaluation
A clinical study was performed to evaluate the use of oral fluid specimens as a specimen
type for detection of SARS-CoV-2 in patients who are suspected of COVID-19. People
Curative SARS-Cov-2 Assay
who were previously tested at a drive through location in Los Angeles where testing was
performed by Curative Inc. were contacted for their willingness to participate in this study.
Tables 7-10 summarize performance of the Curative SARS-CoV-2 test for self-collected
oral fluid swabs, non-healthcare worker (HCW) observed or directed and put into Zymo
DNA/RNA Shield RNA preservative compared to clinician collected nasopharyngeal
swab put into Zymo DNA/RNA Shield RNA preservative:
Clinician Collected Nasopharyngeal Swab (in
Table 7: All Individuals RNA preservative)
(Symptomatic and Asymptomatic)
Positive Negative Total
Self-Collected Oral Positive 15 4 19
Fluid Swab (Non-
HCW Negative 8 17 25
observed/directed) in
Total 23 21 44
RNA preservative
Positive Agreement 65.2% (95% CI 44.9-81.2%)
Negative Agreement
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81.0% (95% CI 60.0-92.3%)
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*1 oral fluid swab sample was QNS (quantity not sufficient) and was excluded from
analysis
When the test is limited to individuals symptomatic at the time of collection, within 21
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days of symptom onset, but not observed and directed by HCW during collection, the test
performance is as follows:
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Table 8: Symptomatic at time of
collection
Self-Collected Oral Positive
Clinician Collected Nasopharyngeal Swab (in
Positive
15
RNA preservative)
Negative
1
Total
16
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Fluid Swab (Non-
HCW
observed/directed) in
RNA preservative
Negative
Positive Agreement
Total
5
20
3
4
75.0% (95% CI 53.1-88.8%)
8
24
Negative Agreement 75.0 % (95% CI 30.1-95.5%)
When the test is limited to individuals symptomatic at time of collection and less than14
days since symptom onset, but not observed and directed by HCW during collection, the
test performance is as follows:
Curative SARS-Cov-2 Assay
Table 9: Symptomatic at time of
Clinician Collected Nasopharyngeal Swab (in
collection, >14 days since symptom
RNA preservative)
onset
Positive Negative Total
Self-Collected Oral Positive 4 1 5
Fluid Swab (Non-
HCW Negative 3 0 3
observed/directed) in
Total 7 1 8
RNA preservative
Positive Agreement 57.1 % (95% CI 25.0-84.2%)
Negative Agreement 0/1
When testing is limited to asymptomatic individuals at time of collection, but not observed
and directed by HCW during collection, the test performance is as follows:
Clinician Collected Nasopharyngeal Swab (in
Table 10: Asymptomatic at time of RNA preservative)
collection
Self-Collected Oral
Fluid Swab (Non-
Positive
Negative
Positive
0
3
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Negative
3
14
Total
3
17
HCW
observed/directed) in
RNA preservative
Total
Positive Agreement
Negative Agreement
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3
0/3
17
82.4 % (95% CI 59.0-93.8%)
20
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*1 oral fluid swab sample was QNS (quantity not sufficient) and was excluded from
analysis
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In the table above, all three false negatives were from individuals tested as asymptomatic
at time of collection, but previously symptomatic, within 17 days of symptom onset (7, 14,
and 17 days).
The data above does not support use of the Curative SARS-CoV-2, for any individuals for
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self-collected oral fluid samples when not directly observed and directed by a trained
healthcare worker during collection and at the site of collection. A modification to the
original clinical study acceptance criteria was made. Subjects more than 14 days from
symptom onset at time of collection, and subjects who were no longer symptomatic, were
excluded from the final clinical study. A total of 52 subjects were enrolled. Upon
obtaining consent, a clinician drove to the subjects’ home with a testing kit, the written
research study information form, and the sample collection materials. Testing kits
included components for collecting 4 different sample types and all samples were
collected into DNA/RNA Shield (Zymo Research).
Subjects were instructed to complete self-collection of the oral fluid specimen and nasal
specimen while observed and directed by the study clinician and package the sample into
the collection bag. Then the clinician collected a nasopharyngeal sample from the subject.
All three specimens were collected in the same visit within a 30-minute window.
There was 100% positive and negative agreement between the results obtained from
testing of clinician observed and directed, self-collected oral fluid swabs put into Zymo
Curative SARS-Cov-2 Assay
DNA/RNA Shield and those obtained from clinician collected nasopharyngeal swabs put
into Zymo DNA/RNA Shield. There was 97% positive agreement and 100% negative
agreement between the results obtained from testing of clinician observed and directed,
self-collected nasal swabs put into Zymo DNA/RNA Shield and those obtained from
clinician collected nasopharyngeal swabs put into Zymo DNA/RNA Shield. A summary
of the results of the study is presented in Table 11.
Table 11. Summary of qualitative results obtained from parallel testing of
nasopharyngeal swabs, nasal swabs and oral fluid specimens.
Nasopharyngeal Swab (In RNA Preservative)
Positive Negative Total
Oral Fluid Swab Positive 34 0 34
(observed and Negative 0 18 18
directed) in RNA
Total 34 18 52
preservative
Positive Agreement 100 % (34/34)
Negative Agreement
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100 % (18/18)
Nasal Swab (observed Positive
and directed) in RNA Negative
preservative Total
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Nasopharyngeal Swab (In RNA Preservative)
Positive
32*
1
33
Negative
0
18
18
Total
32
19
51*
Positive Agreement
Negative Agreement O 97.0 % (32/33)
100 % (18/18)
*one nasal swab sample was QNS (quantity not sufficient) and is excluded
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A modified second study was performed to evaluate the use of oral fluid specimens as a
specimen type for detection of SARS-CoV-2 in patients who are suspected of COVID-19.
The study was modified to collect the nasopharyngeal swabs into Viral Transport Media
which was sent to another laboratory (CMB Laboratories, Cypress, CA) and run on the
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CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel which has been authorized by the
FDA under an Emergency Use Authorization and validated by CMB Laboratories. Oral
fluid swab and nasal swab samples were collected as before and processed at Curative Inc.
CDC Sample Handling Guidelines were followed for collection, transport and processing
of nasopharyngeal swabs samples in viral transport media. A further 28 participants were
enrolled in this modified study.
There was 100% positive and negative agreement between the results obtained from
testing of oral fluid specimens or nasal swabs and those obtained from nasopharyngeal
swabs. A summary of the results of the study is presented in Table 12.
Table 12. Summary of qualitative results obtained from parallel testing of clinician
collected nasopharyngeal swabs (following CDC 2019-nCoV Real-Time RT-PCR
Diagnostic Panel) compared to nasal swabs and oral fluid specimens self-collected
while observed and directed by a clinician.
Curative SARS-Cov-2 Assay
Nasopharyngeal Swab (In VTM)
Positive Negative Total
Oral Fluid Swab Positive 16 0 16
(observed and Negative 0 12 12
directed) in RNA
Total 16 12 28
preservative
Positive Agreement 100 % (16/16)
Negative Agreement 100 % (12/12)
Nasopharyngeal Swab (In VTM)
Positive Negative Total
Nasal Swab Positive 16 0 16
(observed and Negative 0 12 12
directed) in RNA
Total 16 12 28
preservative
Positive Agreement 100 % (16/16)
Negative Agreement 100 % (12/12)
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Clinical Confirmation
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The first 5 positive and the first 5 negative oral fluid specimens as determined by the
Curative SARS-CoV-2 Assay were sent to the UCLA Clinical Microbiology Laboratory
and processed using the Thermo Fisher Scientific, Inc. TaqPath COVID-19 Combo Kit.
There was 100% (5/5) positive and 100% (5/5) negative agreement for the specimens
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tested. The results are acceptable and support use of the Curative SARS-CoV-2 Assay for
testing clinical specimens only according to the intended use, limitations, and only with
the reagents, instruments, and processes described herein
WARNINGS:
•
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This test has not been FDA cleared or approved;
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•
This test has been authorized by FDA under an EUA for use by the
authorized laboratory;
This test has been authorized only for the detection of nucleic acid from
SARSCoV-2, not for any other viruses or pathogens; and
• This test is only authorized for the duration of the declaration that
circumstances exist justifying the authorization of emergency use of in vitro
diagnostic tests for detection and/or diagnosis of COVID-19 under Section
564(b)(1) of the Act, 21 U.S.C. § 360bbb-3(b)(1), unless the authorization is
terminated or revoked sooner.
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