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PF-07302048: Report on Method Validation of a Cepheid Xpert® Xpress PCR Assay to Detect SARS -CoV -2
VR-MVR -10080, Ver. 3.0
PFIZER CONFIDENTIAL
Page 1
Title: Report on Method Validation of a Cepheid Xpert ® Xpress PCR Assay to Detect
SARS -CoV -2
Study Number: C4591001
Parent Compound Number(s): PF-07302048
Alternative Compound Identifiers: N/A
Pfizer Vaccine Research and Development
401 N. Middletown Rd.
Pearl River, NY
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Page 2Title: Report on Method Validation of a Cepheid Xpert ® Xpress PCR Assay to Detect
SARS -CoV -2
PRINCIPAL INVESTIGATOR: Katherine Schneider, Senior Principal Scientist and Head,
Molecular Diagnostic Assay Development
CONTRIBUTING SCIENTIST (S):Jason Bennett, Tyler Garretson,
Joanna Wresilo,
PREPARED BY:
APPROVED BY:
Warren Kalina , PhD
Director, Clinical and Diagnostic Assay Development
Charles Tan, PhD
Senior Director, Earl y Clinical Development Biostatistics
David Cooper , PhD
Executive Director & Head, High -throughput Clinical I mmunoassay s & Diagnostics
Michael Pride , PhD
Executive Director Biology , Clinical and Diagnostic Assay Development
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Page 3Title: Report on Method Validation of a Cepheid Xpert ® Xpress PCR Assay to Detect
SARS -CoV -2
SYNOPSIS
This report summarizes the method validation for the Cepheid Xpert ®Xpress SARS -CoV -2
PCR test performed usi ng the GeneXpert ® and GeneXpert® Infinity instruments at the Pear l
River, NY Pfizer Vaccine Research & Development laboratories . The purpose of the Xpert®
Xpress SARS -CoV -2 test is to objectively score nasal swabs from persons suspected of virus
infection as either positive or negative for SARS -CoV -2. This document summarizes the
detection limits, clinical sensitivity ,and clinical specificit y of the PCR test when used in
Pfizer Vaccine Research & Development labor atories. The validation was performed with
simulated virus samples, nasal swabs scored SARS -CoV -2 positive or negative by a
predicate PCR test, pre-COVID -19 nasal swab samples, and live SARS -CoV -2. This report
documents that the Xpert® Xpress SARS -CoV -2 PCR test met pre -specified acceptance
criteria outlined in protocol VR -MVP -10076 and is suitable for its intended use as a
diagnostic assay for detection of SARS -CoV -2 in clinical samples in support of clinical trials
to evaluate the efficacy of Pfizer’s S ARS -CoV -2 vaccine candidate ,or in other SARS -CoV -2
related epidemiological studies.
Key Validation Outcomes
Clinical Perform ance Parmenter Observed Result Expected Results
Detection Limit of Live Virus 100% detection at 0.01 focus
forming units ( FFU)/mL0.01 FFU/mL
Detection Limit of AccuPlex™
SARS -CoV -2100% detection at 250 copies/mL 250 copies/mL
Agreement in Positive Samples 100% 100%
Agreement in Pre- COVID -19
negative samples100% 100%
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Page 4TABLE OF CONTENTS
SYNOPSI S................................ ................................ ................................ ................................ .3
LIST OF TABLES ................................ ................................ ................................ ..................... 5
1. OBJECTI VES ................................ ................................ ................................ ........................ 6
2. INTRODUCTION ................................ ................................ ................................ ................. 6
3. GLOSSARY ................................ ................................ ................................ .......................... 7
4. MATERIAL S AND MET HODS ................................ ................................ ........................... 7
4.1. L ive SARS -CoV -2 Reporter Virus ................................ ................................ ........... 8
4.2. Clinical Specimens from SARS- CoV -2 Positive Donors ................................ ......... 8
4.3. Clinical Specimens from SARS- CoV -2 Negative Donors ................................ ........ 8
4.4. Pre -Pandemic Nasal Swabs from Healthy Donors ................................ .................... 9
4.5. Methods................................ ................................ ................................ ..................... 9
4.6. Data Handling ................................ ................................ ................................ ........... 9
4.6.1. Data Proc essing and Outputs via I nstrument Software ................................ 9
5. RESUL TS AND DI SCU SSION ................................ ................................ .......................... 10
5.1. Detection Limits................................ ................................ ................................ ......11
5.1.1. Detection Limits Study Design ................................ ................................ ...11
5.1.2. Statistical Analy sis................................ ................................ ...................... 11
5.1.3. Acceptance Criteria ................................ ................................ .................... 11
5.1.4. Detection Limits Results.............................................................................11
5.2. Clinical Performance................................ ................................ ............................... 12
5.2.1. C linical Performance Study Design ................................ ............................ 12
5.2.2. Statistical Analy sis................................ ................................ ...................... 12
5.2.2.1. Percent Positive Agreement ................................ ...................... 12
5.2.2.2. Percent Negative Agreement ................................ ..................... 13
5.2.3. Clinical Performance Acceptance Criteria ................................ ................. 13
5.2.4. Clinical Performance Results ................................ ................................ .....13
5.2.4.1. Clinical Performance / Simulated Samples –AccuPlex™
SARS -CoV -2 Reference Material ................................ ..................... 13
5.2.4.2. Clinical Performance –Patient Samples ................................ ...13
5.2.4.3. Clinical Performance –Pre-COVID -19 Samples...................... 14
6. CONCLUSION ................................ ................................ ................................ .................... 15
7. DEVIATIONS ................................ ................................ ................................ ..................... 15
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Page 58. SUPPORTING DOCUME NTATION ................................ ................................ ................. 16
9. SUPPL EMENTAL INFO RMATION ................................ ................................ ................. 16
10. SU PPORTIVE TABLES ................................ ................................ ................................ ...18
LIST OF TABLES
Table 1. Terms and Definitions ................................ ................................ ................ 7
Table 2. Reagents and Sources ................................ ................................ ................. 8
Table 3. Xpert® Xpress SARS- CoV -2 Possible Results ................................ ........ 10
Table 4. List of Attachments ................................ ................................ ................... 10
Table 5. Detection Limit on AccuPlex™ SARS -CoV -2 Reference Material ........ 12
Table 6. Detection Limit on L ive Virus ................................ ................................ ..12
Table 7. Clinical Performance on Simulated Positive Samples .............................. 13
Table 8. Clinical Performance on Positive Patient Samples ................................ ...14
Table 9. Cts and Confidence Intervals for Positive Clinical Samples .................... 14
Table 10. Clinical Performance on Negative Patient Samples ................................ .14
Table 11. Clinical Performance on Pre -COVID -19 Samples................................ ...15
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Page 6Title: Report on Method Validation of a Cepheid Xpert ® Xpress PCR Assay to Detect
SARS -CoV -2
Study Number: N/A
Functional Area: Vaccine Research and Development
Test Facility : Pfizer Vaccine Research & Development
401 North Middletown Road,
Pearl River, NY 10965
Study/Testing Initiation Date: 20 July 2020
Study/Testing Completion Date: 25 July 2020
1.OBJECTIVES
This report summarizes the successful implementation of protocol VR- MVP-10076 [1]to
validate the FDA -EUA approved Cepheid Xpert® Xpress SARS -CoV -2 RT-PCR assay as
performed on the GeneXpert famil y of instruments for its intended use in Pfizer’s clinical
trials designed to evaluate the efficacy of Pfizer’s SARS -CoV -2 vaccine candidate andother
epidemiological studies to identify subjects infected with COVID -19. The assay method is
described in VR -TM-10295 [2] (Method for I dentification of SARS -CoV -2 in Nasal Swab
Using the Cepheid Xpert ®Xpress RT -PCR Assay Performed on the GeneXpert Molecular
Diagnostic S ystem). Assay sensiti vity, specificity ,and limit of detection have been
evaluated and overall assay performance isdescribed and summarized in this report.
2.INTRODUCTION
Pfizer , in collaboration with BioNTech, has rapidly developed a promising vaccine candidate
to protect agai nst COVID -19. To establish efficacy of the vaccine candidate through a
clinical trial, Pfizer selected a commerciall y available diagnostic assay for detection of
SARS -CoV -2 RNA in clinical trial volunteers. Thediagnostic test, Xpert ®Xpress
SARS -CoV -2 (Cepheid, Sunny vale, CA) is currently being marketed under an FDA
Emergency Use Authorization (EUA) issued March 20, 2020 [3]. The performance
characteristics of the Xpert® Xpress SARS -CoV -2 test are described in the package insert for
this device , which ha sbeen updated as Cepheid generates additional data [4]. Several recent
publications also describe the assay ’s performance [5, 6, 7, 8, 9]. The results of a
multi-center study of sensitivity and accuracy of the Xpert ®Xpress SARS -CoV -2test
substantiated Cepheid’s diagnostic performance claim of detection of 0.01 plaque forming
units per mL of live virus and detection of SARS- CoV -2 while exclusiv elynot detecting
other human coronaviruses [ 5]. In addition, the concordance of positive and negative sample
results in the Xpert ®Xpress test to other EUA SARS -CoV -2 tests has been published , with
percent positive agreement and percent ne gative agreement consistentl y above 95% [6, 7, 8,
9].
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Page 7The method validation protocol, VR- MVP -10076 (Method Validation Protocol for the
Cepheid SARS -CoV -2 PCR Assay [1]), describes the methods used to evaluate the
sensitivity , specificit y and limit of detection of the FDA -EUA approved Cepheid
Xpert ®Xpress SARS -CoV -2 test performed on the GeneXpert family of instruments. Given
that the assay is FDA -EUA approved, the protocol was based on information prov ided in
Cepheid’s EUA ( March 20, 2020 [3, 4]) and other publications [7, 9]to demonstrate that the
assay meets the sensitivity , specificit y, and LOD described in EUA 200047/A001 when
perform ed in the testing faciliti es at Pfizer Vaccine Research and Development,
Pearl River, NY.
This report summarizes the results and conclusions of the executed validation protocol.
3.GLOSSARY
Table 1.Terms and Definitions
Term Definition
Ct value Cyclethreshold is defined as the number of cycles required for the fluorescent signal
(asmeasured in the reaction curve) to exceed the background fluorescence in the assay. The
Ct value is the point (number of cycles) where the reaction curve crosses the thre shold.
LOD Limit of detection –the minimum FFU/mL that can be reliably classified as being positive
with a confidence level of 95%
FFU Focus Forming Unit. A single infectious SARS -CoV -2 viral particle, as measured by the
replication of its progeny on a localized area of a cell monolayer, detected by fluorescence
in a virus titration .
PCC Probe Check Control. The PCC verifies reagent rehydration, PCR tube filling, and confirms
that all reaction components are present in the cartridge including monito ring for probe
integrity and dye stability.
SPC Sample Processing Control. A target included in the Cepheid SARS -CoV -2 assay. The
SPC verifies that sample processing is adequate and detects sample -associated inhibition of
the real -time PCR assay, ensures that the PCR reaction conditions (temperature and time)
are appropriate for the amplification reaction, and that the PCR reagents are functional.
4.MATERIALS AND METHOD S
Documents VR -TM-10295 [2] and VR -SOP-LC-11286 [ 10] describe a laboratory method for
detection of SARS -CoV -2 in nasal swabs usi ng the FDA EUA diagnostic Xpert® Xpress
SARS -CoV -2 Assay (Cepheid, Sunny vale, CA). The Xpert® Xpress SARS -CoV -2 testis
marketed as a rapid, automated in vitro diagnostic test for the qualitative detection of
SARS -CoV -2 gene sequences (N2 and E) in nasal swab samples . The test is intended to be
used in Pfizer’s clinical trials and other epidemiology studies to identify subjects infected
with COVI D-19. Detection of RNA sequences specific for SARS -CoV -2 is carried out b y
real-time multiplex RT-PCR following a single -step sample processing protocol and was
performed according to the manufacturer's instructions.
The reagents described in Table 2were used in the validation. The equipment used in the
validation included GeneXpert IV units
which were controlled b y a
and operated as a , manually loaded instrument. In addition, GeneXpert IV unit
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Page 8 was operated manually in a laboratory , and GeneXpert Infinity was
used with automated cartridge loading.
Table 2.Reagents and Sources
Reagent Use Source
AccuPlex™ SARS -CoV -2
Reference Material Kit, 0505 -0126External Run Control and spike for
LOD ExperimentsSeraCare, Milford, MA
LOD Experiments
Nasal swabs from SARS -CoV -2
positive donorsClinical Sensitivity Experim ents
Nasal swabs from SARS -CoV -2
negative donorsClinical Specificity Experiments
Nasal swabs from healthy donors
collected pre -pandemic Clinical Specificity Experiments Pfizer Vaccine Research &
Development , Pearl River NY
Xpert® Xpress SARS -CoV -2
AssayAssay Kits Cepheid, Sunnyvale, CA
4.1.Live SARS -CoV -2 Reporter Virus
The live
virus was used to confirm the limit of detection of the Xpert ®Xpress test
(Supportive Table 10.2).
4.2.Clinical Specimens from SARS -CoV -2 Positive Donors
Remnant clinical specimens ( Supportive Table 10.4)were purchased from for use in
experiments to assess the Xpert ®Xpress SARS -CoV -2 percent positive agreement to other
molecular -based assay s. The specimens were certified by the vendor to have received
positive results in a predicate PCR assay , the SARS- CoV -2 PCR test.
4.3.Clinical Specimens from SARS -CoV -2 Negative Donors
Remnant clinical specimens ( Supportive Table 10.5) were purchased from for use in
experiments to assess the Xpert ®Xpress SARS -CoV percent negative agreement to other
molecular -based assay s. The specimens were certified by the vendor to have received
negative results in a predicate PCR assay , the SARS -CoV -2 PCR test.
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Page 94.4.Pre-Pandemic Nasal Swabs from Healthy Donors
Pfizer Vaccine Research & Development, Pearl River collected nasal swabs from healthy
donors (Supportive Table 10.6) in the
. The swabs were stored at -80°C before testing. The specimens were
used in experiments to assess the Xpert ®Xpress SARS -CoV test rate of false positive
results.
4.5.Methods
Test method VR -TM-10295 [2] describes the procedures to perform the C epheid
Xpert® Xpress SARS -CoV -2 Assay on nasal swabs collected from patients suspected of
having COVID -19. Barcoded samples are received frozen from Sample Management and
processed for testing as follows (see VR-TM-10295 [2]for details):
1.Scan the barcode of the specimen vial .
2.Thaw the patient sample containing nasal swab and media (~1 mL) at room temperature .
3.Vortex the patient sample tube and roll the swab against the side of the tube to express as
much media as possible from the swab. Remove the swab from the tube and discard.
4.Using a transfer pipette from the Cepheid assay kit, squeeze the top bulb of the pipette
completely and place the pipette tip in the specimen transport tube. Release the top bulb
of the pipette to fill the pipette with sample from the patient specimen tube.
5.Squeeze the top bulb of the pipette completel y again to empty the contents of the pipette
into the large cartridge opening and close the cartridge lid.
6.Scan the barcode of the assay cartridge, and load into the instrument/sample module.
Initiate the test via the Cepheid GeneXpert Dx version 4.7b or higher or Xpertise
software.
4.6.Data Handling
4.6.1. Data Processing and Outputs via Instrument Softw are
1.Data are presented in qualitative (“Positive”, “Negative”) and semi -quantitative
(Ct value) format for the 2 assay targets (N2 and E). For the validation, t he results were
interpreted automatically by the GeneXpert Dx software and shown in the View R esults
window. The Xpert ®Xpress SARS -CoV -2 test provided results based on the detection
of the two gene targets according to the algorithm shown in Table 3.
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Page 10Table 3.Xpert ®Xpress SARS -CoV -2 Possible Results
a.Sample processing control .+/-indicates that the positive coronavirus result is accepted as valid for either a
positive or negative result from the SPC .A strong positive result for either N2 or E can consume the
available PCR reagents in the cartridge and prevent the SPC from amplifying.
2.Semi -Quantitative data in the form of Ct values from positive and negative samples were
captured for each sample run. The Ct values were used to determine average values,
standard deviations and 95% confidence intervals for some validation data .
5. RESULTS AND DISCUSSIO N
Attachments in support of this validation are described in Table 4. Detection limit, clinical
sensitivity and specificity data used for the valida tion anal ysis are listed in
VR-MVR -10080 -ATT01 (seeSection 8).
Table 4.List of Attachments
Nam e Content
VR-MVR -10080 -ATT01 (see Section 8 ) Data Listing
VR-MVR -10080 -ATT02(see Section 8 ) Staff Member Roles and Batch Listing
VR-MVR -10080 -Supplemental Info [14] Cepheid Letter of Authorization
VR-MVR -10080 -Supplemental Info 02 [15] Xpert Xpress SARS -CoV- 2 Assay Cut -off
The strategy , design, and acceptance criteria for the validation were described in
VR-MVP -10076 [ 1],Method Validation Protocol for the Cepheid SARS -CoV -2 PCR Assay .
The validation was intended to demonstrate that the Xpert ®Xpress SARS -CoV -2 test could
be used with acceptable sensitivity ,specificit y, and detection limits when performed in the
Pearl River ,NY Pfizer Vaccine Research & Development laboratories. To meet that goal,
simulated samples were prepared from pooled nasal swab media or sterile
spiked with AccuPlex ™SARS -CoV -2 Reference Material. Live virus
samples were created b y (Section 4.1 )in , and
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Page 11remnant clinical samples were purchased. Finally , pre-COVID -19 samples collected from
healthy volunteers in were tested All samples were tested according to
methods outlined in VR- TM-10295 [ 2] and VR -SOP-LC-11286 [ 10].
The following sections describe the experimental designs , statistical analy ses, acceptance
criteria, and results for confirming the limit of detection and evaluating sensitivity and
specificit y of the Xpert ®Xpress SARS -CoV -2 test.
5.1.Detection Limits
5.1.1. Detection Limits Study Design
The LOD of the Xpert ®Xpress SARS -CoV -2 test was confirmed to the manufacturer’s
claims using both AccuPlex ™SARS -CoV -2 Reference Material and live
(Section 4.1). For all experiments, Xpert ®Xpress
SARS -CoV -2 test kit was used. For Accu Plex™ SARS -CoV -2 Reference Material ,
of AccuPlex ™SARS -CoV -2 was prepared in pooled negative nasal swab clinical matrix
(Section 4.3). A total of samples were prepared (Supportive Ta ble 10.1).
were prepared at 250 copies/mL , which is the L OD of the Xpert ®Xpress
SARS -CoV -2 test claimed in the Cepheid EUA . An additional samples were prepared at
, and another samples at .
For live virus, samples were
prepared (Supportive Table 10.2) , at 0.01 FFU per mL, the LOD ofthe Xpert ®Xpress
SARS -CoV -2 test claimed in the Cepheid EUA ;samples at ,
and samples at .
5.1.2. Statistical Analysis
TheLOD is defined as the minimum amount of input AccuPlex ™SARS -CoV -2 Reference
material or live reporter virus that y ields a 95% probability of a positive result. The
confirmation of LOD requires an independent percent positive rate that is at or above the
estimated LOD level.
5.1.3. Acceptance Criteria
Results are considered descriptive in nature and compared to results provided in Cepheid’s
EUA document (EUA# 200047/A001 ) [1, 4].
5.1.4. Detection Limits Results
The data collected to confirm the detection limits on simulated samples
(Supportive Table 10.1)is summarized in Table 5. On AccuPlex SARS -CoV -2,
samples at were detected. were detected, and
samples at were detected. This result confirms the published results of
detection at 250 copies/mL established by Cepheid [ 4] and confirmed by an independent
clinical testing lab [ 7].
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Page 12Table 5.Detection Limit on AccuPlex ™SARS -CoV -2 Reference Material
Description N Positive Positive
Rate (%)
80.0
1xLOD (250 copies/ml AccuPlex ™) 100.0
100.0
The data collected ondetection limits oflive virus in samples (Supportive Table 10.2)is
summarized in Table 6. On live virus, samples at 0.01 FFU/mL were detected. at
were detected, and samples at were detected. Theseresult s
confirm the published results of detection at 0.01 FFU/mL established b y Cepheid [ 4].
Table 6.Detection Limit on Live Virus
Description N Positive Positive
Rate (%)
75.0
LOD (0.01 FFU/ml Live virus) 100.0
100.0
5.2.Clinical Performance
5.2.1. Clinical Performance Study D esign
Similar to the design of the detection limit experiments, the clinical accuracy of the
Xpert ®Xpress SARS -CoV -2 PCR assay was confirmed to the manufacturer’s claims using
both simulated samples prepared with AccuPlex™ SARS -CoV -2 reference material and with
remnant clinical samples.
5.2.2. Statistical Analysis
5.2.2.1. Percent Positive Agreement
For both simulated samples and clinical samples, the percent positive agreement and its 95%
lower confidence bound were calculated for each target concentration. The mean Ct values
and 95% confidence were calculate for each of the E and N2 nucleic acid targets.
The Positive Percent Agreement (PPA) wasdefined as the percentage of the positive samples
detected b y the assay :
PPA = 100*Number of Agreed Positives/Total Number Positive Samples by Predicate
Method.
The one -sided lower 95% confidence bound for PPA wasdetermined b y the exact
method.
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Page 135.2.2.2. Percent Negative Agreement
The percent negative agreement and its 95% lower confidence bound were calculated for
negative samples .
The Negative Percent Agreement (NPA) wasdefined as the percentage of the negative
samples confirmed b y the assay :
NPA = 100*Number of Agreed Negatives/Total Number Negative Samples by Predicate
Method.
The one -sided lower 95% confidence bound for NPA wasdetermined b y the exact
method.
5.2.3. Clinical Performance Acceptance Criteria
Results are considered descriptive in nature and compared to results provided in Cepheid’s
EUA document (EUA#200047/A001) [ 1, 4].
5.2.4. Clinical Performance Results
5.2.4.1. Clinical Performance / Simulated Samples –AccuPlex™ SARS -CoV -2
Reference Material
The data collected to assess the assay ’s clinical accuracy on simulated samples are
summarized in Table 7.
All of the samples above the LOD were detected which
confirm sthe clinical accuracy of the assay established by the manufacturer [ 4] and multiple
publishe d studies [5,6, 7, 9].
Table 7.Clinical Performance on Simulated Positive Samples
Description N Positive Positive
% Agreem entOne- Sided
95% CI
Lower Limit (%)
5.2.4.2. Clinical Performance –Patient Samples
The data collected to assess the assay ’sclinical accuracy on positive samples is summarized
in Table 8 and Table 9. remnant clinical samples (Supportive Table 10.4), previousl y
determined to be positive using the SARS -CoV -2 PCR test as a predicate
PCR method ,were tested with one lot of Xpert ®Xpress SARS -CoV -2 test kit. Overall, a ll
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Page 14 samples were detected in the Xpert® Xpress SARS- CoV -2 test ( Table 8), and both gene
targets returned mean Ct values below Cepheid’s published positivity cutoff (Ct < 45).
Table 8. Clinical Performance on Positive Patient Samples
Description N Positive Positive
%Agreem entOne- Sided
95% CI
Lower Limit (%)
Pos N asalSwab 100.0 90.5
Table 9. Cts and Confidence Intervals for Positive Clinical Samples
Target N MeanCt Standard
Deviation95% CI
E 24.5 6.62 (22.1, 27.0)
N2 26.7 6.02 (24.4, 28.9)
The data collected to assess theassay ’sclinical accuracy on negative samples is summarized
in Table 10. remnant clinical samples (Supportive Table 10.5),previousl y
determined to be negative u sing the SARS -CoV -2 PCR test as a predicate
PCR method ,were tested using one lot of Xpert ®Xpress SARS -CoV -2 test.
out of samples were negative in the Xpert® Xpress SARS -CoV -2 test. One sample was
unable to be tested due to a cartridge error during processing. One sample had a Ct of41 for
the N2 target onl yand is considered positive per VR -SOP-LC-11286 [10].
Table 10.Clinical Performance on Negative Patient Samples
Description N Negative Negative
%Agreem entOne- Sided
95% CI
Lower Limit (%)
Neg Nasal Swab 96.4 84.1
5.2.4.3. Clinical Performance –Pre-COVI D-19 Samples
The single negative patient sample described in Section 5.2.4.2 with anN2target positivity
created concerns about the false positive rate of the Xpert ®Xpress SARS -CoV -2 test. To
address this question, samples collected from healthy individuals in
(Supportive Table 10.6), before SARS- CoV -2 infection was identified in the US, were tested
using one lot of Xpert® Xpress SARS -CoV -2 kit.
All samples produced negative results in the Xpert® Xpress SARS -CoV -2 test,
giving a false positive rate of 0% (Table 11).
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Page 15Table 11.Clinical Performance on Pre -COVID -19 Samples
Description N Negative Negative
%Agreem entOne- Sided
95% CI
Lower Limit (%)
Pre-COVID -19swab 100.0 95.8
6.CONCLUSION
This validation report provides documented evidence that the Xpert ®Xpress SARS -CoV -2
test, when performed b y qualified personnel in accordance with standard operating
procedures VR -TM-10295 [2] and VR -SOP-LC-11286 [10] is suitable for its intended
purpose of testing of nasal swabs from persons with suspected infection or exposure to
SARS -CoV -2. This assay will be used to test samples from Pfizer’s clinical efficacy trial of
a SARS -CoV -2 vaccine candidate and other epidemiological studies.
7.DEVIATIONS
1.VR-MVP -10076 ‘Method Validation Protocol for the Cepheid SARS -CoV -2 PCR
Assay ” section 6.3 ‘ Clinical Specimens from SARS- CoV -2 Positive Donors ’ states
remnant c linical specimens were purchased for use in experiments to define the LOD and
percent agreement of the assay . This was an error. Those specimens were used to define
the percent agreement only and not to defne the LOD.
2.A deviation to the rules for sample scoring documented in VR-MVP -10076 [ 1] occurred
in this validation. VR -MVP -10076 indicate dPfizer Vaccine Research & Development
will consider a sample positive for SARS -CoV -2 only if both the N2 and E gene targets
are detected . After reviewing the data for the LOD confirmation shown in VR -MVR -
10080 -ATT01 (see Section 8)it became evident that following that rule would
inappropriatel y exclude actual positive samples. Although the E target has potential to
detect the original SARS coronavirus and bat coronavirus, our results demonstrate that
spiked, known SARS -CoV -2 samples can be scored E+/N2- at concentrations close to the
limit of detection. In consultation with Cepheid and after reviewing additional published
data [9] Pfizer Vaccine Research & Development decided to follow the scoring
recommendations in the manufacturer’s instructions for use [ 4]. The impact of this
deviation wasbetter data agreement between LOD and percent sensitivity and specificit y.
3. During testing of clinical performance, the robotic arm that feeds cartridges into the
analysis instrument had a mechanical failure, which resulted in 12 samples sitting more
than 30 minutes before processing started. There was no negative clinical matrix
available to create additional samples for repeat testing, so new samples we re created in
sterile . To address concerns that the matrix change would confound the results, a
new sample set with samples at the LOD,
was prepared and tested. Samples were
negative nasal swab media, and were sterile .The
impact of thedeviation was an unacceptabl y low N for analysis. Creation of the new
sample set permitted retesting with a higher N for anal ysis of clinical performance.
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Page 164.The valid ation protocol specified that negative clinical samples would be tested as part
of clinical performance/ percent negative agreement. Only samples could be obtained
in time for the validation, and one of the samples was received from the vendor
conta ining 130 µl, an insufficient volume for testing. The impact of the deviation is that
the percent negative agreement calculation was limited to samples. The impact was
mitigated b y testing pre -COVID -19 swabs in addition to the negative swabs.
5.VR-MVP-10076 “Method Validation Protocol for the Cepheid SARS -CoV -2 PCR
Assay ” section 6.4 stated remnant clinical specimens were purchased for use in
experiments to define the L OD, sensitivity and percent agreement of the assay . Shipping
and purchasing delay sdue to the pandemic resulted in the remnant clinical specimens not
arriving in time for the validation experim ents. The experiments to define LOD and
sensitivity used as matrix instead of remnant clincal specimens.
6. During validation testing an additi onal nasal swabs samples were tested that pre -date
the existence of COVID -19 by approximately 2.5 y ears. These samples would be
considered better predictors of negative results when assessing assay clinical specificity ;
therefore, these samples were added to the validation testing and documented in this
report as a separate anal ysis of assay clinical specificity .
7.VR-MVP -10076 “Method Validation Protocol for the Cepheid SARS -CoV -2 PCR
Assay ” section 7.1.1 states “SARS -CoV -2 prepared in pooled NP swab clinical matrix”.
It was an oversight that the protocol did not specifically state that the pooled clinical
matrix was negative for SARS -CoV -2, as specified in Section 5.1.1 of this report.
8.SUPPORTING DOCUMENTATION
1. VR-MVR -10080 -ATT01, Supplemental I nformation for VR -MVR -10080 -Validation
Data Listing .
2. VR-MVR -10080 -ATT02, Supplemental I nformation for VR -MVR -10080 – Roles and
Responsibilities for Method Validation .
9.SUPPLEMENTA LINFORMATION
1. VR-MVP-10076, Method Validation Protocol for the Cepheid SARS- CoV -2 PCR
Assay .
2. VR-TM-10295, Method for Identification of SARS -CoV -2 in Nasal Swab using the
Cepheid Xpert® Xpress RT -PCR Assay Performed on the GeneXpert Molecular
Diagnostic S ystem.
3. USDHHS , Response to Cepheid application for Emergency Use Authorization
200047/A001. March 20, 2020.
4. Xpert® Xpress SARS- CoV -2 Instructions For Use. 302 -3562, Rev C. April 2020.
Downloaded from https:/ /www.cepheid.com/ /en_US/package- inserts/1615 on
June 8, 2020.
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Page 175. Loeffelholz MJ, Alland D, Butler -WuSM, et al. Multicenter evaluation of the Cepheid
Xpert Xpress SARS- CoV -2 test. J Clin Microbiol 2020; 58(8) (Jul): e00926-20.
6. Lieberman JA, Pepper G, N accache SN et al. Comparison of commerciall y available
and laboratory -developed assays for in vitro detection of SARS -CoV -2 in clinical
laboratories. J Clin Microbiol 2020 ;58(8) (Jul): e00821 -20.
7. Zhen W, Smith E, Manji R ,et al. Clinical evaluation of three sample -to-answer
platforms for detection of SARS -CoV -2. J Clin Microbiol 2020; 58(8) (Jul): e00783-20.
8. Moran A, Beavis KG, Matushek SM, et al. Detection of SARS- CoV -2 by use of the
Cepheid Xpert Xpress SARS -CoV -2 and Roche cobas SARS- CoV -2 assays. J Clin
Microbiol 2 020;58(8) (Jul): e00772-20.
9. Lowe CF, Matic N, Ritchie G et al. Detection of low levels of SARS- CoV -2 RNA from
nasophary ngeal swabs using three commercial molecular assay s. J Clin Virol
2020 ;128:104387.
10. VR-SOP-LC-11286 , Data Review for Cepheid SARS -CoV -2 RT-PCR Assay .
11. VR-SOP-LC-11294, Preparation and Assessment of SARS -CoV -2 Stocks.
12.
13.
14. VR-MVR -10080 -Supplemental I nfo-Cepheid Letter of Authorization .
15. VR-MVR -10080 -Supplemental I nfo02 -Xpert Xpress SARS -CoV -2 Assay Cut-off.
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Page 1810.SUPPORTIVE TABLES
10.1. Detection Limit Simulated Samples ...............................................................................18
10.2. Detection Limit L ive Virus Samples ................................ ................................ .............. 19
10.3. Clinical Performance Simulated Samples................................ ................................ .......19
10.4. Positive Clinical Samples ................................ ................................ ............................... 20
10.5. Negative Clinical Samples ................................ ................................ .............................. 21
10.6. Pre-COVID -19 Samples ................................ ................................ ................................ .21
10.1. Detection Limit Simulated Samples
Sample Number Sample ID Sample Number Sample ID
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Page 1910.2. Detection Limit Live Virus Samples
Sample Number Sample ID Sample Number Sample ID
10.3. Clinical Performance Simulated Samples
Sample
NumberSample ID Sample
NumberSample ID Sample
NumberSample ID
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Page 2010.3. Clinical Performance Simulated Samples
Sample
NumberSample ID Sample
NumberSample ID Sample
NumberSample ID
10.4. Positive Clinical Samples
Sample Number Sample ID Sample Number Sample ID
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Page 2110.5. Negative Clinical Samples
Sample Number Sample ID Sample Number Sample ID
a.This sample was not tested because it was received from the vendor w ith insufficient volume.
10.6. Pre-COVID -19 Samples
Sample
NumberSample ID Sample
NumberSample ID Sample
NumberSample ID
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Page 2210.6. Pre-COVID -19 Samples
Sample
NumberSample ID Sample
NumberSample ID Sample
NumberSample ID
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