Download Ebola Zaire (EZ1) JBAIDS pre-EUA

REVIEW MEMORANDUM FOR
PRE-EMERGENCY USE AUTHORIZATION
A. PURPOSE FOR SUBMISSION
Emergency Use Authorization (EUA) request for distribution of the Escherichia coli
(AAF) Real-Time PCR (TaqMan) Assay to Department of Defense (DoD) qualified
laboratories to aid in the presumptive diagnosis of E. coli O104:H4 infection. The
assay is for the in vitro qualitative detection of E. coli O104:H4 bacteria from stool of
individuals at risk for exposure to E. coli O104:H4 bacteria or with signs and
symptoms of infection with E. coli.
B. MEASURAND
Detection of a specific nucleic acid sequence from the genome of E. coli O104:H4.
C. APPLICANT
The Office of The Surgeon General (OTSG), in conjunction with the DoD.
D. PROPRIETARY AND ESTABLISHED NAMES
Established Name – E. coli (AAF) Real-Time PCR (TaqMan-MGB) Assay.
E. PRODUCT DESCRIPTION
1. The Light Cycler 480 (LC480) is a real-time polymerase chain reaction (PCR)
platform. The DoD Critical Reagent Program (CRP) E. coli AAF Assay will
consist of one primer/probe set: AAF F275/R345/p321A-MGB along with the
assay master mix, Negative Template Controls (NTC), and Positive Template
Controls (PTC). The Qiagen EZ1 Virus Mini Kit v 2.0 will be purchased
separately from the assay for sample processing.
2. The LC480 instrument is programmed to perform heating and cooling cycles that
drive the PCR process. The heating and cooling cycles are generated using a
heating block and cooling element. Fluorescence emission is monitored over
five excitation and six emission filters of the instrument can be used in any
combination, and the instrument software interprets the change in fluorescence to
determine whether the target DNA is present. When the organism is present, E.
coli O104:H4 DNA is amplified using specific primers. The amplicon is detected
by fluorescence using a specific hydrolysis probe. The hydrolysis probe is a short
oligonucleotide that hybridizes to an internal sequence of the amplified fragment
during the annealing phase of the PCR cycle. This probe has the 5' and 3' ends
labeled with a reporter dye and a quenching dye, respectively. After the probe
hybridizes to the specific DNA target, the Taq polymerase enzyme hydrolyzes the
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probe while replicating the target-specific DNA. The hydrolysis separates the two
fluorophores, thus allowing the reporter dye to fluoresce. The level of
fluorescence from each unknown sample and control is measured by the LC480
instrument. LC480 software analyzes fluorescence amplification curves and
reports results as "Positive" or "Negative."
3. The LC480 system consists of the following components:
LC480 thermocycler
Specific reagents for the target organism (stored at 2-8˚C)
Desktop computer loaded with specific, user-friendly software
4. DNA is extracted from stool using a Qiagen EZ1 Virus Mini Kit v 2.0, purchased
separately from the assay, prior to running on the LC480.
The Qiagen EZ1 Virus Mini Kit v 2.0 simplifies purification of bacterial nucleic
acid from stool with a magnetic bead procedure. Nucleic acid binds specifically to
the Qiagen magnetic beads, and nucleic acid is eluted in the buffer provided with
the kit.
The resulting nucleic acid is analyzed on the LC480 instrument, using provided E.
coli AAF master mix with appropriate controls in place.
The LC480 instrument, when programmed correctly, will call reactions positive,
negative, or uncertain. The amplification conditions have been determined and
programmed into a LC480 macro that is distributed via CD/DVD with the E. coli
testing materials. The LC480 System Manual and LC480 Macro Usage Document
on the DVD provide guidance for importing a macro and analyzing a PCR run.
See Table 7 for interpretation of sample results.
5. The AAF PCR Assay uses the following:
Table 1. AAF PCR Assay
AAF P Set:
Forward primer 5′- TTgCCTTAACAgATCTTCCAgTgA -3′
Reverse primer 5′- gCCgTCTgACTCTAATACCCAAA -3′
Probe 5′- TTgCAAgTCACCACCgTT -3′- NFQMGB
AAF (U12894.1)
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6. Control material to be used with the AAF PCR Assay includes:
Table 2. AAF PCR Assay Controls
Material Used
Supplier & Catalog
Number
Nuclease-free Water
Ambion nucleasefree water (Cat
#AM9937 or
equivalent)
Used in Amplification step to
show no contamination in
loading samples
Nuclease-free Water
Ambion Nucleasefree water (Cat
#AM9937 or
equivalent)
Used in Amplification step to
confirm target amplification
E. coli O104:H4
DNA
DoD Critical
Reagents Program
(CRP)
Control
Type
When
RNTC (Reagents no
template control)
Negative
Control
Used in Amplification step to
show no reagent
contamination
SNTC ( Sample
negative control)
Negative
Control
AAF PTC (Positive
Template Control)
Positive
Control
F. TEST PRINCIPLE
The AAF PCR Assay is a real-time polymerase chain reaction (PCR) test. The E. coli
AAF primer and probe set is designed to detect DNA from the E. coli O104:H4
bacteria in stool specimens from patients.
PCR assays utilize a thermocyclic heating and cooling of the reaction to
logarithmically amplify a specific region of DNA. The probe anneals to a specific
target sequence located between the forward and reverse primers. During the
extension phase of the PCR cycle, the 5’ nuclease activity of Taq polymerase
degrades the probe, causing the reporter dye to separate from the quencher dye,
generating a fluorescent signal. With each cycle, additional reporter dye molecules
are cleaved from their respective probes, removing the quenching effect and
increasing the fluorescence intensity. Fluorescence intensity is monitored at each
PCR cycle.
1. A Reagent “no template” Control (RNTC) is required to ensure the highest level
of confidence that the amplification reagent used in the reactions is free of
contamination. This RNTC contains water in place of an extracted sample and
should not show increased fluorescence during cycling.
2. The PTCs are required to confirm that the amplification reagents are capable of
amplifying the intended targets, if present. For the PTC, E. coli DNA with the
AAF gene amplicon region is added to the amplification reagents and should yield
a positive result.
3. A Negative Sample “no template” Control (SNTC) is required to ensure that no
cross-contamination occurred during sample loading. Water is added in place of
an extracted sample as with the NTC, but the tube is left open during sample
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loading. Also, if aerosolized or free floating amplicon is present, it causes this
control to be positive, indicating that some of the samples could be false positives.
G. INTENDED USE
The AAF PCR Assay on the LC480 platform is a real-time PCR assay used in
conjunction with clinical and epidemiological information.
For the qualitative detection of E. coli DNA in stool from individuals at risk for
exposure to E. coli or with signs and symptoms of infection with E. coli.
Warning:
Testing with the AAF PCR Assay should not be performed unless the individuals
are at risk for exposure to E. coli O104:H4 or with signs and symptoms of
infection with E. coli and meet clinical and epidemiologic criteria for testing
suspect specimens.
Results are for the presumptive identification of E. coli O104:H4. The definitive
identification of the E. coli requires additional testing and confirmation
procedures in consultation with public health or other authorities for whom
reporting is required. The diagnosis of an E. coli O104:H4 infection must be
made based on history, signs, symptoms, exposure likelihood, and other
laboratory evidence in addition to the identification of the E. coli O104:H4 by this
test.
Negative results do not preclude E. coli O104:H4 infection and should not be used
as the sole basis for patient management decisions.
The AAF PCR Assay is intended for use by trained clinical laboratory personnel
who have received advanced training on the LC480. The level of E. coli that
would be present in stool from individuals with early infection is unknown. Due
to the difficulty in obtaining clinical specimens, these assays were not evaluated
with stool from individuals with E. coli O104:H4 infection.
H. UNMET NEED ADDRESSED BY THE PRODUCT (to be completed at time of
emergency)
The Secretary of Health and Human Services (HHS) has declared a public health
emergency because of the [describe emergency if information is available e.g.
outbreak of the E. coli O104:H4 or detection of E. coli O104:H4 in X# of patients…]
Diagnostic devices are an essential and critical element of public health protection
and on [Month, Day, Year] the Secretary of HHS declared an emergency, justifying
the emergency use authorization of certain in vitro diagnostic devices for detection of
E. coli O104:H4.
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At this time, there are no other alternative approved, cleared, adequate, or available
diagnostic tools that can detect the E. coli O104:H4. This EUA is designed to utilize
the AAF PCR Assay to expand testing capabilities within the DoD by providing a
diagnostic tool to qualified laboratories for the detection of the E. coli O104:H4
during this public health emergency. Positive presumptive samples will be packaged
and sent to the U.S. Army Medical Research Institute of Infectious Diseases
(USAMRIID) or World Health Organization (WHO) laboratory network for
confirmatory or definitive analysis (e.g., sequencing, culture, etc...).
The FDA consulted with the Centers for Disease Control and Prevention (CDC) and
the National Institutes of Health (NIH) on the public health need for diagnostic
devices to detect E. coli O104:H4. The FDA’s conclusion that there is a current
public health need for such devices, i.e., that there is no adequate, approved, and
available alternative to the AAF PCR Assay is consistent with opinions expressed by
the CDC and the NIH.
I. APPROVAL/CLEARANCE STATUS:
The AAF PCR Assay is not cleared, Clinical Laboratory Improvement Amendments
(CLIA) waived, approved, or subject to an approved investigational device
exemption.
J. PRODUCT MANUFACTURING:
1.
The DoD CRP/Naval Medical Research Center (NMRC) will manufacture the
AAF PCR Assay. Assay production is in accordance with ISO Guide 34.
Material manufactured by CRP will be bottled and kitted by NMRC.
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2.
Components manufactured by CRP and supplied with the test include:
Table 3. AAF PCR Assay Components
3.
Components
Part Number
Quantity per vial
AAF master mix containing primers,
probe and reaction buffer
TBD
TBD
Platinum Taq Polymerase
Invitrogen
TBD
AAF Positive Control (AAF PTC)—
store at -20°C
TBD
TBD
Components required but not included with the test:
Table 4. AAF PCR Assay Required Equipment
Freezer, -20°C
Refrigerator, 4°C
Pipettes (ranges from 0.5 µl-1000 µl)
Vortexer (VWR 58810-163 or equivalent)
Qiagen EZ1
LC480
LC480 Software (Version 1.5 or later)
Class II Biosafety cabinet (or glove box)
Materials required but not provided in kit:
Table 5. AAF PCR Assay Required Materials
Personal protective equipment: Powder-free gloves, Lab coat, etc…
Tube racks, microcentrifuge tube racks
1.5 mL Nuclease-free microcentrifuge tubes
Assorted aerosol barrier filter, nuclease-free pipette tips
Molecular grade water (nuclease-free water)
DPBS
Qiagen EZ1 Virus Mini Kit v2.0 (Qiagen Cat. No. 955134)
LightCycler 480 96 well plates (Roche)
4.
The AAF PCR Assay has been validated using only the components
referenced above. The AAF PCR Assay was developed using primers and probes
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specific for E. coli O104:H4. The assays were designed to exclusively detect E.
coli O104:H4 in individuals at risk for exposure to E. coli or with signs and
symptoms of infection with E. coli. An array of primers and probes were
designed, ordered, and tested against reference DNA material produced at the
U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) from
the E. coli strain. Primer candidates were used to amplify 1 pg of target template
in the presence of SYBR green dye. Melt curves and agarose gels were analyzed
to eliminate inefficient and/or dimer producing primer pairs. The optimum primer
pair described was chosen because it yielded the highest amount of specific
amplification product in the absence of non-specific extraneous products, the
lowest Ct value and the highest end point fluorescence (EPF).
Stool samples (collected from patients) must first have nucleic acid extracted
using Qiagen EZ1 Virus Mini Kit v 2.0, purchased separately from the assay.
Extracted DNA samples are then run on the LC480 instrument using the master
mix provided in the kit.
A total of 46 samples can be run per LC480 plate in duplicate.
The DNA extraction of samples may take less than one hour, depending on the
number of samples being processed.
Sample set up and LC480 run will take approximately one hour.
Approximately 3 LC480 runs can be completed in a normal work day, resulting in
138 samples analyzed per instrument per day.
K. APPROVED/CLEARED ALTERNATIVE PRODUCTS
Currently, the FDA has not approved/cleared any methods for the detection of the E.
coli O104:H4.
L. INTERPRETATION OF RESULTS
All test controls should be examined prior to the interpretation of patient results. If
the controls are not valid, the patient results cannot be interpreted.
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1. AAF PCR Assay positive and negative controls:
Table 6. AAF PCR Assay Positive and Negative Controls
Expected Test Results
What do Results Mean?
E. coli
-
All Negative Template Controls should be (-). If (+)
reagents are contaminated. Obtain new reagents and
Repeat.
SNTC
-
Sample No Template Loading Control should be (-), if
(+) cross contamination occurred during loading.
Clean working area, reload samples and repeat.
AAF PTC
+
E. coli should be (+). If (-), run is invalid. Rerun. If
still (-), obtain new AAF kit.
Sample
?
Sample will be (+) for AAF for E. coli. Sample will
be (-) if E. coli AAF is not present.
RNTC
In order for a test run to be valid, all NTC reactions must be negative, based on
the instrument call. Control reactions should be interpreted only by the LC480
software, and should not be interpreted subjectively by the operator, based on
displayed amplification curves. If one or more NTC fails, the entire run is invalid
and potential sources of contamination should be identified and corrected. Re-test
the purified sample and controls, and re-analyze.
Failure to achieve a Positive AAF PTC reaction for any of the assays invalidates
the entire run. Obtain new AAF reagents and repeat the run.
2. Examination of Patient Specimen Results:
Unknown Sample Interpretation: When all controls meet the stated requirements
(above), the run is valid. A specimen is presumptively considered positive for E.
coli if the LC480 instrument call for the assay is positive and the specimen should
be sent for confirmatory testing. If one or more controls fail, the test is invalid and
must be repeated.
3. Interpretation of Results:
Interpretation of control and sample calls: The test is considered valid as long as
the control samples have the results identified in this table.
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Table 7. Control & Sample Call Interpretation
Controls
E. coli
RNTC/SNTC
-
Any failures or positive calls invalidate
the run. Re-test from purified sample.
AAF PTC
+
Any failures invalidate the run. Retest
from purified sample. If AAF PTC fails
again, obtain a NEW AAF kit.
Unknown
samples
+
If target assay comes up positive, then
sample is positive for E. coli O104:H4.
-
If a specimen result is negative, the
sample is then considered negative for E.
coli O104:H4.
Unknown
samples
Action
M. SAFETY AND EFFECTIVENES
1. Analytical Sensitivity:
Limit of Detection (LOD): Serial dilutions of nucleic acid purified from E. coli
obtained from the USAMRIID DNA reference panel were tested in triplicate.
The lowest concentration at which all replicates were positive was treated as the
tentative LOD for each test.
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E. coli
Table 8. Tentative LOD: AAF PCR Assay
DNA
Strain tested
quantity/Rxn
100pg
50pg
10pg
5pg
1pg
500fg
100fg
50fg
10fg
Call
rate
3/3
3/3
3/3
3/3
3/3
3/3
3/3
3/3
0/3
Run 1 Ct
25.94
27.03
30.25
31.58
33.71
34.93
36.62
38.71
No Ct
Run 2 Ct
25.94
27.03
30.09
31.12
33.28
34.97
38.03
38.04
No Ct
Run 3 Ct
25.92
27.01
30.3
31.26
33.70
34.60
37.06
38.12
No Ct
Analytical sensitivity LOD studies determine the lowest detectable concentration
of E. coli at which approximately 97% of all (true positive) replicates test
positive.
Table 9. Titers and Strains of Bacterial Stocks for LOD Study
Strain of bacterial
stock
E. coli (Germany) (SPL11.010.01.01, ESCH044)
How were stocks
prepared
Bacterial Stocks were cultured, placed on plastic beads and frozen
at -80 C until further use
The nucleic acid was extracted using Qiagen EZ1 Viral Mini Kit v 2.0.
Page 10 of 23
LOD Estimation
The LOD estimate was confirmed with 1 dilution (60 replicates each) at the initial
estimated LOD. The final LOD of each test was determined to be the lowest
concentration resulting in positive detection of 58 out of 60 replicates (97%). The
rfb LOD was determined with 59/60 positive results at 50fg.
Initial LOD Estimation in Matrix - E. coli Germany
For an initial estimate of LOD, live E. coli in log phase of growth was diluted in a
10 fold dilution series and was then spiked into stool samples. Individual
extracted samples were run in 3 replicates. Results closest to LOD can be found
in Table 10.
Table 10. E. coli O104:H4 Initial LOD Estimation
Assay
AAF
cfu/mL
Results
Ct
Mean Ct (n=3)
SD
33700
3/3
31.37
31.12
31.34
31.28
0.14
3370
3/3
33.71
33.84
34.11
33.89
0.20
380
3/3
38.30
37.24
38.32
37.95
0.62
30
1/3
40.00
x
x
x
x
Refined LOD Estimation—E. coli O104:H4
For a refinement of LOD, the bacteria was spiked into stool at two concentrations
based on LOD estimation (1680 and 3370 cfu/mL) and tested in quadruplicate.
Results from the refined LOD estimation can be found in Table 11. The AAF
assay detected 4/4 samples at 3370 cfu/mL and 4/4 at 1,680 cfu/mL. LOD was
confirmed at 1680 cfu/mL.
Table 11. E. coli O104:H4 Refined LOD Estimation
Assay
AAF
cfu/mL
Results
Ct
Mean Ct
(n=4)
SD
3370
4/4
33.67
33.75
34.55
34.41
34.10
0.45
1680
4/4
34.03
34.87
34.48
34.46
34.46
0.34
Confirmation of LOD Estimation-- E. coli AAF
For a refinement of LOD, the bacteria was spiked into human stool at one
concentration based on LOD estimation at (1680 cfu/mL) and tested with 30
replicates. Results from the refined LOD estimation can be found in Table 11.
The E. coli assay detected 30/30 samples at 1680 cfu/mL. LOD was confirmed at
1680 cfu/mL.
Page 11 of 23
Table 12. Confirmation of E. coli AAF LOD Estimation
Assay
E. coli
AAF
cfu/mL
Results
1680
30/30
Ct
34.03
34.83
34.05
35.02
34.63
34.25
34.11
34.54
34.34
35.67
33.85
34.67
33.88
34.42
36.13
34.35
34.11
34.86
37.19
36.61
36.55
36.66
36.04
34.15
34.24
34.00
33.57
34.52
34.12
34.86
Mean
Ct
(n=4)
SD
34.81
0.98
The Analytical Sensitivity was confirmed with 30 replicates in stool matrix to be
1680 cfu/mL.
2. Analytical Specificity
Bacterial Cross-Reactivity: Bacterial cross-reactivity of the AAF PCR
Assay was evaluated by testing purified nucleic acid of bacteria that
potentially could be infecting the majority of the population. No crossreactivity was observed in the human DNA or any of the bacteria tested
(Table 13).
Table 13. Bacteria Cross Reactivity: E. coli AAF real-time PCR Assay
Purified DNA from Bacteria
Concentration
(pg DNA/Rxn)
Acinetobacter baumanni
100
Genomic
Equivalents
per Reaction
2.5e04
Actinomyces naeslundii
100
Actinobacillus pleuropneumoniae
100
Aeromonas hydrophila
100
Alcaligenes xylosoxidans
100
Bacillus anthracis
Bacillus (Geobacillus)
stearothermophilus
Bacillus thuringiensis
Concentration
(pg/mL)
Ct
Value
2.0e04
N/D
3.5e04
2.0e04
N/D
4.1e04
2.0e04
N/D
2.0e04
N/D
1.0e05
2.0e04
N/D
100
5.4e05
2.0e04
N/D
100
5.2e08
2.0e04
100
8.9e05
2.0e04
N/D
Bacillus subtilis var niger
100
2.4e04
2.0e04
N/D
Bacillus cereus
100
1.9e04
2.0e04
N/D
Bacteroides fragilis
100
1.8e04
2.0e04
N/D
Bartonella henselae
100
5.1e04
2.0e04
N/D
Bifidobacterium infantis
100
3.5e04
2.0e04
N/D
Bordetella bronchiseptica
100
1.8e04
2.0e04
N/D
Bordetella pertussis
100
2.4e04
2.0e04
N/D
Borrelia burgdorferi
100
1.1e05
2.0e04
N/D
Brucella melitensis
100
8.2e04
2.0e04
N/D
N/D
Page 12 of 23
Purified DNA from Bacteria
Concentration
(pg DNA/Rxn)
Budvicia aquatica
100
Genomic
Equivalents
per Reaction
3.0e04
Burkholderia mallei
100
Campylobacter jejuni
Concentration
(pg/mL)
Ct
Value
2.0e04
N/D
2.8e04
2.0e04
N/D
100
5.8e04
2.0e04
N/D
Candida albicans
100
1.0e05
2.0e04
N/D
Chryseobacterium meningosepticum
100
1.8e04
2.0e04
N/D
Clostridium botulinum type A
100
2.4e04
2.0e04
N/D
Clostridium perfringens
100
1.4e06
2.0e04
N/D
Clostridium difficile
100
2.3e04
2.0e04
N/D
Comamonas (Delftia) acidovorans
100
1.4e04
2.0e04
N/D
Corynebacterium diphtheriae
100
3.9e04
2.0e04
N/D
Coxiella burnetii
100
4.9e04
2.0e04
N/D
Deinococcus radiodurans
100
2.4e05
2.0e04
N/D
Enterobacter aerogenes
100
1.9e04
2.0e04
N/D
Enterobacter (Paoea) agglomerans
100
2.1e04
2.0e04
N/D
Enterococcus faecalis
Escherichia coli (serotype not
O104:H4)
Francisella tularensis
100
3.3e04
2.0e04
N/D
100
1.8e04
2.0e04
100
4.8e04
2.0e04
N/D
Haemophilus influenzae
100
5.4e04
2.0e04
N/D
Klebsiella pneumoniae
100
1.7e04
2.0e04
N/D
Legionella pneumophila
100
2.9e04
2.0e04
N/D
Listeria monocytogenes
100
3.3e04
2.0e04
N/D
Moraxella catarrhalis
100
5.2e04
2.0e04
N/D
Mycoplasma pneumoniae
100
1.2e05
2.0e04
N/D
Neisseria meningitidis
100
4.6e04
2.0e04
N/D
Pasteurella multocida
100
4.3e04
2.0e04
N/D
Plesiomonas hydrophila
100
2.0e04
N/D
Porphyromonas gingivalis
100
4.2e04
2.0e04
N/D
Proteus mirabilis
100
2.4e04
2.0e04
N/D
Providencii stuartii
100
2.0e04
N/D
Pseudomonas aeruginosa
100
1.6e04
2.0e04
N/D
Rhizobium radiobacter
Salmonella choleraesuis serovar
paratyphi
Salmonella enterica serovar
typhimurium
Serratia marcescens
100
2.4e04
2.0e04
N/D
100
2.0e04
2.0e04
N/D
2.0e04
N/D
100
1.8e04
2.0e04
N/D
Shigella flexneri
100
2.1e04
2.0e04
N/D
Staphylococcus epidermidis
100
3.7e04
2.0e04
N/D
Staphylococcus aureus
100
3.4e04
2.0e04
N/D
100
N/D
Page 13 of 23
Purified DNA from Bacteria
Concentration
(pg DNA/Rxn)
Stenotrophomonas maltophilia
100
Genomic
Equivalents
per Reaction
2.1e04
Streptococcus pyogenes
100
Streptococcus pneumoniae
Concentration
(pg/mL)
Ct
Value
2.0e04
N/D
5.1e04
2.0e04
N/D
100
4.8e04
2.0e04
N/D
Ureaplasma parvum (urealyticum)
100
1.3e05
2.0e04
N/D
Vibrio cholerae
100
3.1e04
2.0e04
N/D
Yersinia pseudotuberculosis
100
2.1e04
2.0e04
N/D
Yersinia enterocolitica
100
2.1e04
2.0e04
N/D
Yersinia pestis (CO92;PW)
100
2.1e04
2.0e04
N/D
Human DNA Cross-Reactivity: Human DNA cross-reactivity of the EZ1 PCR Assay was
evaluated by testing purified human DNA at a concentration of 100 pg (31 human genome
equivalents) per reaction. No cross-reactivity with human DNA was observed (Table 13).
Table 13. Human DNA Cross Reactivity: E. coli AAF PCR Assay
Purified DNA from Human
Human DNA
Concentration
(pg/Rxn)
100
Genomic
Equivalents
per Reaction
31
Concentration
(pg/mL)
Ct
Value
2.0e04
N/D
The AAF assay was tested against human DNA, E. coli not O104:H4, as well as 61 other
bacterial DNA samples with no cross reactivity.
Page 14 of 23
2. Clinical Studies
LC480 Mock Clinical Trial for Inactivated E. coli Spiked into Stool
Results
Mock Clinical Study. Data from the limit of detection (LOD) study confirmed
that the LOD of the AAF assay for E. coli was 1680 cfu/mL. The Pre-EUA E.
coli (Target 1) Real-Time PCR Assay (TaqMan)(AAF PCR) performance
characteristics were established using contrived specimens in a randomized
single-blind trial comprising negative and positive samples. Samples were
spiked accordingly by persons separate from testing personnel. Briefly, 45
independent stool specimens were spiked with E. coli at the concentrations shown
on Table 14, relating to fifteen samples each of 1.5x and 3.0x LOD as well as a
high concentration sample (MAX). The cfu concentration of bacteria was
determined prior to dilution. Another 100 stool specimens were spiked with
DPBS alone. Operators were blind to the identities of the samples. All blinded
spiked specimens were extracted using the Qiagen EZ1 Viral Mini Kit, and
analyzed on the LC480 instrument using the E. coli AAF assay kit, according to
instructions in the product insert. The blinded spiking key was unmasked after
the results were complete. The results of the analysis are presented in Table 15.
Summary statistics from the mock clinical study are presented in Table 16.
Page 15 of 23
Table 14: Blinded Clinical Study: E. coli AAF Real-time PCR Assay using the LC480
Analyzer
Target
AAF
Ct
values
Agreement
with Key
1
N/D
Yes
2
N/D
Yes
3
N/D
Yes
4
N/D
Yes
5
N/D
Yes
40
Yes
7
N/D
Yes
8
N/D
Yes
Sample Key
6
3.0x LOD
Bacteria
concentration
cfu/mL
5070
AAF
9
3.0x LOD
5070
33.37
Yes
10
1.5x LOD
2540
33.33
Yes
11
N/D
Yes
12
N/D
Yes
13
N/D
Yes
14
N/D
Yes
15
3.0x LOD
5070
34.24
Yes
16
3.0x LOD
5070
35.89
Yes
17
MAX
100000
23.64
Yes
18
N/D
Yes
19
N/D
Yes
20
N/D
Yes
21
N/D
Yes
32.47
Yes
23
N/D
Yes
24
N/D
Yes
25
N/D
Yes
26
N/D
Yes
27
N/D
Yes
28
N/D
Yes
29
N/D
Yes
34.15
Yes
N/D
Yes
34.02
Yes
N/D
Yes
22
30
3.0x LOD
1.5x LOD
5070
2540
31
32
33
3.0x LOD
5070
Page 16 of 23
34
N/D
Yes
35
N/D
Yes
36
1.5x LOD
2540
33.34
Yes
37
3.0x LOD
5070
34.98
Yes
38
1.5x LOD
2504
33.79
Yes
39
N/D
Yes
40
N/D
Yes
41
N/D
Yes
27.62
Yes
43
N/D
Yes
44
N/D
Yes
23.54
Yes
N/D
Yes
42
45
MAX
MAX
100000
100000
46
47
3.0x LOD
5070
32.01
Yes
48
MAX
100000
23.95
Yes
49
N/D
Yes
50
N/D
Yes
51
N/D
Yes
52
N/D
Yes
32.58
Yes
54
N/D
Yes
55
N/D
Yes
53
3.0x LOD
5070
56
1.5x LOD
2540
36.05
Yes
57
1.5x LOD
2540
33.57
Yes
58
N/D
Yes
59
N/D
Yes
60
3.0x LOD
5070
34.89
Yes
61
1.5x LOD
2540
36.71
Yes
62
N/D
Yes
63
N/D
Yes
64
N/D
Yes
65
N/D
Yes
66
N/D
Yes
67
N/D
Yes
68
N/D
Yes
69
N/D
Yes
70
N/D
Yes
71
N/D
Yes
Page 17 of 23
72
N/D
Yes
73
N/D
Yes
74
N/D
Yes
34.27
Yes
76
N/D
Yes
77
N/D
Yes
25.57
Yes
79
N/D
Yes
80
N/D
Yes
81
N/D
Yes
75
78
1.5x LOD
MAX
2540
100000
82
1.5x LOD
2540
33.60
Yes
83
MAX
100000
23.80
Yes
84
N/D
Yes
85
N/D
Yes
86
N/D
Yes
24.63
Yes
N/D
Yes
35.58
Yes
90
N/D
Yes
91
N/D
Yes
34.98
Yes
93
N/D
Yes
94
N/D
Yes
95
N/D
Yes
96
N/D
Yes
97
N/D
Yes
98
N/D
Yes
99
N/D
Yes
24.89
Yes
101
N/D
Yes
102
N/D
Yes
103
N/D
Yes
104
N/D
Yes
N/D
NO
N/D
Yes
87
MAX
100000
88
89
92
100
105
1.5x LOD
3.0x LOD
MAX
1.5x LOD
2540
5070
100000
2540
106
107
1.5x LOD
2540
36.71
Yes
108
3.0x LOD
5070
37.36
Yes
N/D
Yes
109
Page 18 of 23
110
N/D
Yes
111
N/D
Yes
112
N/D
Yes
113
N/D
Yes
114
N/D
Yes
115
1.5x LOD
2540
38.10
Yes
116
3.0x LOD
5070
36.29
Yes
117
N/D
Yes
118
N/D
Yes
119
N/D
Yes
120
N/D
Yes
121
N/D
Yes
122
3.0x LOD
5070
35.93
Yes
123
1.5x LOD
2540
33.84
Yes
124
MAX
100000
23.43
Yes
125
N/D
Yes
126
N/D
Yes
127
N/D
Yes
128
N/D
Yes
129
MAX
100000
23.63
Yes
130
3.0x LOD
5070
32.04
Yes
131
N/D
Yes
132
N/D
Yes
29.10
Yes
N/D
Yes
36.52
Yes
N/D
Yes
24.79
Yes
N/D
Yes
24.92
Yes
N/D
Yes
27.00
Yes
142
N/D
Yes
143
N/D
Yes
144
N/D
Yes
25.50
Yes
133
MAX
100000
134
135
1.5x LOD
2540
136
137
MAX
100000
138
139
MAX
100000
140
141
145
MAX
MAX
100000
100000
Page 19 of 23
Table 15. Mock Clinical Trial Summary Statistics
Inactivated E. coli Spiked Samples
E. coli AAF Real-time PCR Detection on the
LC480 Instrument
Positive
Negative
Total
Comparator Assays
Positive
Negative
44
0
44
1
100
101
Total
45
100
145
95% CI
Positive Percent Agreement
Negative Percent Agreement
44/45
100/100
97.8%
100.0%
96.3-98.5%
99.3-100%
Page 20 of 23
Conclusion
The AAF assay correctly identified 44 of 45 specimens (97.8%) spiked with E.
coli at the concentrations shown. All negative samples, 100 of the 100 (100%)
were correctly identified as negative for E. coli.
N. RISKS AND BENEFITS:
Risks
The AAF PCR Assay has been designed to try to minimize the likelihood of false test
results. However, should false results occur, they may present risks to individuals:
False positive result
o
A false positive result in the context of the current public health
emergency could lead to misallocation of resources used for surveillance and
prevention.
False negative result:
o
Although a negative result does not rule out E. coli O104:H4 infection,
a false negative has the potential to result in a delay of providing or lack of
supportive care.
o
A false negative can also result in a waste of health care resources, as
additional evaluations are pursued in the effort to establish the true diagnosis.
Benefits
The chief benefit associated with the AAF PCR Assay is the continued testing of
human specimens for E. coli O104:H4.
True positive results provide presumptive support for the diagnosis of an E. coli
O104:H4 infection. It establishes the E. coli as the true cause of the individual’s
symptoms, prevents further workup for other possible causes and saves healthcare
resources.
The test additionally benefits overall public health. If people infected with the E. coli
O104:H4 are immediately isolated from other people and appropriate precautions
are taken, this can prevent others from getting sick. By having this test done, the
chance of spreading the bacteria to others is reduced. Also, using the test can help
healthcare providers learn more about E. coli O104:H4 and stop its spread. From
an epidemiological standpoint, a true positive result can aid in determining the
food source of the infection to prevent further spread.
Page 21 of 23
True negative results benefit both physicians and individuals by ruling out a diagnosis
and allowing other possible illness testing to be pursued.
Risk Assessment
Potential risk for sample contamination that could result in a false positive can be
mitigated by the use of controls and strictly adhering to good laboratory practices
and SOPs.
Risk of individual discomfort or harm during sample collection is minimal.
Negative results may not preclude an E. coli O104:H4 infection. It is emphasized that
the AAF PCR test should not be used as the sole basis for treatment or other
patient management decisions. The clinical features of the illness and the type
and risk of exposure must be considered pivotal to making patient management
and isolation decisions. A negative test should not be interpreted as definitive for
the individual not having an E. coli O104:H4 infection.
Risk-Benefit Assessment
Results from validation testing thus far seem to indicate that the AAF PCR Assay can
meet the current need for E. coli testing by providing dependable results from human
specimens that are positive (or presumptively positive, as appropriate) for E. coli
O104:H4.
Thus, it is expected that the risk of false positive or negative results with the AAF
PCR Assay have been mitigated by demonstrating analytical performance (analytical
sensitivity and specificity) and performance with contrived clinical specimens
consisting of E. coli spiked into stool.
The risks posed by use of the AAF PCR Assay are mitigated by:
Limited distribution to DoD qualified laboratories.
Oversight of public health experts in emergency response activities.
Use of the AAF PCR Assay in conjunction with other laboratory, epidemiological,
and clinical evaluation tools.
Based on these factors, the potential benefits from the use of the AAF PCR Assay are
expected to outweigh the risks.
O. FACT SHEET FOR HEALTHCARE PROVIDERS AND PATIENTS:
See Attachment 1 and 2.
P. INSTRUCTIONS FOR USE/PROPOSED LABELING/PACKAGE INSERT:
See Attachment 3.
Page 22 of 23
Q. RECORD KEEPING AND REPORTING INFORMATION TO FDA:
The Joint JPMO/CBMS will distribute the reagents to qualified DoD laboratories that
have PCR capability on the LC480 instrument to test stool specimens. Final results
will be reported to the healthcare provider. The submitting health care provider will
be responsible for communicating test results to the patient with the appropriate
interpretation.
Healthcare providers and patients will be requested to report adverse device events to
the FDA via Medwatch.
Page 23 of 23