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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 Page 1 of 23 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) Page 2 of 23 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 Page 3 of 23 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. Page 4 of 23 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. Page 5 of 23 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 Page 6 of 23 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. Page 7 of 23 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. Page 8 of 23 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. Page 9 of 23 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