Predicate Devices

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The description focuses on a qualitative nucleic acid-based test using PCR and analysis software, with no mention of AI or ML in the device description, intended use, or performance studies.

Therapeutic

No
This device is an in vitro diagnostic test designed to detect antibiotic resistance markers, not to treat or cure a disease or condition.

Diagnostic

Yes

The "Intended Use / Indications for Use" section explicitly states that the Acuitas® AMR Gene Panel is a "qualitative nucleic acid-based multiplex in vitro diagnostic test for detection and differentiation of antibiotic resistance markers." The text further clarifies that the results "aid in the identification of potentially antimicrobial-resistant organisms" and "may be used as an aid to clinicians in the management of patients with known or suspected antibiotic non-susceptible or resistant bacterial infections." These statements clearly indicate the device's diagnostic purpose.

SaMD (Software as a Medical Device)

No

The device description explicitly states that the Acuitas® AMR Gene Panel is an in vitro diagnostic test kit containing necessary reagents for PCR and detection, and it utilizes hardware components like the QIAGEN® EZ1® Advanced XL System and the OpGen Qualified QuantStudio™ 5 Real-Time PCR System. While it includes software for data analysis, it is not solely software.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

The "Intended Use / Indications for Use" section explicitly states: "The Acuitas® AMR Gene Panel... is a qualitative nucleic acid-based multiplex in vitro diagnostic test for detection and differentiation of antibiotic resistance markers..."

The "Device Description" section also refers to it as a "qualitative nucleic acid-based in vitro diagnostic test".

Furthermore, the description of the OpGen Qualified QuantStudio 5 Real-Time PCR System states that it "is a component of the Acuitas AMR Gene Panel assay and is cleared for in vitro diagnostic use only with the Acuitas AMR Gene Panel and not for any other application."

These statements clearly indicate that the device is intended for use in vitro (outside of a living organism) for diagnostic purposes.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The Acuitas® AMR Gene Panel, performed on the QIAGEN® EZ1® Advanced XL System and the OpGen Qualified QuantStudio™ 5 Real-Time PCR System, is a qualitative nucleic acid-based multiplex in vitro diagnostic test for detection and differentiation of antibiotic resistance markers to one or more antimicrobial agents. The test utilizes realtime polymerase chain reaction (PCR) and is performed on isolated colonies of Pseudomonas aeruginosa. Enteroooccus faecalis, or members of Enterobacterales grown in pure culture on blood agar or MacConkey agar.

Organism identification results must be available prior to reporting results for the Acuitas AMR Gene Panel. Antimicrobial resistance gene results are reported by the Acuitas AMR Gene Panel for the combinations of bacterial pathogens and associated genetic resistance markers indicated in Table 1 below.

Product codes (comma separated list FDA assigned to the subject device)

PMY, OOI

Device Description

The Acuitas® AMR Gene Panel is a qualitative nucleic acid-based in vitro diagnostic test capable of simultaneous detection and identification of select genetic determinants of antimicrobial resistance (AMR) in isolated bacterial colonies grown on blood agar or MacConkey agar. The test detects twenty-eight (28) genetic determinants of resistance to the following nine (9) antibiotic classes: aminoglycosides, carbapenems, cephalosporins, fluoroquinolones, penicillins, polymyxins, sulfonamides, trimethoprim and vancomycin. The assay is performed on pure colonies of Enterobacterales and Pseudomonas aeruginosa grown on blood agar or MacConkey agar along with Enterococcus faecalis grown on blood agar.

The Acuitas AMR Gene Panel kit contains all of the necessary reagents for PCR and detection in order to amplify and detect DNA from pure colonies of Enterobacterales (Citrobacter freundii complex (Citrobacter braakii, Citrobacter freundii, Citrobacter werkmanii, Citrobacter youngae), Citrobacter koseri, Enterobacter cloacae complex (Enterobacter asburiae, Enterobacter cloacae, Enterobacter hormaechei, Enterobacter kobei, Enterobacter ludwigii), Escherichia coli, Klebsiella pneumoniae, Klebsiella quasipneumoniae, Klebsiella aerogenes, Klebsiella michiganensis, Klebsiella oxytoca, Klebsiella variicola, Morganii, Proteus mirabilis, Providencia rettgeri, Providencia stuartii, Raoultella ornithinolytica, Raoultella planticola, Serratia marcescens) and Pseudomonas aeruginosa grown on blood agar or MacConkey agar, as well as Enterococcus faecalis grown on blood agar from clinical specimens. The test kit includes PCR plates (96-well) with dried primers and probes for analysis of four (4) isolates (24 wells per isolate).

The Acuitas AMR Gene Panel assay employs automated deoxyribonucleic acid (DNA) extraction on the QIAGEN® EZ1® Advanced XL System and multiplex real-time PCR on an OpGen Qualified Applied Biosystems™ QuantStudio™ 5 Real-Time PCR System ("OpGen Qualified QuantStudio 5") for use with the Acuitas AMR Gene Panel. The QIAGEN EZ1 DSP Virus Kit has been selected for use as the sample preparation method for the Acuitas AMR Gene Panel test.

After colony isolation, the user prepares a 0.5 McFarland suspension for each bacterial isolate and performs DNA extraction. DNA is extracted on the QIAGEN EZ1 Advanced XL System according to manufacturer instructions incorporating the Assay Control within the extraction process. A sample of extracted DNA eluate is transferred to a Reagent Reservoir trough to which Acuitas AMR Gene Panel Master Mix is added. Extracted DNA/Master Mix is transferred to each of 24 wells on the Acuitas AMR Gene Panel PCR plate per test sample. The contents of each well are mixed, and the plate is sealed and transferred to the OpGen Qualified QuantStudio 5 for use with the Acuitas AMR Gene Panel for real-time multiplex reaction and detection using the Acuitas AMR Gene Panel PCR Template File.

Data are exported from the OpGen Qualified QuantStudio 5 and imported into the Acuitas AMR Gene Analysis Software, a spreadsheet application that analyzes the data and generates a report for viewing and printing. Each test report indicates detection of applicable antimicrobial resistance gene variants as "Detected", "Not Detected" or "NA/NR".

The Applied Biosystems QuantStudio 5 Real-Time PCR System is not intended for clinical diagnostic purposes. The OpGen Qualified QuantStudio 5 for use with the Acuitas AMR Gene Panel is a component of the Acuitas AMR Gene Panel assay and is cleared for in vitro diagnostic use only with the Acuitas AMR Gene Panel and not for any other application. The OpGen Qualified QuantStudio 5 for use with the Acuitas AMR Gene Panel may only be used with the Acuitas AMR Gene Panel after the instrument has been qualified for use by OpGen, Inc.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Not Found

Indicated Patient Age Range

Not Found

Intended User / Care Setting

Not Found

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

Reproducibility: A panel of 300 uniquely labeled samples composed of ten (10) unique isolates. The panel was provided to three (3) testing sites, one (1) of which was OpGen. The panel was rotated across two (2) operators, two (2) OpGen Qualified QuantStudio 5 instruments and one (1) EZ1 Instrument per site over 20 days. Three (3) unique lots of all materials were used and rotated at each testing site.

Analytical Reactivity (Inclusivity): Analytical sensitivity was evaluated by testing a panel of two hundred and ninety-eight (298) isolates covering all antimicrobial resistance genes. Each isolate in the test panel was tested once, except for isolates harboring rare resistance genes, which were replicated to achieve at least 18 positive data points per gene target assessed. The Acuitas AMR Gene Panel results were compared with species identification by well-established automated species identification methods and AMR gene detection by Whole Genome Sequencing (WGS).

Analytical Specificity (Cross-Reactivity): Evaluated for a total of four hundred and twenty-three (423) isolates. One hundred twenty-five (125) isolates were tested in duplicate from a single 0.5 McFarland bacterial suspension. Additionally, cross-reactivity at the AMR gene level was evaluated for two-hundred and ninety-eight (298) isolates from the Analytical Reactivity (Inclusivity) Study. Results were compared with species identification by well-established automated species identification methods and AMR gene detection by Whole Genome Sequencing.

Fresh versus Frozen: A panel of nine (9) isolates covering all detected species and majority of the antimicrobial resistance genes in the Acuitas AMR Gene Panel. Each of the study isolates was cultured on blood agar from which 0.5 McFarland suspensions were prepared and aliquoted. 500 µL 0.5 McFarland aliquots were prepared for each isolate and each positive and negative external control. Positive and negatives controls prepared on Day 1 of T=0 testing were used throughout the duration of study. Ten (10) replicates of each isolate were evaluated at each of two testing events (i.e., T=0, T=56). Testing for each timepoint was split across three (3) days, with each of three (3) operators testing ten (10) replicates of a single isolate, one (1) positive control and one (1) negative control on each day of testing.

Sample Stability - Extracted DNA: Five (5) isolates harboring most of the AMR genes detected by the Acuitas AMR Gene Panel. These were tested by one operator across two (2) OpGen Qualified QuantStudio 5 instruments. Three (3) replicates of each isolate were evaluated for each testing event. The isolates were extracted and stored under various conditions (T=0, T=6 hours at 15-25 °C, T=6 hours at 15-25 °C then 7 days at 2-8 °C, T=7 days at 2-8 °C). Five (5) isolates were cultured on blood agar. Fourteen (14) 0.5 McFarland suspensions per isolate were prepared and DNA extracts per isolate were pooled and aliquoted (500 µL) for storage. Three (3) 150µL replicates of extracted DNA were evaluated per isolate per storage condition.

Sample Stability - Frozen DNA: Five (5) isolates harboring most of the AMR genes detected by the Acuitas AMR Gene Panel. These were tested by one (1) operator across two (2) OpGen Qualified QuantStudio 5 instruments. Three (3) replicates of each isolate were evaluated for each testing event. Fifteen (15) 0.5 McFarland suspensions were prepared per isolate and DNA extracts per each isolate were pooled and aliquoted into unique 150 µL aliquots. Three (3) DNA aliquots per isolate were tested immediately (T=0). Remaining aliquots were stored at -15 to -25 °C for 14 or 30 days before testing.

Sample Stability - Plated DNA: Five (5) isolates harboring most of the AMR genes detected by the Acuitas AMR Gene Panel. These were tested by one (1) operator across two (2) OpGen Qualified QuantStudio 5 instruments. Three (3) replicates of each isolate were evaluated for each testing event. Seven (7) 0.5 McFarland suspensions were prepared and extracted per isolate. Extracts per isolate were pooled and aliquoted (500 µL). Extracted DNA samples (150 µL) were combined with Acuitas AMR Gene Panel Master Mix (CP3402) and added to Acuitas AMR Gene Panel assay plates (CP3230). Prepared plates were stored for zero or 6 hours at 2-8 °C and tested.

Media Equivalency: Fifty-two (52) isolates harboring most of the AMR genes detected by the Acuitas AMR Gene Panel. Thirteen (13) isolates per organism (E. coli, K. pneumoniae, P. aeruginosa, and P. mirabilis) were cultured on both blood agar and MacConkey agar. E. faecalis was not tested as it does not grow on MacConkey agar. A 0.5 McFarland suspension was prepared from pure colonies on the blood and MacConkey agar for testing with the Acuitas AMR Gene Panel.

Carry-over/Cross-Contamination: A panel of twelve (12) isolates representing organisms targeted by the Acuitas AMR Gene Panel test, harboring a variety of the targeted resistance genes, and a negative control isolate. Two (2) operators prepared and blinded a panel of six (6) positive isolates and six (6) negative control isolates (a S. aureus isolate negative for all resistance markers) for testing by the other operator. Isolates were cultured on blood agar, 0.5 McFarland suspensions were prepared, and DNA was extracted. Positive and negative control samples were tested in an alternating fashion on the Acuitas AMR Gene Panel assay plates.

Clinical Performance: A multi-site investigational clinical study using bacterial isolates. Four geographically diverse sites participated in the testing of isolates either prospectively collected or stocked and de-identified. The study included 1,307 isolate samples (1,224 clinical stock isolate samples and 83 prospective isolate samples). Isolates harboring rare resistance genes were replicated with a goal of achieving 50 unique testing events per gene target. Each of the 83 prospective isolate samples were unique and enrolled at clinical sites. Isolates enrolled were selected based on identification (Enterobacterales, Pseudomonas aeruginosa, and Enterococcus faecalis) and documented non-susceptibility to at least one antibiotic class. For testing, well-isolated colonies grown on blood agar were diluted to a 0.5 McFarland standard equivalent suspension. Reference methods included organism identification by automated methods (for Gram-negative) or MALDI-TOF MS (for Gram-positive), Antimicrobial Susceptibility Testing (AST) by broth microdilution, and Whole Genome Sequencing (WGS).

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Reproducibility:

Study Type: Reproducibility study
Sample Size: 300 uniquely labeled samples, 10 unique isolates.
Key Results: Overall agreement per sample (All Sites) ranged from 96% to 100%. Total agreement ranged from 97.8% to 100% for detected results and 99.4% to 100% for not detected results across individual assay targets.

Analytical Reactivity (Inclusivity):

Study Type: Analytical Sensitivity (Inclusivity) study
Sample Size: 298 isolates.
Key Results: Data showed detected and not detected status for various AMR genes. Also indicated other AMR gene variants predicted to be detected or likely/potentially detectable by Acuitas AMR Gene Panel based on in silico analysis.

Analytical Specificity (Cross-Reactivity):

Study Type: Analytical Specificity (Cross-Reactivity) study
Sample Size: 423 isolates (125 in duplicate, 298 from Inclusivity study).
Key Results: Determined organisms without cross-reactivity and those demonstrating cross-reactivity (Shigella species with E. coli ID assay). No cross-reactivity observed with genes/variants not targeted by the panel.

Fresh versus Frozen:

Study Type: Sample Stability (Fresh vs. Frozen) study
Sample Size: 9 isolates, 10 replicates per isolate for each testing event.
Key Results: 100% agreement observed between T=0 and frozen T=56 time points. Some initial discrepancies were resolved upon re-testing.

Sample Stability - Extracted DNA:

Study Type: Sample Stability (Extracted DNA) study
Sample Size: 5 isolates, 3 replicates per isolate for each testing event.
Key Results: 100% agreement for all samples and storage conditions compared with the T=0 timepoint, supporting claims for extracted DNA storage: up to 6 hours at room temperature; up to 7 days at 2-8 °C; up to 6 hours at room temperature followed by up to 7 days at 2-8 °C.

Sample Stability - Frozen DNA:

Study Type: Sample Stability (Frozen DNA) study
Sample Size: 5 isolates, 3 replicates per isolate for each testing event.
Key Results: 100% agreement between the T=0 timepoint and tested storage conditions, supporting claims for frozen DNA storage up to 30 days at -15 °C to -25 °C.

Sample Stability - Plated DNA:

Study Type: Sample Stability (Plated DNA) study
Sample Size: 5 isolates, 3 replicates per isolate for each testing event.
Key Results: 100% agreement between T=0 and T=6 hours at 2-8 °C, supporting stability of prepared plates when stored for 6 hours at 2-8 °C.

Media Equivalency:

Study Type: Media Equivalency study
Sample Size: 52 isolates comprising E. coli, K. pneumoniae, P. aeruginosa, and P. mirabilis.
Key Results: Percent agreement for detection of AMR genes by Acuitas AMR Gene Panel versus Whole Genome Sequencing for each isolate replicate from blood and MacConkey agar. The study did not uncover evidence of a media effect between blood and MacConkey agar. Initial isolate agreement ranged from 62% to 92% across the four organisms, with improvements to 100% in most cases after retests and adjudication.

Carry-over/Cross-Contamination:

Study Type: Carry-over/Cross-Contamination study
Sample Size: 12 isolates (6 positive, 6 negative controls).
Key Results: 100% agreement between observed and expected positive/negative results without evidence of carry-over/cross-contamination.

Clinical Performance:

Study Type: Multi-site investigational clinical study
Sample Size: 1,307 isolate samples (1,224 clinical stock, 83 prospective).
Key Results: Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) for AMR genes ranged from 94.4% to 100% and 96.5% to 100%, respectively.

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

Clinical Performance Data (PCR/WGS):

AAC: PPA 98.1% (610/622), NPA 98.3% (536/545)
AAD: PPA 98.5% (128/130), NPA 96.5% (192/199)
ANT: PPA 99.0% (203/205), NPA 99.2% (628/633)
APH: PPA 97.5% (39/40), NPA 99.8% (443/444)
armA: PPA 100.0% (8/8), NPA 100.0% (147/147)
CMY: PPA 98.4% (126/128), NPA 99.6% (688/691)
CTX-M-1: PPA 96.7% (264/273), NPA 99.0% (929/938)
CTX-M-2: PPA 100.0% (35/35), NPA 99.8% (801/803)
CTX-M-9: PPA 98.6% (73/74), NPA 99.9% (781/782)
DFR: PPA 98.8% (167/169), NPA 99.4% (646/650)
DHA: PPA 100.0% (36/36), NPA 100.0% (293/293)
E. coli gyrA Mutant: PPA 98.2% (160/163), NPA 99.4% (167/168)
IMP: PPA 100.0% (72/72), NPA 100.0% (257/257)
KPC: PPA 97.4% (75/77), NPA 99.6% (1130/1134)
MCR-1: PPA 94.4% (51/54), NPA 100.0% (281/281)
NDM: PPA 98.2% (56/57), NPA 99.5% (801/805)
OXA-1: PPA 96.4% (240/249), NPA 99.1% (910/918)
OXA-9: PPA 100.0% (58/58), NPA 99.9% (760/761)
OXA-48: PPA 95.2% (59/62), NPA 99.1% (448/452)
PER: PPA 98.8% (81/82), NPA 99.6% (265/266)
P. aeruginosa gyrA Mutant: PPA 95.0% (265/279), NPA 98.5% (67/68)
RMT: PPA 96.9% (31/32), NPA 100.0% (297/297)
SHV: PPA 100.0% (12/12), NPA 99.6% (668/671)
Sul1: PPA 99.1% (420/424), NPA 96.7% (232/240)
Sul2: PPA 97.6% (489/501), NPA 96.5% (307/318)
TEM: PPA 98.5% (600/609), NPA 97.7% (559/572)
vanA: PPA 100.0% (57/57), NPA 100.0% (36/36)
VEB: PPA 100.0% (89/89), NPA 99.3% (411/414)
VIM: PPA 97.8% (91/93), NPA 99.8% (409/410)

Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.

K152614

Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).

Not Found

Regulation Number and Section

§ 866.1640 Antimicrobial susceptibility test powder.

(a)
Identification. An antimicrobial susceptibility test powder is a device that consists of an antimicrobial drug powder packaged in vials in specified amounts and intended for use in clinical laboratories for determining in vitro susceptibility of bacterial pathogens to these therapeutic agents. Test results are used to determine the antimicrobial agent of choice in the treatment of bacterial diseases.(b)
Classification. Class II (performance standards).

Summary

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Image /page/0/Picture/0 description: The image contains the logo of the U.S. Food and Drug Administration (FDA). The logo consists of two parts: the Department of Health & Human Services logo on the left and the FDA acronym with the full name of the agency on the right. The FDA part of the logo is in blue, with the acronym in a square and the full name written out to the right of the square.

September 30, 2021

OpGen, Inc. % Randy Prebula Partner; Hogan Lovells Hogan Lovells, US LLP Columbia Square 555 Thirteenth Street, NW Washington, District of Columbia 20004

Re: K191288

Trade/Device Name: Acuitas AMR Gene Panel Regulation Number: 21 CFR 866.1640 Regulation Name: Antimicrobial Susceptibility Test Powder Regulatory Class: Class II Product Code: PMY, OOI Dated: October 13, 2020 Received: October 13, 2020

Dear Randy Prebula:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal

1

statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (QS)

regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely,

Ribhi Shawar, Ph.D. (ABMM) Chief General Bacteriology and Antimicrobial Susceptibility Branch Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics and Radiological Health Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K191288

Device Name Acuitas AMR Gene Panel

Indications for Use (Describe)

The Acuitas® AMR Gene Panel, performed on the QIAGEN® EZ1® Advanced XL System and the OpGen Qualified QuantStudio™ 5 Real-Time PCR System, is a qualitative nucleic acid-based multiplex in vitro diagnostic test for detection and differentiation of antibiotic resistance markers to one or more antimicrobial agents. The test utilizes realtime polymerase chain reaction (PCR) and is performed on isolated colonies of Pseudomonas aeruginosa. Enteroooccus faecalis, or members of Enterobacterales grown in pure culture on blood agar or MacConkey agar.

Organism identification results must be available prior to reporting results for the Acuitas AMR Gene Panel. Antimicrobial resistance gene results are reported by the Acuitas AMR Gene Panel for the combinations of bacterial pathogens and associated genetic resistance markers indicated in Table 1 below.

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Type of Use (Select one or both, as applicable)
-------------------------------------------------

X Prescription Use (Part 21 CFR 801 Subpart D)

Over-The-Counter Use (21 CFR 801 Subpart C)

CONTINUE ON A SEPARATE PAGE IF NEEDED.

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[continued from Form FDA 3881, page 1]

Organism	Reported AMR Gene Marker
Citrobacter freundii
complex a	CTX-M-1, CTX-M-9, KPC, NDM, OXA-48
Citrobacter koseri	KPC, OXA-48
Enterobacter
cloacae complex b	CTX-M-1, CTX-M-9, KPC, TEM d
Enterococcus
faecalis	vanA
Escherichia coli	AAC, ANT, CMY, CTX-M-1, CTX-M-2, CTX-M-9, DFR, gyrA Mutant c, KPC, MCR-1 e, OXA-1, OXA-9,
SHV d, Sul1, Sul2, TEM d
Klebsiella
aerogenes	CTX-M-1, CTX-M-9, KPC, NDM, OXA-48
Klebsiella
michiganensis	CTX-M-1, CTX-M-9, KPC, NDM, OXA-48
Klebsiella oxytoca	CTX-M-1, CTX-M-9, KPC, NDM, OXA-48
Klebsiella
pneumoniae	AAC, AAD, APH, CMY, CTX-M-1, CTX-M-9, DFR, DHA, IMP, KPC, NDM, OXA-1, OXA-9, OXA-48,
RMT, Sul1, Sul2, TEM d
Klebsiella
quasipneumoniae	CTX-M-1, CTX-M-9, KPC, NDM, OXA-48
Klebsiella variicola	CTX-M-1, CTX-M-9, KPC, NDM, OXA-48
Morganella morganii	CTX-M-1, KPC, NDM, OXA-48
Proteus mirabilis	AAC, ANT, APH, armA, CMY, CTX-M-1, CTX-M-2, CTX-M-9, DFR, KPC, NDM, OXA-1, OXA-9, OXA-
48, Sul2, TEM d, VEB, VIM
Providencia rettgeri	NDM
Providencia stuartii	NDM
Pseudomonas
aeruginosa	AAC, ANT, CTX-M-1, CTX-M-2, gyrA Mutant c, KPC, NDM, OXA-1, PER, SHV d, TEM d, VEB, VIM
Raoultella
ornithinolytica	KPC, NDM, OXA-48
Raoultella planticola	KPC
Serratia
marcescens	CTX-M-1, CTX-M-9, KPC, NDM, OXA-48

Table 1 - Antimicrobial Resistance Gene Markers (Genetic Determinants) Associated with Bacterial Species

a Citrobacter freundii complex = C. freundii, C. werkmanii and C. youngae.

Bnterobacter cloacae complex = E. asburiae, E. cloacae, E. hormaechei, E. kobei and E. ludwigii.

^ PCR assays associated with fluoroquinolone resistance detect and differentiate wild type and mutants of gyraseA at amino acid position 87 for E. coli and position 83 for P. aeruginosa.

d PCR assays for SHV and TEM detect sequence variants for the two genes, respectively, at amino acid positions 156 and 104 associated with wild type penicillin resistance and mutations associated with ESBL phenotypes.

e The panel includes an assay for the detection of the mobilized colistin genetic determinant MCR-1 in E. coli.

The Acuitas AMR Gene Panel includes assays for the detection and reporting of genetic resistance markers associated with resistance to select drugs in the following antibiotic groups: aminoglycosides, carbapenems, cephalosporins, fluoroquinolones, penicillins, suffonamides, trimethoprim, and vancomycin, to aid in the identification of potentially antimicrobial-resistant organisms. The panel includes an assay for the detection of the mobilized colistin genetic determinant MCR-1, a marker of public health importance associated with reduced inhibitory activity of polymyxins.

[continued on page 3]

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[continued from page 2]

The results of the Acuitas AMR Gene Panel for detection and identification of genetic determinants associated with antimicrobial resistance are used along with the Acuitas AMR Gene Panel Electronic User Guide (EUG). In certain cases, this information may be used as an aid to clinicians in the management of patients with known or suspected antibiotic non-susceptible or resistant bacterial infections. The EUG contains information on the appropriateness of reporting resistance markers detected by the Acuitas AMR Gene Panel for claimed organisms based on the strength of the collective, totality of scientific evidence delineating the level of association between molecular marker detection with phenotypic, clinical resistance. Test results are not conclusive or prescriptive for labeled use of any specific antimicrobial drug product, and therefore, this test cannot be used in place of or to postpone or delay phenotypic antimicrobial susceptibility testing.

A "Detected" or "Not Detected" result does not rule out the presence of other antimicrobial resistance markers not detected by the Acuitas AMR Gene Panel. A "Not Detected" result for a genetic marker of antimicrobial resistance does not indicate susceptibility to associated antimicrobial drugs or drug classes, as multiple mechanisms of resistance may exist.

5

510(K) SUMMARY

OpGen, Inc.

Acuitas® AMR Gene Panel

INTRODUCTION l.

This summary of 510(k) safety and effectiveness information is being submitted in accordance with the requirements of 21 CFR 807.92. According to the requirements of 21 CFR 807.92, the following information provides sufficient detail to understand the basis for a determination of substantial equivalence. The assigned 510(k) number is K191288.

A. SUBMITTER

OpGen, Inc. 9717 Key West Avenue, Suite 100 Rockville, MD 20850

Phone: 301-869-9683 Fax: 301-869-9684

Email: regulatory@opgen.com

Contact Information:

Johannes Bacher, Chief Operating Officer OpGen, Inc. 9717 Key West Avenue, Suite 100 Rockville, MD 20850 US

Phone: 301-869-9683 Fax: 301-869-9684

Date Prepared: September 29, 2021

B. NAME OF DEVICE

Acuitas® AMR Gene Panel

C. COMMON OR USUAL NAME

Acuitas AMR Gene Panel

6

D. REGULATORY INFORMATION

1. Regulation Section:

21 CFR 866.1640 (Antimicrobial susceptibility test powder)

2. Classification:

Class II

3. Product Code:

PMY - System, Nucleic Acid Amplification Test, DNA, Carbapenem Non-Susceptible Gram Negative Organism, Colony

OOI - Real-time nucleic acid amplification system

PREDICATE DEVICE ய்

The Acuitas AMR Gene Panel is substantially equivalent to the Cepheid Xpert® Carba-R [510(k) # K152614].

DEVICE DESCRIPTION ட்

The Acuitas® AMR Gene Panel is a qualitative nucleic acid-based in vitro diagnostic test capable of simultaneous detection and identification of select genetic determinants of antimicrobial resistance (AMR) in isolated bacterial colonies grown on blood agar or MacConkey agar. The test detects twenty-eight (28) genetic determinants of resistance to the following nine (9) antibiotic classes: aminoglycosides, carbapenems, cephalosporins, fluoroquinolones, penicillins, polymyxins, sulfonamides, trimethoprim and vancomycin. The assay is performed on pure colonies of Enterobacterales and Pseudomonas aeruginosa grown on blood agar or MacConkey agar along with Enterococcus faecalis grown on blood agar.

The Acuitas AMR Gene Panel kit contains all of the necessary reagents for PCR and detection in order to amplify and detect DNA from pure colonies of Enterobacterales (Citrobacter freundii complex (Citrobacter braakii, Citrobacter freundii, Citrobacter werkmanii, Citrobacter youngae), Citrobacter koseri, Enterobacter cloacae complex (Enterobacter asburiae, Enterobacter cloacae, Enterobacter hormaechei, Enterobacter kobei, Enterobacter ludwigiì), Escherichia coli, Klebsiella pneumoniae, Klebsiella quasipneumoniae, Klebsiella aerogenes, Klebsiella michiganensis, Klebsiella oxytoca, Klebsiella variicola, Morganii, Proteus mirabilis, Providencia rettgeri, Providencia stuartii, Raoultella ornithinolytica, Raoultella planticola, Serratia marcescens) and Pseudomonas aeruginosa grown on blood agar or MacConkey agar, as well as Enterococcus faecalis grown on blood agar from clinical specimens. The test kit includes PCR plates (96-well) with dried primers and probes for analysis of four (4) isolates (24 wells per isolate).

The Acuitas AMR Gene Panel assay employs automated deoxyribonucleic acid (DNA) extraction on the QIAGEN® EZ1® Advanced XL System and multiplex real-time PCR on an OpGen Qualified Applied Biosystems™ QuantStudio™ 5 Real-Time PCR System ("OpGen Qualified QuantStudio 5")

7

for use with the Acuitas AMR Gene Panel. The QIAGEN EZ1 DSP Virus Kit has been selected for use as the sample preparation method for the Acuitas AMR Gene Panel test.

After colony isolation, the user prepares a 0.5 McFarland suspension for each bacterial isolate and performs DNA extraction. DNA is extracted on the QIAGEN EZ1 Advanced XL System according to manufacturer instructions incorporating the Assay Control within the extraction process. A sample of extracted DNA eluate is transferred to a Reagent Reservoir trough to which Acuitas AMR Gene Panel Master Mix is added. Extracted DNA/Master Mix is transferred to each of 24 wells on the Acuitas AMR Gene Panel PCR plate per test sample. The contents of each well are mixed, and the plate is sealed and transferred to the OpGen Qualified QuantStudio 5 for use with the Acuitas AMR Gene Panel for real-time multiplex reaction and detection using the Acuitas AMR Gene Panel PCR Template File.

Data are exported from the OpGen Qualified QuantStudio 5 and imported into the Acuitas AMR Gene Analysis Software, a spreadsheet application that analyzes the data and generates a report for viewing and printing. Each test report indicates detection of applicable antimicrobial resistance gene variants as "Detected", "Not Detected" or "NA/NR".

The Applied Biosystems QuantStudio 5 Real-Time PCR System is not intended for clinical diagnostic purposes. The OpGen Qualified QuantStudio 5 for use with the Acuitas AMR Gene Panel is a component of the Acuitas AMR Gene Panel assay and is cleared for in vitro diagnostic use only with the Acuitas AMR Gene Panel and not for any other application. The OpGen Qualified QuantStudio 5 for use with the Acuitas AMR Gene Panel may only be used with the Acuitas AMR Gene Panel after the instrument has been qualified for use by OpGen, Inc.

G. INTENDED USE / INDICATIONS FOR USE

The Acuitas® AMR Gene Panel, performed on the QIAGEN® EZ1® Advanced XL System and the OpGen Qualified QuantStudio™ 5 Real-Time PCR System, is a qualitative nucleic acid-based multiplex in vitro diagnostic test for detection and differentiation of antibiotic resistance markers to one or more antimicrobial agents. The test utilizes real-time polymerase chain reaction (PCR) and is performed on isolated colonies of Pseudomonas aeruginosa, Enterocccus faecalis, or members of Enterobacterales grown in pure culture on blood agar or MacConkey agar.

Organism identification results must be available prior to reporting results for the Acuitas AMR Gene Panel. Antimicrobial resistance gene results are reported by the Acuitas AMR Gene Panel for the combinations of bacterial pathogens and associated genetic resistance markers indicated in Table 1 below.

Organism	Reported AMR Gene Marker
Citrobacter freundii complexa	CTX-M-1, CTX-M-9, KPC, NDM, OXA-48
Citrobacter koseri	KPC, OXA-48
Enterobacter cloacae complexb	CTX-M-1, CTX-M-9, KPC, TEMd
Enterococcus faecalis	vanA
Escherichia coli	AAC, ANT, CMY, CTX-M-1, CTX-M-2, CTX-M-9, DFR, gyrA Mutantc, KPC, MCR-1e, OXA-1, OXA-9, SHVd, Sul1, Sul2, TEMd
Klebsiella aerogenes	CTX-M-1, CTX-M-9, KPC, NDM, OXA-48
Klebsiella michiganensis	CTX-M-1, CTX-M-9, KPC, NDM, OXA-48

Table 1 - Antimicrobial Resistance Gene Markers (Genetic Determinants) Associated with Bacterial Species

8

Organism	Reported AMR Gene Marker
Klebsiella oxytoca	CTX-M-1, CTX-M-9, KPC, NDM, OXA-48
Klebsiella pneumoniae	AAC, AAD, APH, CMY, CTX-M-1, CTX-M-9, DFR, DHA, IMP, KPC, NDM, OXA-1, OXA-9, OXA-48, RMT, Sul1, Sul2, TEMd
Klebsiella quasipneumoniae	CTX-M-1, CTX-M-9, KPC, NDM, OXA-48
Klebsiella variicola	CTX-M-1, CTX-M-9, KPC, NDM, OXA-48
Morganella morganii	CTX-M-1, KPC, NDM, OXA-48
Proteus mirabilis	AAC, ANT, APH, armA, CMY, CTX-M-1, CTX-M-2, CTX-M-9, DFR, KPC, NDM, OXA-1, OXA-9, OXA-48, Sul2, TEMd, VEB, VIM
Providencia rettgeri	NDM
Providencia stuartii	NDM
Pseudomonas aeruginosa	AAC, ANT, CTX-M-1, CTX-M-2, gyrA Mutantc, KPC, NDM, OXA-1, PER, SHVd, TEMd, VEB, VIM
Raoultella ornithinolytica	KPC, NDM, OXA-48
Raoultella planticola	KPC
Serratia marcescens	CTX-M-1, CTX-M-9, KPC, NDM, OXA-48

Citrobacter freundii complex = C. freundii, C. braakii, C. werkmanii and C. youngae.

b Enterobacter cloacae complex = E. asburiae, E. cloacae, E. hormaechei, E. kobei and E. ludwigii.

& PCR assays associated with fluoroquinolone resistance detect and differentiate wild type and mutants of gyraseA at amino acid position 87 for E. coli and position 83 for P. aeruginosa.

d PCR assays for SHV and TEM detect sequence variants for the two genes, respectively, at amino acid positions 156 and 104 associated with wild type penicillin resistance and mutations associated with ESBL phenotypes.

® The panel includes an assay for the detection of the mobilized colistin genetic determinant MCR-1 in E. coli.

The Acuitas AMR Gene Panel includes assays for the detection and reporting of genetic resistance markers associated with resistance to select drugs in the following antibiotic groups: aminoglycosides, carbapenems, cephalosporins, fluoroquinolones, penicillins, sulfonamides, trimethoprim, and vancomycin, to aid in the identification of potentially antimicrobial-resistant organisms. The panel includes an assay for the detection of the mobilized colistin genetic determinant MCR-1, a marker of public health importance associated with reduced inhibitory activity of polymyxins.

The results of the Acuitas AMR Gene Panel for detection and identification of genetic determinants associated with antimicrobial resistance are used along with the Acuitas AMR Gene Panel Electronic User Guide (EUG). In certain cases, this information may be used as an aid to clinicians in the management of patients with known or suspected antibiotic non-susceptible or resistant bacterial infections. The EUG contains information on the appropriateness of reporting resistance markers detected by the Acuitas AMR Gene Panel for claimed organisms based on the strength of the collective, totality of scientific evidence delineating the level of association between molecular marker detection with phenotypic, clinical resistance. Test results are not conclusive or prescriptive for labeled use of any specific antimicrobial drug product, and therefore, this test cannot be used in place of or to postpone or delay phenotypic antimicrobial susceptibility testing.

A "Detected" or "Not Detected" result does not rule out the presence of other antimicrobial resistance markers not detected by the Acuitas AMR Gene Panel. A "Not Detected" result for a genetic marker of antimicrobial resistance does not indicate susceptibility to associated antimicrobial drugs or drug classes, as multiple mechanisms of resistance may exist.

H. PREDICATE DEVICE

Acuitas AMR Gene Panel assay is substantially equivalent to Cepheid Xpert® Carba-R, [510(k) K152614]. The Acuitas AMR Gene Panel assay and the Xpert Carba-R Assay both detect target gene sequences from antibiotic-resistant bacteria and use real-time PCR amplification and fluorogenic target-specific hybridization detection.

9

The performance characteristics of the Acuitas AMR Gene Panel with bacterial isolates were determined in a multi-site investigational clinical study by comparing the Acuitas AMR Gene Panel to the results of Whole Genome Sequencing (WGS) and Antimicrobial Susceptibility Testing (AST). Table 2 compares the Acuitas AMR Gene Panel to the Cepheid Xpert® Carba-R and outlines the similarities and differences between the two systems.

Item	Proposed Device	Predicate Device
Sample Types	Acuitas® AMR Gene Panel
Bacterial Isolates	Xpert® Carba-R Assay (K152614)
Bacterial Isolates
Organisms
Indicated	Enterobacterales, Pseudomonas
aeruginosa, and Enterococcus faecalis	Enterobacteriaceae, Acinetobacter
baumannii, Pseudomonas aeruginosa
Assay Targets	AAC, AAD, ANT, APH, armA, CMY,
CTX-M-1, CTX-M-2, CTX-M-9, DFR,
DHA, E. coli gyrA mutant, IMP, KPC,
MCR-1, NDM, OXA-1, OXA-9, OXA-48,
P. aeruginosa gyrA mutant, PER, RMT,
SHV, Sul1, Sul2, TEM, vanA, VEB and
VIM	blaKPC, blaNDM, blaVIM, blaOXA-48,
and blaIMP
Similarities
Result Types	Qualitative	Same
Analyte	DNA	Same
Technological
Principles	Automated Nucleic acid amplification
(DNA); real-time PCR	Fully automated nucleic acid
amplification (DNA); real-time PCR
Interpretation of
Results	Diagnostic software on a Personal
Computer (PC)	Same
Differences
Organism
Detected	Enterobacterales, Pseudomonas
aeruginosa and Enterococcus faecalis	Enterobacteriaceae, Acinetobacter
baumannii, Pseudomonas aeruginosa
Target Antibiotic
Classes	Aminoglycosides, Carbapenems,
Cephalosporins, Fluoroquinolones,
Penicillins, Polymyxins, Sulfonamides,
Trimethoprim, and Vancomycin	Carbapenems
ltem	Proposed Device	Predicate Device
	Acuitas® AMR Gene Panel	Xpert® Carba-R Assay (K152614)
Gene Sequence	AAC, AAD, ANT, APH, armA, CMY,
CTX-M-1, CTX-M-2, CTX-M-9, DFR,
DHA, E. coli gyrA mutant, IMP, KPC,
MCR-1, NDM, OXA-1, OXA-9, OXA-48,
P. aeruginosa gyrA mutant, PER, RMT,
SHV, Sul1, Sul2, TEM, vanA, VEB and
VIM	blaKPC, blaNDM, blaVIM, blaOXA-48,
and blaIMP
Extraction Method	QIAGEN® EZ1® Advanced XL DNA
Extraction	Integrated with GeneXpert® System
Instrument	OpGen Qualified QuantStudio™ 5
Real-Time PCR System1	Cepheid GeneXpert® System
Controls	One Assay Control, External Controls
Available	Internal Sample Processing Control
(SPC) and Probe Check Control
(PCC); External Controls available
Software	Spreadsheet application which takes
output from the OpGen Qualified
QuantStudio 5 and generates report on
the gene sequence	Automated test report using diagnostic
software
Time to obtain
results from start
of test	Approximately 2.5 hours	Approximately 50 minutes to results

Table 2 - Comparison of Similarities and Differences of the Acuitas AMR Gene Panel with the Predicate Device

10

' The Applied Biosystems QuantStudio 5 Real-Time PCR System is not intended for clinical diagnostic purposes. The OpGen Qualified QuantStudio 5 for use with the Acuitas AMR Gene Panel is a component of the Acuitas AMR Gene Panel assay and is cleared for in vitro diagnostic use only with the Acuitas AMR Gene Panel and not for any other application. The OpGen Qualified QuantStudio 5 for use with the Acuitas AMR Gene Panel may only be used with the Acuitas AMR Gene Panel after the instrument has been qualified for use by OpGen, Inc.

ll. PERFORMANCE DATA

A. SELECTED NON-CLINICAL STUDIES

1. Reproducibility

Reproducibility of the Acuitas AMR Gene Panel was evaluated using a panel of 300 uniquely labeled samples composed of ten (10) unique isolates. The panel was provided to three (3) testing sites, one (1) of which was OpGen. The panel was rotated across two (2) operators, two (2) OpGen Qualified QuantStudio 5 instruments and one (1) EZ1 Instrument per site over 20 days. Three (3) unique lots of all materials were used and rotated at each testing site.

Overall results for each sample tested in the reproducibility study are summarized in Table 3 - Acuitas AMR Gene Panel Reproducibility of Study Panel – by Isolate. Total agreement per sample (All Sites) ranged from 96% to 100%.

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	Site 1			Site 2				Site 3
Isolate	Op1	Op2	Site 1	Op1	Op2	Site 2	Op1	Op2	Site 3
L00000068-001
(E. coli)	14/15
93%	15/15
100%	29/30
97%	14/15
93%	14/15
93%	28/30
93%	15/15
100%	14/15
93%	29/30
97%		86/90
96%
L00015886-001
(E. coli)	13/14b
93%	15/15
100%	28/29
97%	15/15
100%	15/15
100%	30/30
100%	15/15
100%	15/15
100%	30/30
100%		88/89
99%
L00009154-001
(E. coli)	15/15
100%	15/15
100%	30/30
100%	15/15
100%	14/15
93%	29/30
97%	15/15
100%	15/15
100%	30/30
100%		89/90
99%
L00009721-001
(K. pneumoniae)	15/15
100%	15/15
100%	30/30
100%	15/15
100%	14/15
93%	29/30
97%	14/15
93%	15/15
100%	29/30
97%		88/90
98%
L00007800-001
(K. pneumoniae)	15/15
100%	15/15
100%	30/30
100%	15/15
100%	14/15
93%	29/30
97%	15/15
100%	14/15
93%	29/30
97%		88/90
98%
L00008624-001
(P. aeruginosa)	15/15
100%	14/15
93%	29/30
97%	15/15
100%	15/15
100%	30/30
100%	14/15
93%	14/15c
93%	28/30
93%		87/90
97%
L00013504-001
(P. mirabilis)	15/15
100%	15/15
100%	30/30
100%	15/15
100%	14/15
93%	29/30
97%	15/15
100%	14/15
93%	29/30
97%		88/90
98%
L00013200-001d
(P. mirabilis)	15/15
100%	15/15
100%	30/30
100%	14/15
93%	14/14e
100%	28/29
97%	15/15
100%	15/15
100%	30/30
100%		88/89
99%
L00022926-001
(E. faecalis)	15/15
100%	15/15
100%	30/30
100%	15/15
100%	15/15
100%	30/30
100%	15/15
100%	15/15
100%	30/30
100%		90/90
100%
L00006246-001f
(S. aureus)	15/15
100%	15/15
100%	30/30
100%	15/15
100%	15/15
100%	30/30
100%	15/15
100%	14/15f
93%	29/30
97%		89/90
99%

Table 3 - Acuitas AMR Gene Panel Reproducibility of Study Panel - by Isolate ª

3 Table includes reported AMR gene results per organism as described in Table 1 along with species ID results for E. coll and P. aeruginosa as used in conjunction with mutant gyrase results for these two organisms.

b One replicate of L00015886-001 had invalid results due to an Assay Control Failure. The replicate was repeated using the same lot, instrument, and operator as the original testing event. The repeat data agreed results. Neither initial nor repeat results are reported for this replicate.

& Amplification present for P. aeruginosa gyraseA assay called negative P. aeruginosa ID result in one sample.

4 CTX-M-14 (AF252622) was reported by WGS for L00013200-001-300187, which are the correct WGS results for L00013200-001.

& One replicate of L00013200-001 had invalid results due to an Assay Control Failure. The replicate was repeated using the same lot, instrument, and operator as the original testing event. The repeat data had 100% agreement with the expected results. Neither initial nor repeat results are reported for this replicate.

f The Acuitas AMR Gene Panel is not intended for S. aureus, which served as a negative control in this study. S. aureus was evaluated for all AMR genes in Table 1 except for gyrase gene targets. One replicate sample of L00006246-001 was false positive for the AAC assay.

Overall results for each assay target in the reproducibility test panel are summarized in Table 4 -Acuitas AMR Gene Panel Reproducibility of Study Panel - by Gene Target. Total agreement ranged from 97.8% to 100% for detected results and 99.4% to 100% for not detected results across the individual assay targets.

12

| Acuitas
AMR
Gene
Target | Expected
Results | Site 1 | | | Site 2 | | | Site 3 | | | Total %
Agreement
by Target |
|---------------------------------------------------------|---------------------|-----------------|-----------------|------------------|-----------------|-----------------|------------------|-----------------|------------------|------------------|-----------------------------------|
| AAC | Detected | 60/60
100% | 60/60
100% | 120/120
100% | 60/60
100% | 59/59
100% | 119/119
100% | 60/60
100% | 60/60
100% | 120/120
100% | 359/359
100% |
| AAC | Not
Detected | 74/74
100% | 75/75
100% | 149/149
100% | 75/75
100% | 75/75
100% | 150/150
100% | 75/75
100% | 74/75 d
98.7% | 149/150
99.3% | 448/449
99.80% |
| AAD | Detected | 15/15
100% | 15/15
100% | 30/30
100% | 15/15
100% | 15/15
100% | 30/30
100% | 15/15
100% | 15/15
100% | 30/30
100% | 90/90
100% |
| AAD | Not
Detected | 30/30
100% | 30/30
100% | 60/60
100% | 30/30
100% | 30/30
100% | 60/60
100% | 30/30
100% | 30/30
100% | 60/60
100% | 180/180
100% |
| ANT | Detected | 30/30
100% | 30/30
100% | 60/60
100% | 29/30
96.7% | 30/30
100% | 59/60
98.30% | 30/30
100% | 28/30
93.3% | 58/60
96.7% | 177/180
98.30% |
| ANT | Not
Detected | 74/74
100% | 75/75
100% | 149/149
100% | 75/75
100% | 73/74
98.6% | 148/149
99.3% | 74/75
98.7% | 75/75
100% | 149/150
99.3% | 446/448
99.60% |
| APH | Detected | 15/15
100% | 15/15
100% | 30/30
100% | 15/15
100% | 14/14
100% | 29/29
100% | 15/15
100% | 15/15
100% | 30/30
100% | 89/89
100% |
| APH | Not
Detected | 60/60
100% | 60/60
100% | 120/120
100% | 60/60
100% | 60/60
100% | 120/120
100% | 60/60
100% | 60/60
100% | 120/120
100% | 360/360
100% |
| armA | Detected e | - | - | - | - | - | - | - | - | - | |
| armA | Not
Detected | 45/45
100% | 45/45
100% | 90/90
100% | 45/45
100% | 44/44
100% | 89/89
100% | 45/45
100% | 45/45
100% | 90/90
100% | 269/269
100% |
| CMY | Detected | 30/30
100% | 30/30
100% | 60/60
100% | 30/30
100% | 30/30
100% | 60/60
100% | 30/30
100% | 30/30
100% | 60/60
100% | 180/180
100% |
| CMY | Not
Detected | 88/89
98.9% | 90/90
100% | 178/179
99.4% | 90/90
100% | 89/89
100% | 179/179
100% | 90/90
100% | 90/90
100% | 180/180
100% | 537/538
99.80% |
| CTX-M-1 | Detected | 30/30
100% | 30/30
100% | 60/60
100% | 30/30
100% | 30/30
100% | 60/60
100% | 30/30
100% | 30/30
100% | 60/60
100% | 180/180
100% |
| CTX-M-1 | Not
Detected | 104/104
100% | 105/105
100% | 209/209
100% | 105/105
100% | 104/104
100% | 209/209
100% | 105/105
100% | 105/105
100% | 210/210
100% | 628/628
100% |
| CTX-M-2 | Detected e | - | - | - | - | - | - | - | - | - | |
| CTX-M-2 | Not
Detected | 104/104
100% | 105/105
100% | 209/209
100% | 105/105
100% | 104/104
100% | 209/209
100% | 105/105
100% | 105/105
100% | 210/210
100% | 628/628
100% |
| CTX-M-9 | Detected | 15/15
100% | 15/15
100% | 30/30
100% | 15/15
100% | 14/14
100% | 29/29
100% | 15/15
100% | 15/15
100% | 30/30
100% | 89/89
100% |
| CTX-M-9 | Not
Detected | 104/104
100% | 105/105
100% | 209/209
100% | 105/105
100% | 104/105
99% | 209/210
99.5% | 105/105
100% | 105/105
100% | 210/210
100% | 628/629
99.80% |
| DFR | Detected | 45/45
100% | 45/45
100% | 90/90
100% | 45/45
100% | 44/44
100% | 89/89
100% | 45/45
100% | 45/45
100% | 90/90
100% | 269/269
100% |
| DFR | Not
Detected | 74/74
100% | 75/75
100% | 149/149
100% | 75/75
100% | 75/75
100% | 150/150
100% | 75/75
100% | 75/75
100% | 150/150
100% | 449/449
100% |
| DHA | Detected e | - | - | - | - | - | - | - | - | - | |
| Acuitas
AMR | Expected | Site 1 | | | Site 2 | | | Site 3 | | | Total % |
| Gene
Target | Results | Op 1 a | Op 2 | Site | Op 1 | Op 2 b | Site | Op 1 | Op 2 | Site | Agreement
by Target |
| | Not
Detected | 45/45
100% | 45/45
100% | 90/90
100% | 45/45
100% | 45/45
100% | 90/90
100% | 45/45
100% | 45/45
100% | 90/90
100% | 270/270
100% |
| E. coli
gyrA
Mutant | Detected | 14/15
93.3% | 15/15
100% | 29/30
96.7% | 15/15
100% | 14/15
93.3% | 29/30
96.70% | 15/15
100% | 15/15
100% | 30/30
100% | 88/90
97.80% |
| | Not
Detected | 29/29
100% | 30/30
100% | 59/59
100% | 30/30
100% | 30/30
100% | 60/60
100% | 30/30
100% | 30/30
100% | 60/60
100% | 179/179
100% |
| E. coli ID | Detected | 44/44
100% | 45/45
100% | 89/89
100% | 45/45
100% | 44/45
97.8% | 89/90
98.9% | 45/45
100% | 45/45
100% | 90/90
100% | 268/269
99.6% |
| | Not
Detected e | - | - | - | - | - | - | - | - | - | - |
| IMP | Detected | 15/15
100% | 15/15
100% | 30/30
100% | 15/15
100% | 15/15
100% | 30/30
100% | 15/15
100% | 15/15
100% | 30/30
100% | 90/90
100% |
| | Not
Detected | 30/30
100% | 30/30
100% | 60/60
100% | 30/30
100% | 30/30
100% | 60/60
100% | 29/30
96.7% | 30/30
100% | 59/60
98.3% | 179/180
99.40% |
| KPC | Detected | 15/15
100% | 15/15
100% | 30/30
100% | 15/15
100% | 15/15
100% | 30/30
100% | 15/15
100% | 15/15
100% | 30/30
100% | 90/90
100% |
| | Not
Detected | 119/119
100% | 120/120
100% | 239/239
100% | 120/120
100% | 119/119
100% | 239/239
100% | 120/120
100% | 120/120
100% | 240/240
100% | 718/718
100% |
| | Detected e | - | - | - | - | - | - | - | - | - | - |
| MCR-1 | Not
Detected | 59/59
100% | 60/60
100% | 119/119
100% | 60/60
100% | 60/60
100% | 120/120
100% | 60/60
100% | 60/60
100% | 120/120
100% | 359/359
100% |
| | Detected | 15/15
100% | 15/15
100% | 30/30
100% | 15/15
100% | 15/15
100% | 30/30
100% | 15/15
100% | 15/15
100% | 30/30
100% | 90/90
100% |
| NDM | Not
Detected | 75/75
100% | 75/75
100% | 150/150
100% | 75/75
100% | 74/74
100% | 149/149
100% | 75/75
100% | 75/75
100% | 150/150
100% | 449/449
100% |
| | Detected | 30/30
100% | 30/30
100% | 60/60
100% | 30/30
100% | 29/29
100% | 59/59
100% | 30/30
100% | 30/30
100% | 60/60
100% | 179/179
100% |
| OXA-1 | Not
Detected | 104/104
100% | 105/105
100% | 209/209
100% | 105/105
100% | 105/105
100% | 210/210
100% | 105/105
100% | 105/105
100% | 210/210
100% | 629/629
100% |
| | Detected e | - | - | - | - | - | - | - | - | - | - |
| OXA-9 | Not
Detected | 119/119
100% | 120/120
100% | 239/239
100% | 120/120
100% | 119/119
100% | 239/239
100% | 120/120
100% | 120/120
100% | 240/240
100% | 718/718
100% |
| | Detected | 15/15
100% | 15/15
100% | 30/30
100% | 15/15
100% | 15/15
100% | 30/30
100% | 15/15
100% | 15/15
100% | 30/30
100% | 90/90
100% |
| OXA-48 | Not
Detected | 60/60
100% | 60/60
100% | 120/120
100% | 59/60
98.3% | 59/59
100% | 118/119
99.2% | 60/60
100% | 60/60
100% | 120/120
100% | 358/359
99.70% |
| P.
aeruginosa
gyrA
Mutant | Detected | 15/15
100% | 14/15
93.3% | 29/30
96.7% | 15/15
100% | 15/15
100% | 30/30
100% | 15/15
100% | 14/15
93.3% | 29/30
96.7% | 88/90
97.80% |
| | Not
Detected e | - | - | - | - | - | - | - | - | - | - |
| Acuitas
AMR
Gene
Target | Expected
Results | Site 1 | | | Site 2 | | | Site 3 | | | Total %
Agreement
by Target |
| | | Op 1 a | Op 2 | Site | Op 1 | Op 2 b | Site | Op 1 | Op 2 | Site | |
| P.
aeruginosa
ID | Detected | 15/15
100% | 15/15
100% | 30/30
100% | 15/15
100% | 15/15
100% | 30/30
100% | 15/15
100% | 14/15
93.3% | 29/30
96.7% | 89/90
98.9% |
| | Not
Detected e | - | - | - | - | - | - | - | - | - | - |
| PER | Detected | 15/15
100% | 15/15
100% | 30/30
100% | 15/15
100% | 15/15
100% | 30/30
100% | 15/15
100% | 15/15
100% | 30/30
100% | 90/90
100% |
| | Not
Detected | 15/15
100% | 15/15
100% | 30/30
100% | 15/15
100% | 15/15
100% | 30/30
100% | 15/15
100% | 15/15
100% | 30/30
100% | 90/90
100% |
| | Detected e | - | - | - | - | - | - | - | - | - | - |
| RMT | Not
Detected | 45/45
100% | 45/45
100% | 90/90
100% | 45/45
100% | 45/45
100% | 90/90
100% | 45/45
100% | 45/45
100% | 90/90
100% | 270/270
100% |
| | Detected e | - | - | - | - | - | - | - | - | - | - |
| SHV | Not
Detected | 74/74
100% | 75/75
100% | 149/149
100% | 75/75
100% | 75/75
100% | 150/150
100% | 75/75
100% | 75/75
100% | 150/150
100% | 449/449
100% |
| | Detected | 60/60
100% | 60/60
100% | 120/120
100% | 60/60
100% | 60/60
100% | 120/120
100% | 60/60
100% | 60/60
100% | 120/120
100% | 360/360
100% |
| Sul1 | Not
Detected | 29/29
100% | 30/30
100% | 59/59
100% | 30/30
100% | 30/30
100% | 60/60
100% | 30/30
100% | 30/30
100% | 60/60
100% | 179/179
100% |
| | Detected | 104/104
100% | 105/105
100% | 209/209
100% | 105/105
100% | 103/104
99% | 208/209
99.5% | 105/105
100% | 105/105
100% | 210/210
100% | 627/628
99.80% |
| Sul2 | Not
Detected | 15/15
100% | 15/15
100% | 30/30
100% | 15/15
100% | 15/15
100% | 30/30
100% | 15/15
100% | 15/15
100% | 30/30
100% | 90/90
100% |
| | Detected | 104/104
100% | 105/105
100% | 209/209
100% | 105/105
100% | 103/104
99% | 208/209
99.5% | 105/105
100% | 105/105
100% | 210/210
100% | 627/628
99.80% |
| TEM | Not
Detected | 30/30
100% | 30/30
100% | 60/60
100% | 30/30
100% | 30/30
100% | 60/60
100% | 30/30
100% | 30/30
100% | 60/60
100% | 180/180
100% |
| | Detected | 15/15
100% | 15/15
100% | 30/30
100% | 15/15
100% | 15/15
100% | 30/30
100% | 15/15
100% | 15/15
100% | 30/30
100% | 90/90
100% |
| vanA | Not
Detected | 15/15
100% | 15/15
100% | 30/30
100% | 15/15
100% | 15/15
100% | 30/30
100% | 15/15
100% | 15/15
100% | 30/30
100% | 90/90
100% |
| | Detected | 15/15
100% | 15/15
100% | 30/30
100% | 15/15
100% | 15/15
100% | 30/30
100% | 15/15
100% | 15/15
100% | 30/30
100% | 90/90
100% |
| VEB | Not
Detected | 45/45
100% | 45/45
100% | 90/90
100% | 45/45
100% | 44/44
100% | 89/89
100% | 45/45
100% | 45/45
100% | 90/90
100% | 269/269
100% |
| | Detected | 15/15
100% | 15/15
100% | 30/30
100% | 15/15
100% | 15/15
100% | 30/30
100% | 15/15
100% | 15/15
100% | 30/30
100% | 90/90
100% |
| VIM | Not
Detected | 45/45
100% | 45/45
100% | 90/90
100% | 45/45
100% | 44/44
100% | 89/89
100% | 45/45
100% | 45/45
100% | 90/90
100% | 269/269
100% |

Table 4 - Acuitas AMR Gene Panel Reproducibility of Study Panel - by Gene Target •

13

14

3 One replicate of L00015886-001 had invalid results due to an Assay Control Failure. The replicate was repeated using the same lot, instrument, and operator as the original testing event. The repeat data had 100% agreement with the initial nor repeat results are reported for this replicate.

One replicate of L00013200-001 had invalid results due to an Assay Control Failure. The replicate was repeated using the same lot, instrument, and operator as the original testing event. The repeat data had 100% agreement with the initial nor repeat results are reported for this replicate.

· Amplification present for P. aeruginosa gyraseA assay called negative P. aeruginosa ID result in one sample.

15

f Organism ID only utilized in the context of gyrA mutant detection.

2. Analytical Reactivity (Inclusivity)

The analytical sensitivity of the Acuitas AMR Gene Panel was evaluated by testing a panel of two hundred and ninety-eight (298) isolates covering all antimicrobial resistance genes detected by the Acuitas AMR Gene Panel test. Each isolate in the test panel was tested once, except for isolates harboring rare resistance genes, which were replicated to achieve at least 18 positive data points per gene target assessed. The Acuitas AMR Gene Panel results were compared with species identification by well-established automated species identification methods and AMR gene detection by Whole Genome Sequencing (WGS).

Table 5 - Analytical Reactivity (Inclusivity) Results summarizes AMR genes detected or not detected by Acuitas AMR Gene Panel. Table 5 also indicates AMR gene variants predicted to be detected by in silico analysis but not tested in this study.

			Acuitas AMR Gene Panel
AMR Gene	Number of
Samples
Positive for
Gene by
WGS	Number of
Unique
Isolates
Positive for
Gene by
WGS	AMR Gene(s)
Detected	AMR Gene(s)
Not
Detected	Other AMR Gene Variants Predicted to be Detected
by Acuitas AMR Gene Panel Based on In
Silico Analysisd
AAC	198	162	aac(3)-Ila,
aac(3)-Ild,
aac(3)-IVa,
aac(6')-lb,
aac(6')Ib-cr,
aacA4-8, aacA4		Detectable: aac(3)-IIc, aac(3)-Ile, aac(3)-Ib-aac(6')-Ib,
ant(3")-Ih-aac(6')-Ild
AAD	70	62	aadA1, aadA2		Detectable: aadA13, aadA3, aadA8, aadA8b, aadA7b,
aadA17
Likely Detectable: aadA12, aadA21, aadA22, aadA23
ANT	63	40	aadB		-
APH	15	14	aph(4)-la	aph(4)-
la V01499	-
armA	1	1	armA		-
CMY	26	25	blaCMY-16,
blaCMY-2,
blaCMY-4,
blaCMY-42,
blaCMY-6,
blaCMY-60	blaCMY-
16_FJ855437	Detectable: blaBIL-1, blaCMY-0, blaCMY-102, blaCMY-
108, blaCMY-110, blaCMY-111, blaCMY-112, blaCMY-
113, blaCMY-114, blaCMY-115, blaCMY-118, blaCMY-
12, blaCMY-14, blaCMY-15, blaCMY-17, blaCMY-18,
blaCMY-20, blaCMY-21, blaCMY-22, blaCMY-23,
blaCMY-24, blaCMY-25, blaCMY-27, blaCMY-28,
blaCMY-29, blaCMY-3, blaCMY-30, blaCMY-31,
blaCMY-32, blaCMY-33, blaCMY-34, blaCMY-35,
blaCMY-36, blaCMY-38, blaCMY-39, blaCMY-41,
blaCMY-43, blaCMY-44, blaCMY-45, blaCMY-47,
blaCMY-48, blaCMY-5, blaCMY-50, blaCMY-51,
blaCMY-54, blaCMY-55, blaCMY-56, blaCMY-57,
blaCMY-58, blaCMY-59, blaCMY-61, blaCMY-62,
blaCMY-63, blaCMY-65, blaCMY-66, blaCMY-67,
blaCMY-68, blaCMY-69, blaCMY-7, blaCMY-71,
blaCMY-72, blaCMY-75, blaCMY-76, blaCMY-77,
blaCMY-78, blaCMY-80, blaCMY-81, blaCMY-84,
blaCMY-87, blaCMY-90, blaCMY-94, blaCMY-95
AMR Gene	Number of
Samples
Positive for
Gene by
WGS	Number of
Unique
Isolates
Positive for
Gene by
WGS	AMR Gene(s)
Detected	AMR Gene(s)
Not
Detected	Other AMR Gene Variants Predicted to be Detected
by Acuitas AMR Gene Panel Based on In
Silico Analysisd
CTX-M-1	72	62	blaCTX-M-1,
blaCTX-M-15,
blaCTX-M-55,
blaCTX-M-64	blaCTX-M-
15_DQ302097	blaCMY-99, blaLAT-1, blaCMY-103, blaCMY-117,
blaCMY-79
Likely Detectable: blaCMY-13, blaCMY-26, blaCMY-37,
blaCMY-49, blaCMY-73, blaCMY-79, blaCMY-116,
blaCMY-117,
Potentially Detectable: blaCMY-40, blaCMY-53
CTX-M-2	14	12	blaCTX-M-131,
blaCTX-M-2		Detectable: blaCTX-M-141, blaCTX-M-20, blaCTX-M-31,
blaCTX-M-35, blaCTX-M-43, blaCTX-M-44, blaCTX-M-5,
blaCTX-M-56, blaCTX-M-59, blaCTX-M-76, blaCTX-M-
77, blaCTX-M-92, blaCTX-M-95, blaCTX-M-97
Likely Detectable: blaCTX-M-115, blaCTX-M-124
CTX-M-9	34	32	blaCTX-M-14,
blaCTX-M-14b,
blaCTX-M-27,
blaCTX-M-64,
blaCTX-M-65,
blaCTX-M-9,
blaCTX-M-90		Detectable: blaCTX-M-104, blaCTX-M-106, blaCTX-M-
110, blaCTX-M-111, blaCTX-M-112, blaCTX-M-113,
blaCTX-M-121, blaCTX-M-122, blaCTX-M-123, blaCTX-
M-125, blaCTX-M-126, blaCTX-M-129, blaCTX-M-13,
blaCTX-M-130, blaCTX-M-134, blaCTX-M-147, blaCTX-
M-148, blaCTX-M-159, blaCTX-M-16, blaCTX-M-17,
blaCTX-M-19, blaCTX-M-21, blaCTX-M-24, blaCTX-M-
38, blaCTX-M-46, blaCTX-M-47 blaCTX-M-48, blaCTX-
M-49, blaCTX-M-50, blaCTX-M-51, blaCTX-M-67,
blaCTX-M-81, blaCTX-M-83, blaCTX-M-84, blaCTX-M-
85, blaCTX-M-86, blaCTX-M-87, blaCTX-M-93, blaCTX-
M-98, blaCTX-M-99
DFR	42	37	dfrA17, dfrA5		-
DHA	16	13	blaDHA-1		Detectable: blaDHA-10, blaDHA-13, blaDHA-14,
blaDHA-15, blaDHA-17, blaDHA-18, blaDHA-19,
blaDHA-2, blaDHA-20, blaDHA-21, blaDHA-22, blaDHA-
3, blaDHA-5, blaDHA-6, blaDHA-7, blaDHA-9, blaMOR-2
IMP	25	15	blaIMP-1,
blaIMP-13,
blaIMP-18,
blaIMP-26,
blaIMP-34,
blaIMP-4,
blaIMP-6		Detectable: blaIMP-10, blaIMP-25, blaIMP-3, blaIMP-40,
blaIMP-42, blaIMP-52, blaIMP-14, blaIMP-14a, blaIMP-
19, blaIMP-2, blaIMP-20, blaIMP-24, blaIMP-32, blaIMP-
48, blaIMP-8, blaIMP-28, blaIMP-5
Likely Detectable: blaIMP-15, blaIMP-29
Potentially Detectable: blaIMP-38
KPC	23	22	blaKPC-2,
blaKPC-3	blaKPC-
2_AY034847	Detectable: blaKPC-1, blaKPC-10, blaKPC-11, blaKPC-
12, blaKPC-13, blaKPC-14, blaKPC-15, blaKPC-16,
blaKPC-17, blaKPC-19, blaKPC-22, blaKPC-4, blaKPC-
5, blaKPC-6, blaKPC-8, blaKPC-9
MCR-1	20	13	MCR-1		-
NDM	10	10	blaNDM-1,
blaNDM-7		Detectable: blaNDM-12, blaNDM-2, blaNDM-3, blaNDM-
4, blaNDM-5, blaNDM-6, blaNDM-8, blaNDM-9
Likely Detectable: blaNDM-10
OXA-1	78	56	blaOXA-1,
blaOXA-4	blaOXA-
1_J02967	Detectable: blaOXA-224, blaOXA-31, blaOXA-320
Likely Detectable: blaOXA-47
	Acuitas AMR Gene Panel
AMR Gene	Number of
Samples
Positive for
Gene by
WGS	Number of
Unique
Isolates
Positive for
Gene by
WGS	AMR Gene(s)
Detected	AMR Gene(s)
Not
Detected	Other AMR Gene Variants Predicted to be Detected
by Acuitas AMR Gene Panel Based on In Silico Analysis d
OXA-48	19	19	blaOXA-181,
blaOXA-232,
blaOXA-370,
blaOXA-48	blaOXA-
48_AY236073	Detectable: blaOXA-162, blaOXA-163, blaOXA-199,
blaOXA-204, blaOXA-244, blaOXA-245, blaOXA-247
OXA-9	26	26	blaOXA-9	blaOXA-
9_JF703130	-
PER	20	19	blaPER-1,
blaPER-3		Detectable: blaPER-4, blaPER-5, blaPER-7, blaPER-8
RMT	11	9	rmtB		Detectable: rmtF
Likely Detectable: rmtB2
SHV	4	3	blaSHV-12,
blaSHV-2a		Detectable:
blaSHV-1, blaSHV-100, blaSHV-101,
blaSHV-102, blaSHV-103, blaSHV-104, blaSHV-106,
blaSHV-107, blaSHV-108, blaSHV-109, blaSHV-11,
blaSHV-119, blaSHV-120, blaSHV-121, blaSHV-122,
blaSHV-128, blaSHV-129, blaSHV-13, blaSHV-132,
blaSHV-133, blaSHV-135, blaSHV-137, blaSHV-14,
blaSHV-140, blaSHV-141, blaSHV-142, blaSHV-143,
blaSHV-144, blaSHV-145, blaSHV-147, blaSHV-148,
blaSHV-149, blaSHV-15, blaSHV-150, blaSHV-151,
blaSHV-152, blaSHV-153, blaSHV-154, blaSHV-155,
blaSHV-156, blaSHV-157, blaSHV-158, blaSHV-159,
blaSHV-16, blaSHV-160, blaSHV-161, blaSHV-162,
blaSHV-163, blaSHV-164, blaSHV-165, blaSHV-167,
blaSHV-168, blaSHV-172, blaSHV-173, blaSHV-178,
blaSHV-179, blaSHV-18, blaSHV-183, blaSHV-2,
blaSHV-24, blaSHV-25, blaSHV-26, blaSHV-28, blaSHV-29,
blaSHV-30, blaSHV-31, blaSHV-33, blaSHV-34,
blaSHV-35, blaSHV-36, blaSHV-38, blaSHV-40, blaSHV-41,
blaSHV-42, blaSHV-44, blaSHV-46, blaSHV-48,
blaSHV-49, blaSHV-5, blaSHV-50, blaSHV-51, blaSHV-52,
blaSHV-55, blaSHV-56, blaSHV-57, blaSHV-59,
blaSHV-60, blaSHV-61, blaSHV-62, blaSHV-63, blaSHV-64,
blaSHV-65, blaSHV-66, blaSHV-67, blaSHV-69,
blaSHV-7, blaSHV-70, blaSHV-71, blaSHV-72, blaSHV-73,
blaSHV-74, blaSHV-75, blaSHV-76, blaSHV-77,
blaSHV-78, blaSHV-79, blaSHV-8, blaSHV-80, blaSHV-81,
blaSHV-82, blaSHV-83, blaSHV-85, blaSHV-86,
blaSHV-89, blaSHV-92, blaSHV-94, blaSHV-95, blaSHV-96,
blaSHV-97, blaSHV-98, blaSHV-99, blaOKP-A,
blaSHV-105, blaSHV-110, blaSHV-27, blaSHV-45,
blaSHV-93
Likely Detectable: blaSHV-119, blaSHV-137, blaSHV-144,
blaSHV-167, blaSHV-168, blaSHV-38, blaSHV-51,
blaSHV-70, blaSHV-71, blaSHV-72, blaSHV-73, blaSHV-80,
blaSHV-81
Sul1	137	103	Sul1	Sul1_AY224185	Sul3 (AY047357)§, Sul3 (AB281183)º
Likely Detectable: Sul1 (AM746675)
Potentially Detectable: Sul1 (AY260546)
Sul2	133	99	Sul2	Sul2_GQ421466	-
TEM	173	132	blaTEM-1,
blaTEM-117,
blaTEM-143,
blaTEM-176,
blaTEM-192,
blaTEM-1A,
blaTEM-1B,
blaTEM-1C,
blaTEM-1D,
blaTEM-2		Detectable:
blaTEM-10, blaTEM-101, blaTEM-102,
blaTEM-104, blaTEM-105, blaTEM-108, blaTEM-11,
blaTEM-110, blaTEM-112, blaTEM-114, blaTEM-115,
blaTEM-116, blaTEM-118, blaTEM-12, blaTEM-120,
blaTEM-122, blaTEM-126, blaTEM-127, blaTEM-128,
blaTEM-129, blaTEM-132, blaTEM-135, blaTEM-136,
blaTEM-137, blaTEM-141, blaTEM-144, blaTEM-145,
blaTEM-147, blaTEM-148, blaTEM-150, blaTEM-151,
blaTEM-152, blaTEM-154, blaTEM-155, blaTEM-156,
blaTEM-157, blaTEM-158, blaTEM-159, blaTEM-160, blaTEM-166
AMR Gene	Number of
Samples
Positive for
Gene by
WGS	Number of
Unique
Isolates
Positive for
Gene by
WGS	AMR Gene(s)
Detected	AMR Gene(s)
Not
Detected	Other AMR Gene Variants Predicted to be Detected
by Acuitas AMR Gene Panel Based on In
Silico Analysisd
vanA	21	4	VanA-A	-	blaTEM-169, blaTEM-171, blaTEM-178, blaTEM-182,
blaTEM-183, blaTEM-185, blaTEM-186, blaTEM-187,
blaTEM-188, blaTEM-189, blaTEM-190, blaTEM-193,
blaTEM-194, blaTEM-195, blaTEM-198, blaTEM-20,
blaTEM-201, blaTEM-206, blaTEM-207, blaTEM-209,
blaTEM-216, blaTEM-217, blaTEM-28, blaTEM-29,
blaTEM-30, blaTEM-34, blaTEM-45, blaTEM-47,
blaTEM-48, blaTEM-49, blaTEM-53, blaTEM-54,
blaTEM-55, blaTEM-57, blaTEM-67, blaTEM-68,
blaTEM-70, blaTEM-71, blaTEM-72, blaTEM-75,
blaTEM-76, blaTEM-77, blaTEM-78, blaTEM-79,
blaTEM-80, blaTEM-81, blaTEM-82, blaTEM-83,
blaTEM-84, blaTEM-85, blaTEM-86, blaTEM-91,
blaTEM-93, blaTEM-95, blaTEM-96, blaTEM-97,
blaTEM-98, blaTEM-99, blaTEM-106, blaTEM-107,
blaTEM-109, blaTEM-111, blaTEM-113, blaTEM-121,
blaTEM-123, blaTEM-124, blaTEM-130, blaTEM-131,
blaTEM-133, blaTEM-134, blaTEM-138, blaTEM-139,
blaTEM-142, blaTEM-149, blaTEM-15, blaTEM-153,
blaTEM-16, blaTEM-167, blaTEM-177, blaTEM-184,
blaTEM-197, blaTEM-199, blaTEM-205, blaTEM-21,
blaTEM-211, blaTEM-22, blaTEM-24, blaTEM-3,
blaTEM-43, blaTEM-52, blaTEM-52B, blaTEM-52C,
blaTEM-6, blaTEM-60, blaTEM-63, blaTEM-8, blaTEM-
87, blaTEM-88, blaTEM-89, blaTEM-92, blaTEM-94
Likely Detectable: blaTEM-17, blaTEM-125, blaTEM-146,
blaTEM-90
VEB	14	13	blaVEB-1,
blaVEB-5,
blaVEB-6	-	Detectable: blaVEB-2, blaVEB-3, blaVEB-4, blaVEB-7,
blaVEB-8
VIM	20	18	blaVIM-1,
blaVIM-2,
blaVIM-20		Detectable: blaVIM-12, blaVIM-13, blaVIM-14, blaVIM-19,
blaVIM-26, blaVIM-27, blaVIM-28, blaVIM-29,
blaVIM-32, blaVIM-33, blaVIM-34, blaVIM-35, blaVIM-37,
blaVIM-39, blaVIM-4, blaVIM-42, blaVIM-43, blaVIM-10,
blaVIM-11, blaVIM-15, blaVIM-16, blaVIM-17, blaVIM-23,
blaVIM-24, blaVIM-30, blaVIM-31, blaVIM-8, blaVIM-9,
blaVIM-18, blaVIM-3, blaVIM-36, blaVIM-6, blaVIM-25,
blaVIM-38, blaVIM-5

Table 5 - Analytical Reactivity (Inclusivity) Results 3, e

9The Acuitas AMR Gene Panel is not intended for S. aureus, which served as a negative control in this study. S. aureus was evaluated for all AMR genes in Table 3 except for gyrase gene targets. One replicate sample of L00006246-001 was false positive for the AAC assay.

€ No data available.

16

17

13

18

a Results include combinations of AMR gene variants and organisms tested in the study that are absent from Table 1.

One gene variant of aadA7 (NCBI Accession AF224733) is not consistently detected by the Acuitas AMR Gene Panel due to 4 mismatches with the reverse PCR primer and one mismatch with the PCR probe of the AAD assay. Other aadA7 variants lack the mismatches and are expected to be consistently detected.

· Only these two Sul3 sequences are predicted to be detected by the Acuitas AMR Gene Panel. Other Sul3 accession numbers are not predicted to be detected.

1 Detectable indicates 100% homology of each primer/detector probe(s) with the target sequence. Likely Detectable indicates 4. Some but not all isolates of Shigella dysenteriae cross-react with the E. coll species ID assay of the Acultas AMR Gene Panel.

21

	AMR Gene Variants
aac(2')-la	aph(3')-lla	blaMIR-5	cphA2	mcr-3	QnrVC1
aac(3)-la	aph(3')-llb	blaMOX-3	dfrA1	mecA	rmtC
aac(3)-Ic	aph(3')-Vla	blaOXA-10	dfrA12	mph(A)	rmtD
aac(3)-Id	aph(3')-Vlb	blaOXA-114	dfrA14	mph(C)	spc
aac(6')	aph(3')-XV	blaOXA-17	dfrA15	mph(D)	strA
aac(6')-31	aph(6)-Ic	blaOXA-2	dfrA18	mph(E)	strB
aac(6')-33	ARR-2	blaOXA-5	dfrA23	msr(A)	Sul3a
aac(6')-aph(2")	ARR-3	blaOXA-50	dfrA27	msr(C)	tet(41)
aac(6')-Ic	blaACC-1	blaOXA-56	dfrA29	msr(E)	tet(A)
aac(6')-II	blaACT-12	blaOXA-94	dfrA30	norA	tet(B)
aac(6')-IIc	blaACT-16	blaOXY-1	dfrA31	oqxA	tet(C)
aac(6')-Im	blaACT-25	blaOXY-2	dfrA32	oqxB	tet(D)
aac(6')-Iq	blaACT-27	blaPAO	dfrA3b	qepA	tet(G)
aacA29	blaACT-28	blaSME-4	dfrB1	QnrA1	tet(H)
aadA11	blaACT-35	blaZ	dfrB5	QnrB1	tet(J)
aadA14	blaACT-37	cat	dfrG	QnrB12	tet(K)
aadA16	blaACT-7	cat(pC221)	ere(A)	QnrB19	tet(M)
aadA24	blaADC-25	catA1	ere(B)	QnrB2	tet(X)
aadA5	blaCARB-2	catA2	erm(42)	QnrB34	VanH-A
aadA6	blaCMY-100	catA3	erm(A)	QnrB4	VanR-A
aadA7b	blaHERA-1	catB10	erm(B)	QnrB6	VanS-A
aadD	blaLEN11	catB3	erm(C)	QnrB68	VanX-A
ampH	blaLEN17	catB7	floR	QnrB7	VanY-A
ant(6)-la	blaLEN22	catB8	fosA	QnrB88	VanZ-A
aph(2")-lb	blaLUT-1	cml	hugA	QnrD
aph(3')-la	blaMAL-1	cmlA1	Inu(F)	QnrS1

Table 8 - AMR Gene Variants without Cross-Reactivity

22

AMR Gene Variants
aph(3')-Ic	blaMIR-3	cmx	lsa(A)	QnrS3

a Sul3 sequences in NCBI other than accession numbers AY047357 and AB281183.

b aadA7 is not consistently detected by the AMR Gene Panel due to 4 mismatches for the reverse PCR primer and one mismatch for the PCR probe of the AAD assay.

4. Fresh versus Frozen

The equivalence of fresh bacterial isolates and bacterial suspensions, previously prepared and stored frozen at -20 °C, was established in a Fresh versus Frozen study to assess the performance of both preparations on the Acuitas AMR Gene Panel. A panel of nine (9) isolates covering all detected species and majority of the antimicrobial resistance genes in the Acuitas AMR Gene Panel were tested as a part of this study; refer to Table 9 - Fresh vs. Frozen Test Panel for the complete fresh versus frozen test panel. Each of the study isolates was cultured on blood agar from which 0.5 McFarland suspensions were prepared and aliquoted. 500 µL 0.5 McFarland aliquots were prepared for each isolate and each positive and negative external control. Positive and negatives controls prepared on Day 1 of T=0 testing were used throughout the duration of study.

lmmediately following preparation of the 0.5 McFarland suspensions, ten (10) aliquots of each isolate were extracted for performing the T=0 testing event; this data served as the control data set. The remaining aliquots for each isolate, positive control, and negative control were stored at -20 °C until the final T=56 days testing event. Storage conditions are summarized below in Table 10 - Fresh vs. Frozen Study Storage Conditions.

Ten (10) replicates of each isolate were evaluated at each of two testing events (i.e., T=0, T=56). Testing for each timepoint was split across three (3) days, with each of three (3) operators testing ten (10) replicates of a single isolate, one (1) positive control and one (1) negative control on each day of testing.

| Parent LDW
Number/External ID | Study LDW
Number | Organism | AMR Genes Identified by Whole Genome Sequencing |
|----------------------------------|---------------------|----------------------|------------------------------------------------------------|
| L00000068-001 | L00022847-001 | E. coli | ANT, DFR, E. coli gyrA Mutant, KPC, Sul1, Sul2, TEM |
| L00015886-001 | L00022848-001 | E. coli | Sul2, TEM |
| L00009154-001a | L00022849-001 | E. coli | DFR, Sul1, Sul2, TEM |
| L00009721-001 | L00022850-001 | K. pneumoniae | AAC, AAD, CMY, CTX-M-1, NDM, OXA-48, Sul1, Sul2, TEM |
| L00007800-001 | L00022851-001 | K. pneumoniae | AAC, CTX-M-1, IMP, OXA-1, Sul1, Sul2, TEM |
| L00008624-001 | L00022852-001 | P. aeruginosa | P. aeruginosa gyrA Mutant, PER, VIM |
| L00013504-001 | L00022853-001 | P. mirabilis | AAC, ANT, CMY, Sul2, TEM, VEB |
| L00013200-001b | L00022854-001 | P. mirabilis | AAC, APH, CTX-M-9, DFR, OXA-1, Sul2, TEM |

Table 9 - Fresh vs. Frozen Test Panel
---------------------------------------	--	--

23

| Parent LDW
Number/External ID | Study LDW
Number | Organism | AMR Genes Identified by Whole Genome Sequencing |
|----------------------------------------------------------------------------------------------------------------------------------------|---------------------|--------------------|-------------------------------------------------|
| ATTC BAA-2573 | L00022855-001 | E. faecalis | vanA |
| a. WGS results compiled from different sets of AMR Gene and Gyrase data.
b. WGS results compiled from merged sets of AMR Gene data. | | | |

Prepared aliquots 0.5 McFarland suspensions, described above, of isolates as well as positive and negative controls were stored under conditions summarized in Table 10 - Fresh vs. Frozen Study Storage Conditions and tested with the Acuitas AMR Gene Panel.

Table 10 - Fresh vs. Frozen Study Storage Conditions

Specimen Type	Storage Conditions	Number Isolates Tested	Replicates per Isolate
Fresh 0.5 McFarland	T = 0 days	9	10
Frozen 0.5 McFarland	T = 56 days at -15 to -25 °C	9	10

Overall, 100% agreement was observed between the T=0 and frozen T=56 time points as summarized in Table 11 - Fresh versus Frozen Results.

| Study LDW Number | Organism | Testing Event | Replicates per
Isolate | Replicates Matching
Expected Results |
|------------------|---------------|---------------|---------------------------|-----------------------------------------|
| L00022847-001 | E. coli | T=0 | 10 | 100% |
| | | T=56 | 10 | 100% |
| L00022848-001 | E. coli | T=0 | 10 | 100% |
| | | T=56 | 10 | 100% |
| L00022849-001 | E. coli | T=0 | 10 | 100% |
| | | T=56 | 10 | 100% |
| L00022850-001 | K. pneumoniae | T=0 | 10 | 100% |
| | | T=56 | 10 | 100% |
| L00022851-001 | K. pneumoniae | T=0 | 10 | 100% |
| | | T=56 | 10 | 100% |
| L00022852-001 | P. aeruginosa | T=0 | 10 | 100% |
| | | T=56 | 10 | 90%a |
| L00022853-001 | P. mirabilis | T=0 | 10 | 90%b |

Table 11 - Fresh versus Frozen Results

24

| Study LDW Number | Organism | Testing Event | Replicates per
Isolate | Replicates Matching
Expected Results |
|------------------|--------------|---------------|---------------------------|-----------------------------------------|
| | | T=56 | 10 | 100% |
| L00022854-001 | P. mirabilis | T=0 | 10 | 100% |
| | | T=56 | 10 | 100% |
| L00022855-001 | E. faecalis | T=0 | 10 | 100% |
| | | T=56 | 10 | 100% |

ª One (1) of ten (10) replicates of isolate L00022852-001 at T=56 had a false positive for ANT gene target. The time point for isolate L00022852-001 was repeated, and all ten (10) replicates returned expected gene targets. Substituting original results with retest results would improve the NPA to 100%.

b One of ten (10) replicates of L0022853-001 (T=0) had a false positive for DFR (dfrA17 gene). The time point for isolate L00022853-001 was repeated, and all ten (10) replicates returned expected gene targets. Substituting original results with retest results would improve the NPA to 100%.

5. Sample Stability - Extracted DNA

The performance of the Acuitas AMR Gene Panel was evaluated using extracted DNA stored at 15-25 °C and/or 2-8 °C prior to testing. Five (5) isolates harboring most of the AMR genes detected by the Acuitas AMR Gene Panel (Table 12 - Extracted DNA Stability Test Panel) were tested by one operator across two (2) OpGen Qualified QuantStudio 5 instruments. Three (3) replicates of each isolate were evaluated for each testing event.

| Parent LDW Number/External
ID | Study LDW
Number | Organism | AMR Genes Identified by Whole Genome
Sequencing |
|----------------------------------|---------------------|------------------|---------------------------------------------------------|
| L00005419-001 | L00021522-001 | E. coli | AAC, CTX-M-9, DFR, E. coli gyrA Mutant, Sul1, Sul2, TEM |
| L00003125-002 | L00021523-001 | K.
pneumoniae | AAC, AAD, DHA, NDM, Sul1, Sul2 |
| L00000353-001 | L00021524-001 | P. aeruginosa | None |
| L00012247-001 | L00021525-001 | P. mirabilis | AAC, CTX-M-2, Sul2, TEM |
| L00021516-001/FS10 | L00021521-001 | E. faecalis | None |

Table 12 - Extracted DNA Stability Test Panel

The isolates were extracted and stored under the conditions specified in Table 13 - Extracted DNA Storage Conditions before testing at OpGen by a single operator on two (2) QuantStudio5 instruments.

Storage Conditions	Number of Isolates Tested	Replicates per Isolate
T = 0	5	3

Table 13 - Extracted DNA Storage Conditions

25

Storage Conditions	Number of Isolates Tested	Replicates per Isolate
T = 6 hours at 15-25 °C	5	3
T = 6 hours at 15-25 °C, then 7 days at 2-8 °C	5	3
T = 7 days at 2-8 °C	5	3

The five (5) isolates were cultured on blood agar. Fourteen (14) 0.5 McFarland suspensions per isolate were prepared and DNA extracts per isolate were pooled and aliquoted (500 µL) for storage (Table 13). Three (3) 150µL replicates of extracted DNA were evaluated per isolate per storage condition.

Overall agreement between T=0 and storage conditions are summarized in Table 14 - Extracted DNA Stability Results. There was 100% agreement for all samples and storage conditions compared with the T=0 timepoint, supporting the claim that DNA extracted on the QIAGEN EZ1 Advanced XL System can be stored as follows before testing on the OpGen Qualified QuantStudio 5 instrument:

Up to six (6) hours at room temperature; ●
Up to seven (7) days at 2-8 °C; .
Up to six (6) hours at room temperature followed by up to seven (7) days at 2-8 °C. .

| Study LDW
Number
(Organism) | Testing Event | Replicates
Tested | Replicates
Matching
T=0 Results
(%) |
|-----------------------------------|------------------------------------------------|----------------------|----------------------------------------------|
| L00021521-001
(E. faecalis) | T = 6 hours at 15-25 °C | 3 | 3/3 (100%) |
| | T = 6 hours at 15-25 °C, then 7 days at 2-8 °C | 3 | 3/3 (100%) |
| | T = 7 days at 2-8 °C | 3 | 3/3 (100%) |
| L00021522-001
(E. coli) | T = 6 hours at 15-25 °C | 3 | 3/3 (100%) |
| | T = 6 hours at 15-25 °C, then 7 days at 2-8 °C | 3 | 3/3 (100%) |
| | T = 7 days at 2-8 °C | 3 | 3/3 (100%) |
| L00021523-001
(K. pneumoniae) | T = 6 hours at 15-25 °C | 3 | 3/3 (100%) |
| | T = 6 hours at 15-25 °C, then 7 days at 2-8 °C | 3 | 3/3 (100%) |
| | T = 7 days at 2-8 °C | 3 | 3/3 (100%) |
| L00021524-001
(P. aeruginosa) | T = 6 hours at 15-25 °C | 3 | 3/3 (100%) |
| | T = 6 hours at 15-25 °C, then 7 days at 2-8 °C | 3 | 3/3 (100%) |
| | T = 7 days at 2-8 °C | 3 | 3/3 (100%) |

Table 14 - Extracted DNA Stability Results

26

| Study LDW
Number
(Organism) | Testing Event | Replicates
Tested | Replicates
Matching
T=0 Results
(%) |
|-----------------------------------|------------------------------------------------|----------------------|----------------------------------------------|
| L00021525-001
(P. mirabilis) | T = 6 hours at 15-25 °C | 3 | 3/3 (100%) |
| | T = 6 hours at 15-25 °C, then 7 days at 2-8 °C | 3 | 3/3 (100%) |
| | T = 7 days at 2-8 °C | 3 | 3/3 (100%) |

Sample Stability - Frozen DNA 6.

L00021939-001

L00021940-001

The performance of the Acuitas AMR Gene Panel was also evaluated using extracted DNA stored up to 30 days at -15 °C to -25 °C. Five (5) isolates harboring most of the AMR genes detected by the Acuitas AMR Gene Panel (Table 15 - Frozen DNA Test Panel) were tested by one (1) operator across two (2) OpGen Qualified QuantStudio 5 instruments. Three (3) replicates of each isolate were evaluated for each testing event.

| Parent LDW
Number/External ID | Study LDW Number | Organism | AMR Genes Identified by Whole Genome
Sequencing |
|----------------------------------|------------------|----------------------|-------------------------------------------------------------------|
| L00005419-001 | L00021936-001 | E. coli | AAC, CTX-M-9, DFR, E. coli gyrA Mutant, Sul1,
Sul2, TEM |
| L00003125-002 | L00021937-001 | K. pneumoniae | AAC, AAD, DHA, NDM, Sul1, Sul2 |
| L00000353-001 | L00021938-001 | P. aeruginosa | None |

			Table 15 - Frozen DNA Test Panel
--	--	--	----------------------------------	--

Fifteen (15) 0.5 McFarland suspensions were prepared per isolate and DNA extracts per each isolate were pooled and aliquoted into unique 150 µL aliquots. Three (3) DNA aliquots per isolate were tested immediately (T=0). Remaining aliquots were stored at -15 to -25 °C for 14 or 30 days before testing.

P. mirabilis

E. faecalis

AAC, CTX-M-2, Sul2, TEM

None

Overall agreement was 100% between the T=0 timepoint and tested storage conditions (Table 16 -Frozen DNA Results).

| Study LDW Number | Timepoint | Replicates
Tested | Replicates Matching
Expected Results (%) |
|----------------------------|---------------------|----------------------|---------------------------------------------|
| L00021936-001
(E. coli) | T=14 days at -20 °C | 3 | 3/3 (100%) |
| | T=30 days at -20 °C | 3 | 3/3 (100%) |
| L00021937-001 | T=14 days at -20 °C | 3 | 3/3 (100%) |

Table 16 - Frozen DNA Results

L00012247-001

L00021516-001/ FS10

27

| Study LDW Number | Timepoint | Replicates
Tested | Replicates Matching
Expected Results (%) |
|------------------|---------------------|----------------------|---------------------------------------------|
| (K. pneumoniae) | T=30 days at -20 °C | 3 | 3/3 (100%) |
| L00021938-001 | T=14 days at -20 °C | 3 | 3/3 (100%) |
| (P. aeruginosa) | T=30 days at -20 °C | 3 | 3/3 (100%) |
| L00021939-001 | T=14 days at -20 °C | 3 | 3/3 (100%) |
| (P. mirabilis) | T=30 days at -20 °C | 3 | 3/3 (100%) |
| L00021940-001 | T=14 days at -20 °C | 3 | 3/3 (100%) |
| (E. faecalis) | T=30 days at -20 °C | 3 | 3/3 (100%) |

7. Sample Stability - Plated DNA

This study evaluated the stability of prepared Acuitas AMR Gene Panel plates, containing extracted DNA and Master Mix, after storage for 6 hours at 2-8 °C. Five (5) isolates harboring most of the AMR genes detected by the Acuitas AMR Gene Panel (Table 17 - Prepared Plate Test Panel) were tested by one (1) operator across two (2) OpGen Qualified QuantStudio 5 instruments. Three (3) replicates of each isolate were evaluated for each testing event.

| Original LDW
Number/External ID | LDW Number for Study | Organism | AMR Genes Identified by Whole Genome
Sequencing |
|------------------------------------|----------------------|----------------------|-------------------------------------------------------------------|
| L00005419-001 | L00021931-001 | E. coli | AAC, CTX-M-9, DFR, E. coli gyrA Mutant, Sul1,
Sul2, TEM |
| L00003125-002 | L00021932-001 | K. pneumoniae | AAC, AAD, DHA, NDM, Sul1, Sul2 |
| L00000353-001 | L00021933-001 | P. aeruginosa | None |
| L00012247-001 | L00021934-001 | P. mirabilis | AAC, CTX-M-2, Sul2, TEM |
| L00021516-001/FS10 | L00021935-001 | E. faecalis | None |

Table 17 - Prepared Plate Test Panel

Seven (7) 0.5 McFarland suspensions were prepared and extracted per isolate. Extracts per isolate were pooled and aliquoted (500 µL). Extracted DNA samples (150 µL) were combined with Acuitas AMR Gene Panel Master Mix (CP3402) and added to Acuitas AMR Gene Panel assay plates (CP3230). Prepared plates were stored for zero or 6 hours at 2-8 °C (Table 18 - Prepared Plate Storage Conditions) and tested.

				Table 18 - Prepared Plate Storage Conditions
--	--	--	--	----------------------------------------------

Storage Condition	Number of Isolates Tested	Replicates per Isolate
T = 0	5	3

28

T = 6 hours at 2-8 °C	5	3
-----------------------	---	---

Overall agreement was 100% between T=0 and T=6 hours at 2-8 °C (Table 19 - Prepared Plate Results).

| Study LDW Number (Organism) | Timepoint | Replicates Tested | Replicates matching
T=0 Results (%) |
|-------------------------------|---------------------|-------------------|----------------------------------------|
| L00021931-001 (E. coli) | T = 6 hours, 2-8 °C | 3 | 3/3 (100%) |
| L00021932-001 (K. pneumoniae) | T = 6 hours, 2-8 °C | 3 | 3/3 (100%) |
| L00021933-001 (P. aeruginosa) | T = 6 hours, 2-8 °C | 3 | 3/3 (100%) |
| L00021934-001 (P. mirabilis) | T = 6 hours, 2-8 °C | 3 | 3/3 (100%) |
| L00021935-001 (E. faecalis) | T = 6 hours, 2-8 °C | 3 | 3/3 (100%) |

Table 19 - Prepared Plate Results

8. Media Equivalency

The equivalence of blood agar and MacConkey agar media when used to culture pure bacterial colonies for use with the Acuitas AMR Gene Panel test was established by evaluating the performance of fifty-two (52) isolates harboring most of the AMR genes detected by the Acuitas AMR Gene Panel (Table 20 - Media Equivalency Test Panel).

Table 20 - Media Equivalency Test Panel

| Organism | Species ID and AMR Genes
Detected by Whole Genome Sequencing | Study LDW
Number | Parent LDW
Number |
|---------------|------------------------------------------------------------------------|---------------------|----------------------|
| E. coli | AAC, CMY, CTX-M-1, DFR, E. coli gyrA Mutant, OXA-1, SHV, Sul1,
Sul2 | L00021535-001 | L00009217-001 |
| E. coli | AAC, ANT, CTX-M-1, E. coli gyrA Mutant, Sul1, Sul2, TEM | L00021536-001 | L00008878-001 |
| E. coli | None | L00021537-001 | L00015883-001 |
| E. coli | Sul2 | L00021538-001 | L00015885-001 |
| E. coli | DFR, Sul2, TEM | L00021539-001 | L00009368-001 |
| E. coli | ANT, E. coli gyrA Mutant, KPC, SHV, TEM | L00021540-001 | L00009104-001 |
| E. coli | AAC, DFR, Sul1, Sul2, TEM | L00021541-001 | L00009121-001 |
| E. coli | CMY, CTX-M-1, E. coli gyrA Mutant, Sul2, TEM | L00021542-001 | L00008677-001 |
| E. coli | KPC, TEM | L00021543-001 | L00009056-001 |
| E. coli | None | L00021544-001 | L00015917-001 |
| Organism | Species ID and AMR Genes
Detected by Whole Genome Sequencing | Study LDW
Number | Parent LDW
Number |
| E. coli | AAC, Sul2, TEM | L00021575-001 | L00014163-001 |
| E. coli | AAC, CTX-M-1, DFR, E. coli gyrA Mutant, OXA-1, Sul1, Sul2 | L00021576-001 | L00015876-001 |
| E. coli | None | L00021577-001 | L00015883-001 |
| K. pneumoniae | AAC, AAD, CTX-M-1, DFR, KPC, NDM, Sul1, TEM | L00021545-001 | L00005469-001 |
| K. pneumoniae | AAC, AAD, APH, CTX-M-1, OXA-1, OXA-48, Sul1, TEM | L00021546-001 | L00008681-001 |
| K. pneumoniae | None | L00021547-001 | L00015913-001 |
| K. pneumoniae | None | L00021548-001 | L00015918-001 |
| K. pneumoniae | AAC, KPC, Sul1, TEM | L00021549-001 | L00009602-001 |
| K. pneumoniae | CTX-M-1, RMT, Sul2, TEM | L00021550-001 | L00011112-001 |
| K. pneumoniae | AAC, AAD, KPC, OXA-9, TEM | L00021551-001 | L00007709-001 |
| K. pneumoniae | AAC, CTX-M-1, Sul2, TEM | L00021552-001 | L00007888-001 |
| K. pneumoniae | AAC, CTX-M-1, OXA-1, RMT, Sul1 | L00021553-001 | L00009765-001 |
| K. pneumoniae | None | L00021554-001 | L00000120-001 |
| K. pneumoniae | None | L00021578-001 | L00015918-001 |
| K. pneumoniae | AAC, AAD, CTX-M-1, OXA-1, OXA-48, Sul1 | L00021579-001 | L00015736-001 |
| K. pneumoniae | AAC, CTX-M-1, OXA-1, OXA-48 | L00021580-001 | L00015867-001 |
| P. aeruginosa | AAC, ANT, OXA-1, P. aeruginosa gyrA Mutant, VIM | L00021555-001 | L00008639-001 |
| P. aeruginosa | AAC, P. aeruginosa gyrA Mutant | L00021556-001 | L00004931-001 |
| P. aeruginosa | None | L00021557-001 | L00015786-001 |
| P. aeruginosa | AAC, P. aeruginosa gyrA Mutant | L00021558-001 | L00015788-001 |
| P. aeruginosa | None | L00021559-001 | L00015789-001 |
| P. aeruginosa | None | L00021560-001 | L00015790-001 |
| P. aeruginosa | AAC, P. aeruginosa gyrA Mutant | L00021561-001 | L00010360-001 |
| P. aeruginosa | AAC, CTX-M-1, OXA-1, TEMa | L00021562-001 | L00008666-001 |
| P. aeruginosa | None | L00021563-001 | L00015792-001 |
| P. aeruginosa | P. aeruginosa gyrA Mutant, VIM | L00021564-001 | L00009440-001 |
| Organism | Species ID and AMR Genes
Detected by Whole Genome Sequencing | Study LDW
Number | Parent LDW
Number |
| P. aeruginosa | None | L00021581-001 | L00010871-001b |
| P. aeruginosa | AAC, P. aeruginosa gyrA Mutant, VIM | L00021582-001 | L00015582-001 |
| P. aeruginosa | AAC, P. aeruginosa gyrA Mutant, VIM | L00021583-001 | L00015586-001 |
| P. mirabilis | AAC, APH, CTX-M-9, DFR, OXA-1, Sul2, TEM | L00021565-001 | L00012352-001 |
| P. mirabilis | Sul2, TEM | L00021566-001 | L00015759-001 |
| P. mirabilis | AAC, ANT, CTX-M-9, DFR, Sul2, TEM | L00021567-001 | L00012613-001 |
| P. mirabilis | None | L00021568-001 | L00015801-001 |
| P. mirabilis | AAC, armA, CMY, TEM | L00021569-001 | L00012786-001 |
| P. mirabilis | AAC, APH, OXA-1, Sul2 | L00021570-001 | L00015806-001 |
| P. mirabilis | AAC, CTX-M-2, OXA-9, Sul2, TEM | L00021571-001 | L00012812-001 |
| P. mirabilis | None | L00021572-001 | L00015828-001 |
| P. mirabilis | CMY, NDM, Sul2 | L00021573-001 | L00012564-001 |
| P. mirabilis | None | L00021574-001 | L00015912-001 |
| P. mirabilis | None | L00021584-001 | L00013441-001 |
| P. mirabilis | TEM | L00021585-001 | L00015645-001 |
| P. mirabilis | None | L00021586-001 | L00015912-001 |

29

30

a Re-sequencing of isolate L00021562-001 unambiguously identified the species as P. aeruginosa and detected AMR gene variants P. aeruginosa gyrA Mutant and VEB.

b. WGS results compiled from different sets of AMR gene and Gyrase data.

Thirteen (13) isolates per organism (E. coli, K. pneumoniae, P. aeruginosa, and P. mirabilis) were cultured on both blood agar and MacConkey agar. E. faecalis was not tested as it does not grow on MacConkey agar. A 0.5 McFarland suspension was prepared from pure colonies on the blood and MacConkey agar for testing with the Acuitas AMR Gene Panel.

Percent agreement was determined for detection of AMR genes by the Acuitas AMR Gene Panel versus Whole Genome Sequencing for each isolate replicate from blood and MacConkey agar.

Acuitas AMR Gene Panel results were compiled and analyzed to show percent agreement with the two comparator methods (well-established automated species identification methods and Whole Genome Sequencing) for each combination of organism, AMR gene and agar media, as summarized in Table 21 - Performance from MacConkey and Blood Agar, which represents original results without inclusion or analysis of repeat results. Isolate (testing event) agreement across all AMR genes and both types of agar media ranged from 62 to 92% across the four organisms versus the two comparator

31

methods (not shown directly in Table 23). Incorporation of repeat test results as described in the footnotes of Table 21 would improve isolate agreement as follows: E. coli on blood (100%), E. coli on MacConkey (100%), K. pneumoniae on blood (100%), K. pneumoniae on MacConkey (100%), P. aeruginosa on blood (100%), P. aeruginosa on MacConkey (100%), P. mirabilis on blood (92%), and P. mirabilis on MacConkey (92%).

The study did not uncover evidence of a media effect between blood and MacConkey agar, determining that both are suitable for the Acuitas AMR Gene Panel.

| Organism Marker | Number
of Testing
Events | Blood Agar |----------------------|---- | | (TP/(TP+FN))
(95% CI) | NPA
(TN/(TN+FP))
(95% CI) | PPA
(TP/(TP+FN))
(95% CI) | NPA
(TN/(TN+FP))
(95% CI) |
| | AAC (64.57 - 100) | 100% (6/6)
(60.97 - 100) | 100% (7/7)
(64.57 - 100) | 100% (6/6)
(60.97 - 100) |
| | ANT (34.24 - 100) | 100% (11/11)
(74.12 - 100) | 100% (2/2)
(34.24 - 100) | 100% (11/11)
(74.12 - 100) |
| | CMY (34.24 - 100) | 100% (11/11)
(74.12 - 100) | 100% (2/2)
(34.24 - 100) | 100% (11/11)
(74.12 - 100) |
| | CTX-M-1 (60.97 - 100) | 100% (7/7)
(64.57 - 100) | 100% (6/6)
(60.97 - 100) | 100% (7/7)
(64.57 - 100) |
| | CTX-M-2 (77.19 - 100) | - (77.19 - 100) |
| | CTX-M-9 (77.19 - 100) | - (77.19 - 100) |
| | DFR (51.01 - 100) | 100% (9/9)
(70.08 - 100) | 100% (4/4)
(51.01 - 100) | 100% (9/9)
(70.08 - 100) |
| E. coli (64.57 - 100) | 100% (6/6)
(60.97 - 100) | 100% (7/7)
(64.57 - 100) | 100% (6/6)
(60.97 - 100) |
| | KPC (34.24 - 100) | 100% (11/11)
(74.12 - 100) | 100% (2/2)
(34.24 - 100) | 100% (11/11)
(74.12 - 100) |
| | MCR-1 (77.19 - 100) | - (77.19 - 100) |
| | OXA-1 (51.01 - 100) | 100% (9/9)
(70.08 - 100) | 100% (4/4)
(51.01 - 100) | 100% (9/9)
(70.08 - 100) |
| | OXA-9 (77.19 - 100) | - (77.19 - 100) |
| | SHV (34.24 - 100) | 100% (11/11)
(74.12 - 100) | 100% (2/2)
(34.24 - 100) | 100% (11/11)
(74.12 - 100) |
| | Sul1 (51.01 - 100) | 100% (9/9)
(70.08 - 100) | 100% (4/4)
(51.01 - 100) | 100% (9/9)
(70.08 - 100) |
| | Sul2 (67.56 - 100) | 100% (5/5)
(56.55 - 100) | 100% (8/8)
(67.56 - 100) | 100% (5/5)
(56.55 - 100) |
| | TEM (70.08 - 100) | 100% (4/4)
(51.01 - 100) | 100% (9/9)
(70.08 - 100) | 100% (4/4)
(51.01 - 100) |
| K. pneumoniae | AAC (67.56 - 100) | 100% (5/5)
(56.55 - 100) | 100% (8/8)
(67.56 - 100) | 100% (5/5)
(56.55 - 100) |
| Organism Marker | Number
of Testing
Events | Blood Agar | | (TP/(TP+FN))
(95% CI) | NPA
(TN/(TN+FP))
(95% CI) | PPA
(TP/(TP+FN))
(95% CI) | NPA
(TN/(TN+FP))
(95% CI) |
| | AAD a (51.01 - 100) | 100% (9/9)
(70.08 - 100) | 75% (3/4)
(30.06 -
95.44) | 100% (9/9)
(70.08 - 100) |
| | APH (20.65 - 100) | 100% (12/12)
(75.75 - 100) | 100% (1/1)
(20.65 - 100) | 100% (12/12)
(75.75 - 100) |
| | CMY (77.19 - 100) | - (77.19 - 100) |
| | CTX-M-1 b (48.69 -
97.43) | 100% (6/6)
(60.97 - 100) | 100% (7/7)
(64.57 - 100) | 100% (6/6)
(60.97 - 100) |
| | CTX-M-9 (77.19 - 100) | - (77.19 - 100) |
| | DFR (20.65 - 100) | 100% (12/12)
(75.75 - 100) | 100% (1/1)
(20.65 - 100) | 100% (12/12)
(75.75 - 100) |
| | DHA (77.19 - 100) | - (77.19 - 100) |
| | IMP (77.19 - 100) | - (77.19 - 100) |
| | KPC (43.85 - 100) | 100% (10/10)
(72.25 - 100) | 100% (3/3)
(43.85 - 100) | 100% (10/10)
(72.25 - 100) |
| | NDM (20.65 - 100) | 100% (12/12)
(75.75 - 100) | 100% (1/1)
(20.65 - 100) | 100% (12/12)
(75.75 - 100) |
| | OXA-1 (51.01 - 100) | 100% (9/9)
(70.08 - 100) | 100% (4/4)
(51.01 - 100) | 100% (9/9)
(70.08 - 100) |
| | OXA-9 (20.65 - 100) | 100% (12/12)
(75.75 - 100) | 100% (1/1)
(20.65 - 100) | 100% (12/12)
(75.75 - 100) |
| | OXA-48 (43.85 - 100) | 100% (10/10)
(72.25 - 100) | 100% (3/3)
(43.85 - 100) | 100% (10/10)
(72.25 - 100) |
| | RMT b (9.45 - 90.55) (74.12 - 100) | 100% (2/2)
(34.24 - 100) | 100% (11/11)
(74.12 - 100) |
| | Sul1 a (56.55 - 100) | 100% (8/8)
(67.56 - 100) | 80% (4/5)
(37.55 -
96.38) | 100% (8/8)
(67.56 - 100) |
| | Sul2 (34.24 - 100) | 100% (11/11)
(74.12 - 100) | 100% (2/2)
(34.24 - 100) | 100% (11/11)
(74.12 - 100) |
| | TEM b (43.65 -
96.99) | 100% (7/7)
(64.57 - 100) | 100% (6/6)
(60.97 - 100) | 100% (7/7)
(64.57 - 100) |
| P. aeruginosa | AAC (48.69 -
97.43) | 100% (6/6)
(60.97 - 100) (48.69 -
97.43) | 100% (6/6)
(60.97 - 100) |
| Marker | Number
of Testing
Events | Blood Agar | Organism | (TP/(TP+FN))
(95% CI) | NPA
(TN/(TN+FP))
(95% CI) | PPA
(TP/(TP+FN))
(95% CI) | NPA
(TN/(TN+FP))
(95% CI) |
| | ANT (20.65 - 100) | 100% (12/12)
(75.75 - 100) | 100% (1/1)
(20.65 - 100) | 100% (12/12)
(75.75 - 100) |
| | CTX-M-1 (0 - 79.35) (75.75 - 100) | 0% (0/1)
(0 - 79.35) (75.75 - 100) |
| | CTX-M-2 (77.19 - 100) | - (77.19 - 100) |
| | P.
aeruginosa
gyrAc | 12 d (64.57 - 100) | 100% (5/5)
(56.55 - 100) (48.69 -
97.43) | 100% (5/5)
(56.55 - 100) |
| | KPC (77.19 - 100) | - (77.19 - 100) |
| | NDM (77.19 - 100) | - (77.19 - 100) |
| | OXA-1 (9.45 - 90.55) (74.12 - 100) | 50% (1/2)
(9.45 - 90.55) (74.12 - 100) |
| | PER (77.19 - 100) | - (77.19 - 100) |
| | SHV (77.19 - 100) | - (77.19 - 100) |
| | TEM (0 - 79.35) (75.75 - 100) | 0% (0/1)
(0 - 79.35) (75.75 - 100) |
| | VEBd (66.69 -
98.63) | - (66.69 -
98.63) |
| | VIM (51.01 - 100) | 100% (9/9)
(70.08 - 100) | 100% (4/4)
(51.01 - 100) | 100% (9/9)
(70.08 - 100) |
| P. mirabilis | AACe, f (37.55 -
96.38) | 75% (6/8)
(40.93 -
92.85) | 100% (5/5)
(56.55 - 100) | 100% (8/8)
(67.56 - 100) |
| | ANT (20.65 - 100) | 100% (12/12)
(75.75 - 100) | 100% (1/1)
(20.65 - 100) | 100% (12/12)
(75.75 - 100) |
| | APH (34.24 - 100) | 100% (11/11)
(74.12 - 100) | 100% (2/2)
(34.24 - 100) | 100% (11/11)
(74.12 - 100) |
| | armAg (20.65 - 100) (64.61 -
98.51) | 100% (1/1)
(20.65 - 100) (64.61 -
98.51) |
| | CMY (34.24 - 100) | 100% (11/11)
(74.12 - 100) | 100% (2/2)
(34.24 - 100) | 100% (11/11)
(74.12 - 100) |
| | CTX-M-1h (66.69 -
98.63) | - (77.19 - 100) |
| Organism Marker | Number
of Testing
Events | Blood Agar | | (TP/(TP+FN))
(95% CI) | NPA
(TN/(TN+FP))
(95% CI) | PPA
(TP/(TP+FN))
(95% CI) | NPA
(TN/(TN+FP))
(95% CI) |
| | CTX-M-2 f,i (0 - 79.35) (64.61 -
98.51) | 100% (1/1)
(20.65 - 100) | 100% (12/12)
(75.75 - 100) |
| | CTX-M-9 (34.24 - 100) | 100% (11/11)
(74.12 - 100) | 100% (2/2)
(34.24 - 100) | 100% (11/11)
(74.12 - 100) |
| | DFR (34.24 - 100) | 100% (11/11)
(74.12 - 100) | 100% (2/2)
(34.24 - 100) | 100% (11/11)
(74.12 - 100) |
| | KPC (77.19 - 100) | - (77.19 - 100) |
| | NDM (20.65 - 100) | 100% (12/12)
(75.75 - 100) | 100% (1/1)
(20.65 - 100) | 100% (12/12)
(75.75 - 100) |
| | OXA-1 h (34.24 - 100) (62.26 -
98.38) | 100% (2/2)
(34.24 - 100) | 100% (11/11)
(74.12 - 100) |
| | OXA-9 f (0 - 79.35) (75.75 - 100) | 100% (1/1)
(20.65 - 100) | 100% (12/12)
(75.75 - 100) |
| | OXA-48 h (66.69 -
98.63) | - (77.19 - 100) |
| | Sul2 f (43.65 -
96.99) | 100% (7/7)
(64.57 - 100) | 100% (6/6)
(60.97 - 100) | 100% (7/7)
(64.57 - 100) |
| | TEM f (43.65 -
96.99) | 100% (7/7)
(64.57 - 100) | 100% (6/6)
(60.97 - 100) | 100% (7/7)
(64.57 - 100) |
| | VEB (77.19 - 100) | - (77.19 - 100) |
| | VIM (77.19 - 100) | - (77.19 - 100) | | Resistance
| | MacConkey Agar | |
-----------------------|--------------------------------|-----------------------------------|-------------------------------------|-----------------------------------|-------------------------------------|
| | PPA
| 13 | 100% (7/7)
| 13 | 100% (2/2)
| 13 | 100% (2/2)
| 13 | 100% (6/6)
| 13 | - | 100% (13/13)
| 100% (13/13)
| 13 | - | 100% (13/13)
| 100% (13/13)
| 13 | 100% (4/4)
| E. coli gyrA | 13 | 100% (7/7)
| 13 | 100% (2/2)
| 13 | - | 100% (13/13)
| 100% (13/13)
| 13 | 100% (4/4)
| 13 | - | 100% (13/13)
| 100% (13/13)
| 13 | 100% (2/2)
| 13 | 100% (4/4)
| 13 | 100% (8/8)
| 13 | 100% (9/9)
| 13 | 100% (8/8)
| Resistance
| | MacConkey Agar | |
| | PPA
| 13 | 100% (4/4)
| 13 | 100% (1/1)
| 13 | - | 100% (13/13)
| 100% (13/13)
| 13 | 85.7% (6/7)
| 13 | - | 100% (13/13)
| 100% (13/13)
| 13 | 100% (1/1)
| 13 | - | 100% (13/13)
| 100% (13/13)
| 13 | - | 100% (13/13)
| 100% (13/13)
| 13 | 100% (3/3)
| 13 | 100% (1/1)
| 13 | 100% (4/4)
| 13 | 100% (1/1)
| 13 | 100% (3/3)
| 13 | 50% (1/2)
| 100% (11/11)
| 13 | 100% (5/5)
| 13 | 100% (2/2)
| 13 | 83.3% (5/6)
| 13 | 85.7% (6/7)
| 85.7% (6/7)
| Resistance
| | MacConkey Agar | |
| | PPA
| 13 | 100% (1/1)
| 13 | 0% (0/1)
| 100% (12/12)
| 100% (12/12)
| 13 | - | 100% (13/13)
| 100% (13/13)
| 100% (7/7)
| 85.7% (6/7)
| 13 | - | 100% (13/13)
| 100% (13/13)
| 13 | - | 100% (13/13)
| 100% (13/13)
| 13 | 50% (1/2)
| 100% (11/11)
| 100% (11/11)
| 13 | - | 100% (13/13)
| 100% (13/13)
| 13 | - | 100% (13/13)
| 100% (13/13)
| 13 | 0% (0/1)
| 100% (12/12)
| 100% (12/12)
| 13 | - | 92.3% (12/13)
| 92.3% (12/13)
| 13 | 100% (4/4)
| 13 | 80% (4/5)
| 13 | 100% (1/1)
| 13 | 100% (2/2)
| 13 | 100% (1/1)
| 91.7% (11/12)
| 91.7% (11/12)
| 13 | 100% (2/2)
| 13 | - | 92.3% (12/13)
| 100% (13/13)
| Resistance
| | MacConkey Agar | |
| | PPA
| 13 | 0% (0/1)
| 91.7% (11/12)
| 13 | 100% (2/2)
| 13 | 100% (2/2)
| 13 | - | 100% (13/13)
| 100% (13/13)
| 13 | 100% (1/1)
| 13 | 100% (2/2)
| 90.9% (10/11)
| 13 | 0% (0/1)
| 100% (12/12)
| 13 | - | 92.3% (12/13)
| 100% (13/13)
| 13 | 83.3% (5/6)
| 13 | 83.3% (5/6)
| 13 | - | 100% (13/13)
| 100% (13/13)
| 13 | - | 100% (13/13)
| 100% (13/13)

Table 21 - Performance from MacConkey and Blood Agar

32

33

34

ª False negatives were obtained with one isolate on MacConkey agar for AAD and Sul1. This isolate was repeated on both media, and true positives were obtained for AAD and Sul1 on both media. Substituting original results would improve isolate (testing event) agreement across all AMR genes to 100% for K. pneumoniae on MacConkey media.

b False negatives were obtained with one isolate on blood agar for CTX-M-1, RMT and TEM. Repeat testing of this isolate on both media produced false negatives for CTX-M-1, RMT and TEM on both is not consistent with a media effect. Adjudication testing was performed with this isolate in duplicate on both agar media and true positives for CTX-M-1, RMT and TEM were obtained for all samples. Substituting original results with these adjudication test results would improve isolate (testing event) agreement across all AMR genes to 100% for K. pneumoniae on blood media.

° A false negative was obtained with one isolate on MacConkey agar for P. aeruginosa gyrA mutant. This isolate was repeated on both media, and true positives were obtained for P. aeruginosa gyrA mutant on both media. Substituting original results with retest results would improve isolate (testing event) agreement across all AMR genes to 100% for P. aeruginosa on both media.

1 One isolate was not evaluated for P. aeruginosa gyrA due to a mismatch in species ID from WGS and the expected species. E. coli was reported by whole genome sequencing, but the isolate was expected to be P. aeruginosa. Results of re-sequencing for this isolate agree with the Acuitas AMR Gene panel results for the positives for P. aeruginosa gyrase mutant and VEB with true negatives for all other AMR gene variants on both media.

® False positives were obtained with two isolates on blood agar for AAC. The two isolates were repeated on both media, and true negatives were obtained for AAC for both isolates on both media.

'False negatives were obtained with one isolate on blood agar for AAC, CTX-M-2, OXA-9, Sul2 and TEM. This isolate was repeated on both media, and true positives were obtained for AAC, CTX-M-2, OXA-9, Sul2 and TEM on both media.

35

9 False positives were obtained with one isolate on both media for armA. Substituting original results would improve isolate (testing event) across all AMR genes to 92% (12/13) for P. mirabils on both media. The lack of 100% agreement is due to the false positives on both media for armA, which is not consistent with a media effect. " False positives were obtained with one isolate on blood agar for CTX-M-1, OXA-1 and OXA-48. This isolate was repeated on both media, and true negatives were obtained for CTX-M-1, OXA-1 and OXA-48 on both media. ' A false positive was obtained with one isolate on blood agar for CTX-M-2. This isolate was repeated on both media, and true negatives were obtained for CTX-M-2 on both media.

9. Carry-over/Cross-Contamination

Carry-over/cross contamination between samples tested by the Acuitas AMR Gene Panel was evaluated. A panel of twelve (12) isolates representing organisms targeted by the Acuitas AMR Gene Panel test, harboring a variety of the targeted resistance genes, and a negative control isolate were subject to testing; refer to Table 22 - Carry-over/Cross-Contamination Test Panel.

| Parent LDW
Number/
External ID | Study LDW
Number | Organism | AMR Genes Detected by Whole Genome
Sequencing | Operator |
|--------------------------------------|---------------------|---------------|--------------------------------------------------------|----------|
| L00008802-001 | L00021588-001 | E. coli | AAC, E. coli gyrA Mutant, OXA-1, SHV, Sul1 | 1 |
| L00009106-001 | L00021589-001 | E. coli | AAC, Sul1, TEM | 1 |
| L00000411-001 | L00021590-001 | K. pneumoniae | AAC, AAD, APH, KPC, Sul1, TEM | 1 |
| L00011693-001 | L00021591-001 | P. aeruginosa | AAC, ANT, CTX-M-2, P. aeruginosa gyrA Mutant | 1 |
| L00012604-001 | L00021592-001 | P. mirabilis | AAC, APH, CTX-M-9, DFR, OXA-1, Sul2, TEM | 1 |
| L00014668-001 | L00021593-001 | E. faecalis | None | 1 |
| L00008788-001 | L00021594-001 | E. coli | AAC, CMY, E. coli gyrA Mutant, SHV, Sul1, Sul2,
TEM | 2 |
| L00009581-001 | L00021595-001 | K. pneumoniae | AAC, AAD, CTX-M-1, DFR, OXA-9, Sul1, Sul2, TEM | 2 |
| L00007838-001 | L00021596-001 | K. pneumoniae | AAC, AAD, CTX-M-1, NDM, RMT, Sul2, TEM | 2 |
| L00007586-001 | L00021597-001 | P. aeruginosa | P. aeruginosa gyrA Mutant, VIM | 2 |
| L00000276-001 | L00021598-001 | P. mirabilis | AAC, armA, CTX-M-1, Sul2, TEM | 2 |
| L00021587-001 | L00021599-001 | E. faecalis | None | 2 |

Table 22 - Carry-over/Cross-Contamination Test Panel

Two (2) operators prepared and blinded a panel of six (6) positive isolates and six (6) negative control isolates (a S. aureus isolate negative for all resistance markers) for testing by the other operator. lsolates were cultured on blood agar, 0.5 McFarland suspensions were prepared, and DNA was extracted. Positive and negative control samples were tested in an alternating fashion on the Acuitas AMR Gene Panel assay plates. There was 100% agreement between observed and expected positive/negative results (Table 23 - Carry-over/Cross-Contamination Results) without evidence of carry-over/cross-contamination between samples tested by the Acuitas AMR Gene Panel.

36

| Sample Number
For Study | #
Results | # Positive Results/# Expected Positive
Results (%) | # Negative Results/# Expected Negative
Results (%) |
|-----------------------------------|--------------|-------------------------------------------------------|-------------------------------------------------------|
| L00021588-001 | 17 | 6/6 (100%) | 11/11 (100%) |
| L00021589-001 | 17 | 4/4 (100%) | 13/13 (100%) |
| L00021590-001 | 18 | 6/6 (100%) | 12/12 (100%) |
| L00021591-001 | 14 | 5/5 (100%) | 9/9 (100%) |
| L00021592-001 | 18 | 7/7 (100%) | 11/11 (100%) |
| L00021593-001 | 1 | 0/0 (-) | 1/1 (100%) |
| L00021594-001 | 17 | 8/8 (100%) | 9/9 (100%) |
| L00021595-001 | 18 | 8/8 (100%) | 10/10 (100%) |
| L00021596-001 | 18 | 7/7 (100%) | 11/11 (100%) |
| L00021597-001 | 14 | 3/3 (100%) | 11/11 (100%) |
| L00021598-001 | 18 | 5/5 (100%) | 13/13 (100%) |
| L00021599-001 | 1 | 0/0 (-) | 1/1 (100%) |
| CP3416 -
Negative Control (NC) | 420 | 0/0 (-) | 420/420 (100%) |

Table 23 - Carry-over/Cross-Contamination Results ª

ª Table includes reported AMR gene results per organism as described in Table 1 along with species ID results for E. coli and P. aeruginosa as used in conjunction with mutant gyrase results for these two organisms.

B. CLINICAL PERFORMANCE

1. Introduction

The performance characteristics of the Acuitas AMR Gene Panel with bacterial isolates were determined in a multi-site investigational clinical study by comparing the Acuitas AMR Gene Panel with Whole Genome Sequencing (WGS), species identification by MALDI-TOF MS, and Antimicrobial Susceptibility Testing (AST) by broth microdilution. Four geographically diverse sites participated in the testing of isolates either prospectively collected or stocked and de-identified for use in the Clinical Performance Evaluation.

lsolates included in the study had been previously identified as Enterobacterales, Pseudomonas aeruginosa, or Enterococcus faecalis. Study samples included bacterial isolates grown on blood agar.

The Clinical Performance Evaluation was performed between September 11, 2018 and May 2, 2019 with final results reported for a total of 1,307 isolate samples (1,224 clinical stock isolate samples and 83 prospective isolate samples).

. Isolates harboring rare resistance genes were replicated with a goal of achieving 50 unique testing events per gene target.
. Each of the 83 prospective isolate samples were unique and enrolled at clinical sites. Isolates enrolled in the prospective arm of the clinical performance evaluation were selected based on their identification (enrolling when organisms are one of the bacterial species detected by the test: Enterobacterales, Pseudomonas aeruginosa, and Enterococcus faecalis) and documented non-susceptibility, excluding intrinsic resistance, to at least one (1) of the

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following antibiotic classes, where appropriate: Aminoglycosides, Carbapenems, Cephalosporins (including ß-Lactam Combination Agents), Fluoroquinolones, Penicillins, Polymyxins. Sulfonamides. Trimethoprim, or Vancomvcin.

For testing with the Acuitas AMR Gene Panel, well-isolated colonies that grew on blood agar were diluted to a 0.5 McFarland standard equivalent suspension using the direct colony suspension method per CLSI M071 Approved Standard.

2. Reference Methods

a) Culture ID/AST

Organism identification was confirmed for the Gram-negative organisms (e.g., Enterobacterales and Pseudomonas aeruginosa) isolates in the clinical study using an automated species identification method. Testing by broth microdilution was used to determine Antimicrobial Susceptibility for a given Gram-negative isolate according to which CLSI M100- 28th edition breakpoints and FDA-specified exceptions for the SDO recommended breakpoints available on the FDA's Antibacterial Susceptibility Test Interpretive Criteria3 website were applied.

Orqanism identification for Gram-positive organisms (e.g., Enterocccus faecalis) was performed via MALDI-TOF MS and antimicrobial susceptibility testing by broth microdilution.

Whole Genome Sequencing b)

Detection of AMR genes in the clinical study isolates by the Acuitas AMR Gene Panel was confirmed aqainst WGS results for each isolate using a validated WGS pipeline using the Illumina Hi-Seq 4000 platform. A glycerol stock of each bacterial isolate that was assessed by the Acuitas AMR Gene Panel was sent for sequencing for comparison.

3. Results

Results and performance of the Acuitas AMR Gene Panel for detection of AMR genes versus WGS results in the Clinical Evaluation are detailed in Table 24 - Clinical Performance for the Acuitas AMR Gene Panel (PCR/WGS) which summarizes the Acuitas AMR Gene Panel assays for applicable organisms according to Table 1 - Antimicrobial Resistance Gene Markers Associated with Bacterial Species. Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) for AMR genes ranged from 94.4% to 100% and 96.5 to 100%, respectively.

'Total Unique Strains' in Table 24 - Clinical Performance for the Acuitas AMR Gene Panel (PCR/WGS) is the number of unique strains tested per AMR Gene Target, limited to species for which the AMR gene is reported as indicated in Table 24. A subset of 'Total Unique Strains' were tested in replicate as indicated by 'Total Replicates' in Table 26. WGS results for the unique strains and replicates are indicated in the last four columns of Table 26.

1 Clinical Laboratory Standards Institute (CLSI). M07 Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: 11th Edition. Wayne, PA; 2018.

2 Clinical Laboratory Standards Institute (CLS). M100 Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Eighth Informational Supplement. Wayne, PA; 2018.

3 U.S. Food and Drug Administration (2020). Antibacterial Susceptibility Test Interpretive Criteria. Available from: https://www.fda.gov/drugs/development-resources/antibacterial-susceptibility-test-interpretive-criteria

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| AMR Gene
Target | PPA | | | NPA | | | Total | Total | Positive for AMR
Targets by WGS | | Negative for AMR
Target by WGS | |
|--------------------------------------------|----------------|-------|-------------------|----------------|-------|---------------|--------------------------|-------------------|------------------------------------|-------------------|-----------------------------------|-------------------|
| | TP/
(TP+FN) | % | 95% CI | TN/
(TN+FP) | % | 95% CI | Unique
Strains
(n) | Replicates
(n) | Unique
Strains
(n) | Replicates
(n) | Unique
Strains
(n) | Replicates
(n) |
| AAC | 610/(622) a-c | 98.1 | 96.66-
98.89 | 536/(545) d-e | 98.3 | 96.89-99.13 | 577 | 732 | 315 | 386 | 262 | 346 |
| AAD | 128/(130) | 98.5 | 94.56-
99.58 | 192/(199)f | 96.5 | 92.92-98.29 | 185 | 189 | 98 | 54 | 87 | 135 |
| ANT | 203/(205) | 99.0 | 96.51 -
99.73 | 628/(633)g | 99.2 | 98.16 - 99.66 | 392 | 543 | 64 | 168 | 328 | 375 |
| APH | 39/(40) | 97.5 | 87.12-
99.56 | 443/(444) | 99.8 | 98.74-99.96 | 263 | 280 | 31 | 12 | 232 | 268 |
| armA | 8/(8) | 100.0 | 67.56-
100.00 | 147/(147) | 100.0 | 97.45-100.00 | 78 | 91 | 4 | 8 | 74 | 83 |
| CMY | 126/(128) h | 98.4 | 94.48-
99.57 | 688/(691)i | 99.6 | 98.73-99.85 | 422 | 489 | 55 | 84 | 367 | 405 |
| CTX-M-1 | 264/(273) | 96.7 | 93.85-
98.26 | 929/(938)j | 99.0 | 98.19-99.49 | 621 | 732 | 162 | 143 | 459 | 589 |
| CTX-M-2 | 35/(35) | 100.0 | 90.11-
100.00 | 801/(803) | 99.8 | 99.10-99.93 | 392 | 543 | 21 | 27 | 371 | 516 |
| CTX-M-9 | 73/(74) | 98.6 | 92.73-
99.76 | 781/(782) | 99.9 | 99.28-99.98 | 459 | 489 | 58 | 23 | 401 | 466 |
| DFR | 167/(169) | 98.8 | 95.79-
99.67 | 646/(650) | 99.4 | 98.43-99.76 | 422 | 489 | 90 | 96 | 332 | 393 |
| DHA | 36/(36) | 100.0 | 90.36-
100.00 | 293/(293) | 100.0 | 98.71-100.00 | 185 | 189 | 33 | 6 | 152 | 183 |
| E. coli gyrA
Mutant | 160/(163) | 98.2 | 94.73-
99.37 | 167/(168) | 99.4 | 96.71-99.89 | 155 | 209 | 81 | 101 | 74 | 108 |
| IMP | 72/(72) | 100.0 | 94.93-
100.00 | 257/(257) | 100.0 | 98.53-100.00 | 185 | 189 | 5 | 71 | 180 | 118 |
| KPC | 75/(77) | 97.4 | 91.02-
99.28 | 1130/(1134) | 99.6 | 99.10-99.86 | 621 | 732 | 63 | 21 | 558 | 711 |
| MCR-1 | 51/(54)k | 94.4 | 84.89-
98.09 | 281/(281) | 100.0 | 98.65-100.00 | 159 | 209 | 14 | 48 | 145 | 161 |
| NDM | 56/(57) | 98.2 | 90.71-
99.69 | 801/(805)l | 99.5 | 98.73-99.81 | 448 | 523 | 47 | 17 | 401 | 506 |
| OXA-1 | 240/(249) | 96.4 | 93.27 -
98.09 | 910/(918) | 99.1 | 98.29 - 99.56 | 577 | 732 | 112 | 161 | 465 | 571 |
| OXA-9 | 58/(58) | 100.0 | 93.79-
100.00 | 760/(761) | 99.9 | 99.26-99.98 | 422 | 489 | 47 | 21 | 375 | 468 |
| OXA-48 | 59/(62) | 95.2 | 86.71-
98.34 | 448/(452) | 99.1 | 97.75-99.66 | 293 | 280 | 48 | 27 | 245 | 253 |
| PER | 81/(82) | 98.8 | 93.41-
99.78 | 265/(266) | 99.6 | 97.90-99.93 | 155 | 243 | 9 | 81 | 146 | 162 |
| P. aeruginosa
gyrA Mutant | 265/(279)m | 95.0 | 91.75-
96.99 | 67/(68) | 98.5 | 92.13-99.74 | 154 | 243 | 103 | 216 | 51 | 27 |
| RMT | 31/(32) | 96.9 | 84.26-
99.45 | 297/(297) | 100.0 | 98.72-100.00 | 185 | 189 | 27 | 10 | 158 | 179 |
| SHV | 12/(12) | 100 | 75.75 -
100.00 | 668/(671) | 99.6 | 98.69 - 99.85 | 314 | 452 | 10 | 4 | 304 | 448 |
| Sul1 | 420/(424) | 99.1 | 97.60-
99.63 | 232/(240)n | 96.7 | 93.56-98.30 | 344 | 398 | 226 | 249 | 118 | 149 |
| Sul2 | 489/(501) | 97.6 | 95.86-
98.62 | 307/(318)o | 96.5 | 93.91-98.06 | 422 | 489 | 212 | 331 | 210 | 158 |
| TEM | 600/(609)p | 98.5 | 97.22 -
99.22 | 559/(572)q | 97.7 | 96.15 - 98.67 | 591 | 732 | 277 | 391 | 314 | 341 |
| vanA | 57/(57) | 100.0 | 93.69-
100.00 | 36/(36) | 100.0 | 90.36-100.00 | 43 | 54 | 8 | 52 | 35 | 2 |
| VEB | 89/(89) | 100.0 | 95.86-
100.00 | 411/(414) | 99.3 | 97.89-99.75 | 233 | 334 | 24 | 72 | 209 | 262 |
| VIM | 91/(93) | 97.8 | 92.49-
99.96 | 409/(410) | 99.8 | 98.63-99.96 | 233 | 334 | 22 | 80 | 211 | 254 |

Table 24 - Clinical Performance for the Acuitas AMR Gene Panel (PCR/WGS)

ª One (1) FN result attributed to the presence of an aac(3)-Ila gene variant that had no valid alignment with the primers/probe of the AAC assay harbored by a single K. pneumoniae unique isolate.

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b Two (2) FN results due to testing of two (2) replicates of a single unique E. coli isolate.

° Two (2) FN results due to testing of two (2) replicates of a single unique P. aeruginosa isolate.

d One (1) FP result attributed to the presence of a truncated ac(3)-11 gene harbored by a single unique E. coli isolate.

e One (1) FP result attributed to the presence of a truncated aac(3)-Ib gene in a single unique isolate.

1 Three (3) FP results attributed to an add415 gene variant harbored by three (3) K. pneumoniae isolates with high numbers (23) of mismatches in the reverse primer of the AAD assay, with two (2) isolates of a single unique strain. Two (2) additional FP results from K. pneumoniae isolates demonstrated alignment of the AAD assay primers/probe with high numbers of mismatches (23) in the reverse primer, but no attributable gene variant was detected in the AR database used for analysis.

9 One (1) FP result attributed to high PCR baseline driff and not true amplification of the ANT target assay in one (1) unique P. aeruginosa isolate

1 Two (2) FN results attributed to the presence of CMY gene variants with high numbers of mismatches (201) assay in two (2) E. coli isolates. One (1) isolate harbored a blaCMY-2 gene variant and one (1) isolate harbored a blaCMY-42 gene variant.

i Four (4) FP results attributed to the presence of a CMY gene variant with high numbers of the CMY assay in four (4) E. coli isolates. These four (4) E. coli isolates represented two (2) replicates. Both isolates harbored a blaCMY-4 gene variant. Seven (7) FP results attributed to the presence of a blaCMY-16 gene variant with high numbers of mismatches (≥3) to the reverse primer of the CMY assay in six (6) unique K. pneumoniae isolates, with one isolates. One (1) FP result attributed to the presence of a blaCMY-16 gene variant with high numbers of mismatches (23) to the reverse primer of the CMY assay in one (1) P. mirabilis isolate.

l Seven (7) FP results attributed to the presence of a blaction with perfect alignment to the primers/probe of the CTX-M-1 assay for 7 replicates of 1 unique E. coli isolate that was not originally identified by WGS analysis.

& Three (3) FN results due to testing of three (3) replicates of a single E. coli isolate.

1 Four (4) FP results due to testing of four (4) replicates of a single K. pneumoniae isolate.

™ One (1) FN result attributed to a negative result for the P. aeruginosa ID assay for a single unique P. aeruginosa isolate. Amplification of the P. aeruginosa gvrA Mutant assay was present for this isolate.

" Two (2) FP results due to testing of two (2) replicates of one unique E. coli isolate.

· Four (4) FP results due to testing for four (4) replicates of a single unique K. pneumoniae isolate.

P Two (2) FN results due to testing of two (2) replicates of a single unique E. coli isolate.

9 Four (4) FP results due to testing for four (4) replicates of a single unique K. pneumoniae isolate.

' Replicates are the total number of samples isolates tested multiple times. For example, replicates would equal 5 if three unique isolates were respectively tested in singlicate, duplicate and triplicate.

Please refer to the Acuitas AMR Gene Panel Electronic User Guide (EUG) at www.opgen.com for further complementary performance information as well as details on antibiotic drug classes and for organism/drug/resistance marker combinations for which association with resistance was demonstrated.

CONCLUSIONS lll.

The results of the analytical and clinical performance studies summarized above demonstrate that the Acuitas AMR Gene Panel is safe and effective for its intended use and is substantially equivalent to the predicate device.