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.

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.

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.

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 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") 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.

AI/ML Overview

Here's a summary of the acceptance criteria and study details for the OpGen Acuitas AMR Gene Panel, based on the provided document:

1. Table of Acceptance Criteria and Reported Device Performance

The document provides performance metrics primarily in terms of Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) compared to Whole Genome Sequencing (WGS) for each AMR gene target. While explicit "acceptance criteria" values (e.g., "PPA must be >95%") are not explicitly stated as numbered criteria, the reported performance values implicitly serve as fulfilled acceptance criteria given the 510(k) clearance.

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

2. Sample Size for Test Set and Data Provenance

The "Clinical Performance Evaluation" used a total of 1,307 isolate samples.

1,224 clinical stock isolate samples: These were previously collected and de-identified. The document specifies that they were selected based on identification as Enterobacterales, Pseudomonas aeruginosa, or Enterococcus faecalis and documented non-susceptibility to at least one antibiotic class (excluding intrinsic resistance). The country of origin for these retrospective samples is not explicitly stated but implies a clinical setting (likely within the US given the FDA submission).
83 prospective isolate samples: These were unique and enrolled at clinical sites. These isolates were also selected based on their identification to the target organisms and documented non-susceptibility to at least one of the specified antibiotic classes. The country of origin is not explicitly stated but suggests a clinical setting.
Total unique strains: 591 were evaluated (across all gene targets), with some strains tested in replicate, leading to the 1,307 total samples.

3. Number of Experts and Qualifications for Ground Truth

The document does not mention the use of human experts to establish ground truth in the traditional sense of medical image interpretation or clinical diagnosis. The ground truth for AMR gene detection was established using Whole Genome Sequencing (WGS), which is a highly objective, molecular-level method.

For organism identification, MALDI-TOF MS and automated species identification methods were used. These are standard laboratory techniques and do not involve human expert consensus for "ground truth" in the same way, but rather rely on established molecular and biochemical profiles.

4. Adjudication Method for Test Set

The document does not describe an explicit adjudication method (like 2+1 or 3+1) for the clinical performance evaluation, as the primary ground truth for AMR gene detection was WGS. WGS is considered a definitive molecular method, rendering such adjudication methods unnecessary for this type of test.

However, in the "Media Equivalency" study (Table 21 footnotes, particularly footnote b), it notes an "adjudication testing" event: "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." This suggests that for potentially discordant results in specific analytical studies, further internal verification or re-testing (which they call "adjudication testing") was performed, rather than external expert consensus.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No, a multi-reader multi-case (MRMC) comparative effectiveness study was not done. This device is an in vitro diagnostic (IVD) test for detecting specific genetic markers, not an AI-assisted diagnostic tool for human readers. Therefore, there is no "human readers improve with AI vs without AI assistance" effect size to report.

6. Standalone (Algorithm Only) Performance

Yes, the primary performance evaluation presented is the standalone performance of the Acuitas AMR Gene Panel. The reported PPA and NPA values in "Clinical Performance" (Table 24) compare the device's output (detection of AMR genes) directly against Whole Genome Sequencing (WGS), which serves as the independent and highly accurate ground truth. This is a direct measure of the algorithm/device's performance without human intervention in the interpretation of the results.

7. Type of Ground Truth Used

The primary ground truth for AMR gene detection in the clinical study was Whole Genome Sequencing (WGS).
Other ground truth methods used in the broader studies (clinical and analytical) include:

MALDI-TOF MS for organism identification (Gram-positive).
Automated species identification methods for organism identification (Gram-negative).
Antimicrobial Susceptibility Testing (AST) by broth microdilution to determine phenotypic susceptibility (used for context and selection criteria of isolates, but WGS was the direct comparator for gene detection).

8. Sample Size for Training Set

The document does not explicitly state a sample size for a "training set" in the context of machine learning or AI models with a distinct training phase. This device is a real-time PCR assay and its development would typically involve design and validation of primers/probes and optimization against a collection of known target and non-target sequences/isolates. The "Analytical Reactivity (Inclusivity)" study (298 isolates) and "Analytical Specificity (Cross-Reactivity)" study (423 isolates) describe testing against a comprehensive set of known gene variants and non-target organisms, which collectively contribute to the robustness of the assay design (analogous to a very targeted training/validation approach for molecular assays).

9. How Ground Truth for Training Set Was Established

Given that this is a molecular diagnostic assay (PCR-based) and not an AI/machine learning model in the typical sense, there isn't a "training set" ground truth established in the same manner. The assay is designed to detect specific gene sequences. The "ground truth" for developing such an assay typically relies on:

Known gene sequences: Public databases (e.g., NCBI) for AMR genes.
Sequencing (WGS/Sanger sequencing): To confirm the presence or absence of target genes in isolates used during assay development and optimization.
Phenotypic AST: To correlate gene presence with resistance phenotypes, which helps in the clinical interpretation of the assay.

The analytical studies (inclusivity and specificity) served to validate the comprehensive detection capabilities against a wide range of molecular "ground truths" (confirmed by WGS) and non-targets.

Summary

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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

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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)

[continued on page 2]

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)

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[continued from Form FDA 3881, page 1]

Organism	Reported AMR Gene Marker
Citrobacter freundiicomplex a	CTX-M-1, CTX-M-9, KPC, NDM, OXA-48
Citrobacter koseri	KPC, OXA-48
Enterobactercloacae complex b	CTX-M-1, CTX-M-9, KPC, TEM d
Enterococcusfaecalis	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
Klebsiellaaerogenes	CTX-M-1, CTX-M-9, KPC, NDM, OXA-48
Klebsiellamichiganensis	CTX-M-1, CTX-M-9, KPC, NDM, OXA-48
Klebsiella oxytoca	CTX-M-1, CTX-M-9, KPC, NDM, OXA-48
Klebsiellapneumoniae	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
Klebsiellaquasipneumoniae	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
Pseudomonasaeruginosa	AAC, ANT, CTX-M-1, CTX-M-2, gyrA Mutant c, KPC, NDM, OXA-1, PER, SHV d, TEM d, VEB, VIM
Raoultellaornithinolytica	KPC, NDM, OXA-48
Raoultella planticola	KPC
Serratiamarcescens	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.

[continued on page 3]

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[continued from page 2]

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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

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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

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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.

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	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

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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.

® 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.

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.

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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 PanelBacterial Isolates	Xpert® Carba-R Assay (K152614)Bacterial Isolates
OrganismsIndicated	Enterobacterales, Pseudomonasaeruginosa, and Enterococcus faecalis	Enterobacteriaceae, Acinetobacterbaumannii, 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 andVIM	blaKPC, blaNDM, blaVIM, blaOXA-48,and blaIMP
Similarities
Result Types	Qualitative	Same
Analyte	DNA	Same
TechnologicalPrinciples	Automated Nucleic acid amplification(DNA); real-time PCR	Fully automated nucleic acidamplification (DNA); real-time PCR
Interpretation ofResults	Diagnostic software on a PersonalComputer (PC)	Same
Differences
OrganismDetected	Enterobacterales, Pseudomonasaeruginosa and Enterococcus faecalis	Enterobacteriaceae, Acinetobacterbaumannii, Pseudomonas aeruginosa
Target AntibioticClasses	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 andVIM	blaKPC, blaNDM, blaVIM, blaOXA-48,and blaIMP
Extraction Method	QIAGEN® EZ1® Advanced XL DNAExtraction	Integrated with GeneXpert® System
Instrument	OpGen Qualified QuantStudio™ 5Real-Time PCR System1	Cepheid GeneXpert® System
Controls	One Assay Control, External ControlsAvailable	Internal Sample Processing Control(SPC) and Probe Check Control(PCC); External Controls available
Software	Spreadsheet application which takesoutput from the OpGen QualifiedQuantStudio 5 and generates report onthe gene sequence	Automated test report using diagnosticsoftware
Time to obtainresults from startof 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

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' 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)	14/1593%	15/15100%	29/3097%	14/1593%	14/1593%	28/3093%	15/15100%	14/1593%	29/3097%	86/9096%
L00015886-001(E. coli)	13/14b93%	15/15100%	28/2997%	15/15100%	15/15100%	30/30100%	15/15100%	15/15100%	30/30100%	88/8999%
L00009154-001(E. coli)	15/15100%	15/15100%	30/30100%	15/15100%	14/1593%	29/3097%	15/15100%	15/15100%	30/30100%	89/9099%
L00009721-001(K. pneumoniae)	15/15100%	15/15100%	30/30100%	15/15100%	14/1593%	29/3097%	14/1593%	15/15100%	29/3097%	88/9098%
L00007800-001(K. pneumoniae)	15/15100%	15/15100%	30/30100%	15/15100%	14/1593%	29/3097%	15/15100%	14/1593%	29/3097%	88/9098%
L00008624-001(P. aeruginosa)	15/15100%	14/1593%	29/3097%	15/15100%	15/15100%	30/30100%	14/1593%	14/15c93%	28/3093%	87/9097%
L00013504-001(P. mirabilis)	15/15100%	15/15100%	30/30100%	15/15100%	14/1593%	29/3097%	15/15100%	14/1593%	29/3097%	88/9098%
L00013200-001d(P. mirabilis)	15/15100%	15/15100%	30/30100%	14/1593%	14/14e100%	28/2997%	15/15100%	15/15100%	30/30100%	88/8999%
L00022926-001(E. faecalis)	15/15100%	15/15100%	30/30100%	15/15100%	15/15100%	30/30100%	15/15100%	15/15100%	30/30100%	90/90100%
L00006246-001f(S. aureus)	15/15100%	15/15100%	30/30100%	15/15100%	15/15100%	30/30100%	15/15100%	14/15f93%	29/3097%	89/9099%

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.

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AcuitasAMRGeneTarget	ExpectedResults	Site 1			Site 2			Site 3			Total %Agreementby Target
AAC	Detected	60/60100%	60/60100%	120/120100%	60/60100%	59/59100%	119/119100%	60/60100%	60/60100%	120/120100%	359/359100%
AAC	NotDetected	74/74100%	75/75100%	149/149100%	75/75100%	75/75100%	150/150100%	75/75100%	74/75 d98.7%	149/15099.3%	448/44999.80%
AAD	Detected	15/15100%	15/15100%	30/30100%	15/15100%	15/15100%	30/30100%	15/15100%	15/15100%	30/30100%	90/90100%
AAD	NotDetected	30/30100%	30/30100%	60/60100%	30/30100%	30/30100%	60/60100%	30/30100%	30/30100%	60/60100%	180/180100%
ANT	Detected	30/30100%	30/30100%	60/60100%	29/3096.7%	30/30100%	59/6098.30%	30/30100%	28/3093.3%	58/6096.7%	177/18098.30%
ANT	NotDetected	74/74100%	75/75100%	149/149100%	75/75100%	73/7498.6%	148/14999.3%	74/7598.7%	75/75100%	149/15099.3%	446/44899.60%
APH	Detected	15/15100%	15/15100%	30/30100%	15/15100%	14/14100%	29/29100%	15/15100%	15/15100%	30/30100%	89/89100%
APH	NotDetected	60/60100%	60/60100%	120/120100%	60/60100%	60/60100%	120/120100%	60/60100%	60/60100%	120/120100%	360/360100%
armA	Detected e	-	-	-	-	-	-	-	-	-
armA	NotDetected	45/45100%	45/45100%	90/90100%	45/45100%	44/44100%	89/89100%	45/45100%	45/45100%	90/90100%	269/269100%
CMY	Detected	30/30100%	30/30100%	60/60100%	30/30100%	30/30100%	60/60100%	30/30100%	30/30100%	60/60100%	180/180100%
CMY	NotDetected	88/8998.9%	90/90100%	178/17999.4%	90/90100%	89/89100%	179/179100%	90/90100%	90/90100%	180/180100%	537/53899.80%
CTX-M-1	Detected	30/30100%	30/30100%	60/60100%	30/30100%	30/30100%	60/60100%	30/30100%	30/30100%	60/60100%	180/180100%
CTX-M-1	NotDetected	104/104100%	105/105100%	209/209100%	105/105100%	104/104100%	209/209100%	105/105100%	105/105100%	210/210100%	628/628100%
CTX-M-2	Detected e	-	-	-	-	-	-	-	-	-
CTX-M-2	NotDetected	104/104100%	105/105100%	209/209100%	105/105100%	104/104100%	209/209100%	105/105100%	105/105100%	210/210100%	628/628100%
CTX-M-9	Detected	15/15100%	15/15100%	30/30100%	15/15100%	14/14100%	29/29100%	15/15100%	15/15100%	30/30100%	89/89100%
CTX-M-9	NotDetected	104/104100%	105/105100%	209/209100%	105/105100%	104/10599%	209/21099.5%	105/105100%	105/105100%	210/210100%	628/62999.80%
DFR	Detected	45/45100%	45/45100%	90/90100%	45/45100%	44/44100%	89/89100%	45/45100%	45/45100%	90/90100%	269/269100%
DFR	NotDetected	74/74100%	75/75100%	149/149100%	75/75100%	75/75100%	150/150100%	75/75100%	75/75100%	150/150100%	449/449100%
DHA	Detected e	-	-	-	-	-	-	-	-	-
AcuitasAMR	Expected	Site 1			Site 2			Site 3			Total %
GeneTarget	Results	Op 1 a	Op 2	Site	Op 1	Op 2 b	Site	Op 1	Op 2	Site	Agreementby Target
	NotDetected	45/45100%	45/45100%	90/90100%	45/45100%	45/45100%	90/90100%	45/45100%	45/45100%	90/90100%	270/270100%
E. coligyrAMutant	Detected	14/1593.3%	15/15100%	29/3096.7%	15/15100%	14/1593.3%	29/3096.70%	15/15100%	15/15100%	30/30100%	88/9097.80%
	NotDetected	29/29100%	30/30100%	59/59100%	30/30100%	30/30100%	60/60100%	30/30100%	30/30100%	60/60100%	179/179100%
E. coli ID	Detected	44/44100%	45/45100%	89/89100%	45/45100%	44/4597.8%	89/9098.9%	45/45100%	45/45100%	90/90100%	268/26999.6%
	NotDetected e	-	-	-	-	-	-	-	-	-	-
IMP	Detected	15/15100%	15/15100%	30/30100%	15/15100%	15/15100%	30/30100%	15/15100%	15/15100%	30/30100%	90/90100%
	NotDetected	30/30100%	30/30100%	60/60100%	30/30100%	30/30100%	60/60100%	29/3096.7%	30/30100%	59/6098.3%	179/18099.40%
KPC	Detected	15/15100%	15/15100%	30/30100%	15/15100%	15/15100%	30/30100%	15/15100%	15/15100%	30/30100%	90/90100%
	NotDetected	119/119100%	120/120100%	239/239100%	120/120100%	119/119100%	239/239100%	120/120100%	120/120100%	240/240100%	718/718100%
	Detected e	-	-	-	-	-	-	-	-	-	-
MCR-1	NotDetected	59/59100%	60/60100%	119/119100%	60/60100%	60/60100%	120/120100%	60/60100%	60/60100%	120/120100%	359/359100%
	Detected	15/15100%	15/15100%	30/30100%	15/15100%	15/15100%	30/30100%	15/15100%	15/15100%	30/30100%	90/90100%
NDM	NotDetected	75/75100%	75/75100%	150/150100%	75/75100%	74/74100%	149/149100%	75/75100%	75/75100%	150/150100%	449/449100%
	Detected	30/30100%	30/30100%	60/60100%	30/30100%	29/29100%	59/59100%	30/30100%	30/30100%	60/60100%	179/179100%
OXA-1	NotDetected	104/104100%	105/105100%	209/209100%	105/105100%	105/105100%	210/210100%	105/105100%	105/105100%	210/210100%	629/629100%
	Detected e	-	-	-	-	-	-	-	-	-	-
OXA-9	NotDetected	119/119100%	120/120100%	239/239100%	120/120100%	119/119100%	239/239100%	120/120100%	120/120100%	240/240100%	718/718100%
	Detected	15/15100%	15/15100%	30/30100%	15/15100%	15/15100%	30/30100%	15/15100%	15/15100%	30/30100%	90/90100%
OXA-48	NotDetected	60/60100%	60/60100%	120/120100%	59/6098.3%	59/59100%	118/11999.2%	60/60100%	60/60100%	120/120100%	358/35999.70%
P.aeruginosagyrAMutant	Detected	15/15100%	14/1593.3%	29/3096.7%	15/15100%	15/15100%	30/30100%	15/15100%	14/1593.3%	29/3096.7%	88/9097.80%
	NotDetected e	-	-	-	-	-	-	-	-	-	-
AcuitasAMRGeneTarget	ExpectedResults	Site 1			Site 2			Site 3			Total %Agreementby Target
		Op 1 a	Op 2	Site	Op 1	Op 2 b	Site	Op 1	Op 2	Site
P.aeruginosaID	Detected	15/15100%	15/15100%	30/30100%	15/15100%	15/15100%	30/30100%	15/15100%	14/1593.3%	29/3096.7%	89/9098.9%
	NotDetected e	-	-	-	-	-	-	-	-	-	-
PER	Detected	15/15100%	15/15100%	30/30100%	15/15100%	15/15100%	30/30100%	15/15100%	15/15100%	30/30100%	90/90100%
	NotDetected	15/15100%	15/15100%	30/30100%	15/15100%	15/15100%	30/30100%	15/15100%	15/15100%	30/30100%	90/90100%
	Detected e	-	-	-	-	-	-	-	-	-	-
RMT	NotDetected	45/45100%	45/45100%	90/90100%	45/45100%	45/45100%	90/90100%	45/45100%	45/45100%	90/90100%	270/270100%
	Detected e	-	-	-	-	-	-	-	-	-	-
SHV	NotDetected	74/74100%	75/75100%	149/149100%	75/75100%	75/75100%	150/150100%	75/75100%	75/75100%	150/150100%	449/449100%
	Detected	60/60100%	60/60100%	120/120100%	60/60100%	60/60100%	120/120100%	60/60100%	60/60100%	120/120100%	360/360100%
Sul1	NotDetected	29/29100%	30/30100%	59/59100%	30/30100%	30/30100%	60/60100%	30/30100%	30/30100%	60/60100%	179/179100%
	Detected	104/104100%	105/105100%	209/209100%	105/105100%	103/10499%	208/20999.5%	105/105100%	105/105100%	210/210100%	627/62899.80%
Sul2	NotDetected	15/15100%	15/15100%	30/30100%	15/15100%	15/15100%	30/30100%	15/15100%	15/15100%	30/30100%	90/90100%
	Detected	104/104100%	105/105100%	209/209100%	105/105100%	103/10499%	208/20999.5%	105/105100%	105/105100%	210/210100%	627/62899.80%
TEM	NotDetected	30/30100%	30/30100%	60/60100%	30/30100%	30/30100%	60/60100%	30/30100%	30/30100%	60/60100%	180/180100%
	Detected	15/15100%	15/15100%	30/30100%	15/15100%	15/15100%	30/30100%	15/15100%	15/15100%	30/30100%	90/90100%
vanA	NotDetected	15/15100%	15/15100%	30/30100%	15/15100%	15/15100%	30/30100%	15/15100%	15/15100%	30/30100%	90/90100%
	Detected	15/15100%	15/15100%	30/30100%	15/15100%	15/15100%	30/30100%	15/15100%	15/15100%	30/30100%	90/90100%
VEB	NotDetected	45/45100%	45/45100%	90/90100%	45/45100%	44/44100%	89/89100%	45/45100%	45/45100%	90/90100%	269/269100%
	Detected	15/15100%	15/15100%	30/30100%	15/15100%	15/15100%	30/30100%	15/15100%	15/15100%	30/30100%	90/90100%
VIM	NotDetected	45/45100%	45/45100%	90/90100%	45/45100%	44/44100%	89/89100%	45/45100%	45/45100%	90/90100%	269/269100%

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

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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.

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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 ofSamplesPositive forGene byWGS	Number ofUniqueIsolatesPositive forGene byWGS	AMR Gene(s)Detected	AMR Gene(s)NotDetected	Other AMR Gene Variants Predicted to be Detectedby Acuitas AMR Gene Panel Based on InSilico 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,aadA17Likely 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 ofSamplesPositive forGene byWGS	Number ofUniqueIsolatesPositive forGene byWGS	AMR Gene(s)Detected	AMR Gene(s)NotDetected	Other AMR Gene Variants Predicted to be Detectedby Acuitas AMR Gene Panel Based on InSilico 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-79Likely 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-97Likely 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-5Likely Detectable: blaIMP-15, blaIMP-29Potentially 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-9Likely Detectable: blaNDM-10
OXA-1	78	56	blaOXA-1,blaOXA-4	blaOXA-1_J02967	Detectable: blaOXA-224, blaOXA-31, blaOXA-320Likely Detectable: blaOXA-47
	Acuitas AMR Gene Panel
AMR Gene	Number ofSamplesPositive forGene byWGS	Number ofUniqueIsolatesPositive forGene byWGS	AMR Gene(s)Detected	AMR Gene(s)NotDetected	Other AMR Gene Variants Predicted to be Detectedby 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: rmtFLikely 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-93Likely 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)ºLikely 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 ofSamplesPositive forGene byWGS	Number ofUniqueIsolatesPositive forGene byWGS	AMR Gene(s)Detected	AMR Gene(s)NotDetected	Other AMR Gene Variants Predicted to be Detectedby Acuitas AMR Gene Panel Based on InSilico 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-94Likely 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.

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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 <100% homology of one or both primers with the target sequence (one mismatch in one or both primers) and 100% homology of detector probe(s). Potentially Detectable indicates <95% homology of one or more primers with the target sequence but ≤2 nucleotide mismatches over their entire length along with 100% homology of detector probe(s).

® The test panel for this study was composed of 5 Enterichia odi, 3 Klebsiella pneumoniae ssp ozaenae, 89 Klebsiella pneumoniae, 34 Proteus mirabilis, and 98 Pseudomonas aeruginosa strains.

3. Analytical Specificity (Cross-Reactivity)

The Acuitas AMR Gene Panel was investigated for potential cross-reactivity with AMR genes. Crossreactivity was also evaluated for the test's species identification assays for E. coli and P. aeruginosa, which are used in conjunction with mutant gyrase assays for these two organisms.

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Analytical specificity (cross-reactivity) of the Acuitas AMR Gene Panel was 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 to evaluate analytical specificity at the organism and AMR gene level. Additionally, cross-reactivity at the AMR gene level was evaluated for two-hundred and ninety-eight (298) isolates from the Analytical Reactivity (Inclusivity) Study.

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.

Organisms that did not cross-react with the E. coli or P. aeruginosa species identification assays of the Acuitas AMR Gene Panel are provided in Table 6 - Organisms without Cross-Reactivity.

Bacterial Species
Achromobacter xylosoxidans	Enterococcus gallinarum	Raoultella planticola
Acinetobacter baumannii complex	Enterococcus gilvus	Salmonella enterica
Acinetobacter ursingii	Enterococcus hiraec	Salmonella species
Aeromonas hydrophila	Enterococcus italicus	Serratia marcescens
Candida albicans	Enterococcus pseudoavium	Serratia plymuthica
Citrobacter braakii	Enterococcus raffinosus	Shigella boydiid
Citrobacter freundii complex	Hafnia alvei	Shigella dysenteriaed
Citrobacter koseri	Klebsiella oxytoca	Sphingomonas paucimobilis
Citrobacter youngae	Klebsiella oxytoca ESBL	Staphylococcus aureus
Clostridioides (Clostridium) difficile	Leclercia adecarboxylata	Staphylococcus capitis
Corynebacterium diphtheriae	Moraxella catarrhalisc	Staphylococcus epidermidis
Escherichia fergusoniia	Morganella morganii ssp morganii	Staphylococcus haemolyticus
Escherichia hermanniib	Proteus vulgaris	Staphylococcus hominis
Enterobacter aerogenes	Providencia rettgeri	Staphylococcus lugdunensis
Enterobacter cloacae	Providencia stuartii	Staphylococcus saprophyticus
Enterobacter cloacae complex	Pseudomonas fluorescens	Staphylococcus warneri
Enterobacter hormaecheie	Pseudomonas luteola	Streptococcus agalactiae

Table 6 - Organisms without Cross-Reactivity

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Bacterial Species
Enterococcus disparc	Pseudomonas oryzihabitans	Streptococcus pyogenes
Enterococcus durans	Pseudomonas putida	Yersinia pseudotuberculosis
Enterococcus faecium	Pseudomonas stutzeri

යි One isolate (Parent LDW L00021948-001) was identified as Escherichia fergusonii by ATCC and by WGS whereas the automated species identification method reported it as E. coli. The Acuitas AMR Gene Panel reported true negative for P. aeruginosa species ID and false negative for E. coli species ID with this isolate based on the automated species identification method designation as E. coli. This isolate is included as E. fergusonii based on ATCC and WGS designation.

් One isolate (Parent LDW L00017841-001) was identified as Escherichia hermannii by ATCC and as Enterobacterales by WGS. The automated species identification method showed the isolate as E. coli. The Acuitas AMR Gene Panel reported true negative for P. aeruginosa species ID and false neqative for E. coli species ID with this isolate based on the automated species identification method designation as E. col. This isolate is included as E. hermanni based on ATCC and WGS designation.

C. These isolates are included based on species designations by ATCC and/or WGS.

Isolates demonstrating cross-reactivity with the species identification assays of the Acuitas AMR Gene Panel are summarized in Table 7 - Orqanisms Demonstrating Cross-Reactivity.

External Strain ID	Organism	Positive SpeciesIdentification Assay
AR-0030	Shigella sonnei	E. coli ID
ATCC 8700	Shigella boydii	E. coli ID
ATCC 35966	Shigella boydii	E. coli ID
99-10354	Shigella dysenteriae	E. coli ID
ATCC 9361	Shigella dysenteriae	E. coli ID
ATCC 12022	Shigella flexneri	E. coli ID
ATCC 9199	Shigella flexneri	E. coli ID
ATCC 12025	Shigella flexneri	E. coli ID
ATCC 9290	Shigella sonnei	E. coli ID

Table 7 - Organisms Demonstrating Cross-Reactivity

Shigella boydii, Shigella dysenteriae, Shigella flexneri, and Shigella sonnei cross-reacted with the E. coli ID assay on the Acuitas AMR Gene Panel as expected from in silico analysis, although one (1) isolate of Shigella boydii (Parent LDW L00021962-001, External ID ATCC 9207) and one (1) isolate of Shigella dysenteriae (Parent LDW L00021966-001, External ID ATCC 49347) did not cross-react with the E. coli ID target. All Shigella strains that demonstrated cross-reactivity with the E. coli ID assay target had wild-type gyrase sequences. Therefore, no determination could be made regarding cross-reactivity of Shigella species with the E. coli gyrA Mutant assay.

No cross-reactivity was observed with genes/variants not targeted by the Acuitas AMR Gene Panel. Table 8 - AMR Gene Variants without Cross-Reactivity summarizes AMR genes that did not crossreact with the Acuitas AMR Gene Panel. The genes (or variants) in Table 8 were identified in a crossreactivity panel of organisms by WGS and are not specific targets of the Acuitas AMR Gene Panel.

4. Some but not all isolates of Shigella dysenteriae cross-react with the E. coll species ID assay of the Acultas AMR Gene Panel.

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	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

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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 LDWNumber/External ID	Study LDWNumber	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
---------------------------------------	--	--

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Parent LDWNumber/External ID	Study LDWNumber	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 perIsolate	Replicates MatchingExpected 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

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Study LDW Number	Organism	Testing Event	Replicates perIsolate	Replicates MatchingExpected 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/ExternalID	Study LDWNumber	Organism	AMR Genes Identified by Whole GenomeSequencing
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

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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 LDWNumber(Organism)	Testing Event	ReplicatesTested	ReplicatesMatchingT=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

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Study LDWNumber(Organism)	Testing Event	ReplicatesTested	ReplicatesMatchingT=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 LDWNumber/External ID	Study LDW Number	Organism	AMR Genes Identified by Whole GenomeSequencing
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	ReplicatesTested	Replicates MatchingExpected 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	ReplicatesTested	Replicates MatchingExpected 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 LDWNumber/External ID	LDW Number for Study	Organism	AMR Genes Identified by Whole GenomeSequencing
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

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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 matchingT=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 GenesDetected by Whole Genome Sequencing	Study LDWNumber	Parent LDWNumber
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 GenesDetected by Whole Genome Sequencing	Study LDWNumber	Parent LDWNumber
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 GenesDetected by Whole Genome Sequencing	Study LDWNumber	Parent LDWNumber
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	ResistanceMarker	Numberof TestingEvents	Blood Agar		MacConkey Agar
			PPA(TP/(TP+FN))(95% CI)	NPA(TN/(TN+FP))(95% CI)	PPA(TP/(TP+FN))(95% CI)	NPA(TN/(TN+FP))(95% CI)
	AAC	13	100% (7/7)(64.57 - 100)	100% (6/6)(60.97 - 100)	100% (7/7)(64.57 - 100)	100% (6/6)(60.97 - 100)
	ANT	13	100% (2/2)(34.24 - 100)	100% (11/11)(74.12 - 100)	100% (2/2)(34.24 - 100)	100% (11/11)(74.12 - 100)
	CMY	13	100% (2/2)(34.24 - 100)	100% (11/11)(74.12 - 100)	100% (2/2)(34.24 - 100)	100% (11/11)(74.12 - 100)
	CTX-M-1	13	100% (6/6)(60.97 - 100)	100% (7/7)(64.57 - 100)	100% (6/6)(60.97 - 100)	100% (7/7)(64.57 - 100)
	CTX-M-2	13	-	100% (13/13)(77.19 - 100)	-	100% (13/13)(77.19 - 100)
	CTX-M-9	13	-	100% (13/13)(77.19 - 100)	-	100% (13/13)(77.19 - 100)
	DFR	13	100% (4/4)(51.01 - 100)	100% (9/9)(70.08 - 100)	100% (4/4)(51.01 - 100)	100% (9/9)(70.08 - 100)
E. coli	E. coli gyrA	13	100% (7/7)(64.57 - 100)	100% (6/6)(60.97 - 100)	100% (7/7)(64.57 - 100)	100% (6/6)(60.97 - 100)
	KPC	13	100% (2/2)(34.24 - 100)	100% (11/11)(74.12 - 100)	100% (2/2)(34.24 - 100)	100% (11/11)(74.12 - 100)
	MCR-1	13	-	100% (13/13)(77.19 - 100)	-	100% (13/13)(77.19 - 100)
	OXA-1	13	100% (4/4)(51.01 - 100)	100% (9/9)(70.08 - 100)	100% (4/4)(51.01 - 100)	100% (9/9)(70.08 - 100)
	OXA-9	13	-	100% (13/13)(77.19 - 100)	-	100% (13/13)(77.19 - 100)
	SHV	13	100% (2/2)(34.24 - 100)	100% (11/11)(74.12 - 100)	100% (2/2)(34.24 - 100)	100% (11/11)(74.12 - 100)
	Sul1	13	100% (4/4)(51.01 - 100)	100% (9/9)(70.08 - 100)	100% (4/4)(51.01 - 100)	100% (9/9)(70.08 - 100)
	Sul2	13	100% (8/8)(67.56 - 100)	100% (5/5)(56.55 - 100)	100% (8/8)(67.56 - 100)	100% (5/5)(56.55 - 100)
	TEM	13	100% (9/9)(70.08 - 100)	100% (4/4)(51.01 - 100)	100% (9/9)(70.08 - 100)	100% (4/4)(51.01 - 100)
K. pneumoniae	AAC	13	100% (8/8)(67.56 - 100)	100% (5/5)(56.55 - 100)	100% (8/8)(67.56 - 100)	100% (5/5)(56.55 - 100)
Organism	ResistanceMarker	Numberof TestingEvents	Blood Agar		MacConkey Agar
			PPA(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	13	100% (4/4)(51.01 - 100)	100% (9/9)(70.08 - 100)	75% (3/4)(30.06 -95.44)	100% (9/9)(70.08 - 100)
	APH	13	100% (1/1)(20.65 - 100)	100% (12/12)(75.75 - 100)	100% (1/1)(20.65 - 100)	100% (12/12)(75.75 - 100)
	CMY	13	-	100% (13/13)(77.19 - 100)	-	100% (13/13)(77.19 - 100)
	CTX-M-1 b	13	85.7% (6/7)(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	13	-	100% (13/13)(77.19 - 100)	-	100% (13/13)(77.19 - 100)
	DFR	13	100% (1/1)(20.65 - 100)	100% (12/12)(75.75 - 100)	100% (1/1)(20.65 - 100)	100% (12/12)(75.75 - 100)
	DHA	13	-	100% (13/13)(77.19 - 100)	-	100% (13/13)(77.19 - 100)
	IMP	13	-	100% (13/13)(77.19 - 100)	-	100% (13/13)(77.19 - 100)
	KPC	13	100% (3/3)(43.85 - 100)	100% (10/10)(72.25 - 100)	100% (3/3)(43.85 - 100)	100% (10/10)(72.25 - 100)
	NDM	13	100% (1/1)(20.65 - 100)	100% (12/12)(75.75 - 100)	100% (1/1)(20.65 - 100)	100% (12/12)(75.75 - 100)
	OXA-1	13	100% (4/4)(51.01 - 100)	100% (9/9)(70.08 - 100)	100% (4/4)(51.01 - 100)	100% (9/9)(70.08 - 100)
	OXA-9	13	100% (1/1)(20.65 - 100)	100% (12/12)(75.75 - 100)	100% (1/1)(20.65 - 100)	100% (12/12)(75.75 - 100)
	OXA-48	13	100% (3/3)(43.85 - 100)	100% (10/10)(72.25 - 100)	100% (3/3)(43.85 - 100)	100% (10/10)(72.25 - 100)
	RMT b	13	50% (1/2)(9.45 - 90.55)	100% (11/11)(74.12 - 100)	100% (2/2)(34.24 - 100)	100% (11/11)(74.12 - 100)
	Sul1 a	13	100% (5/5)(56.55 - 100)	100% (8/8)(67.56 - 100)	80% (4/5)(37.55 -96.38)	100% (8/8)(67.56 - 100)
	Sul2	13	100% (2/2)(34.24 - 100)	100% (11/11)(74.12 - 100)	100% (2/2)(34.24 - 100)	100% (11/11)(74.12 - 100)
	TEM b	13	83.3% (5/6)(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	13	85.7% (6/7)(48.69 -97.43)	100% (6/6)(60.97 - 100)	85.7% (6/7)(48.69 -97.43)	100% (6/6)(60.97 - 100)
	ResistanceMarker	Numberof TestingEvents	Blood Agar		MacConkey Agar
Organism			PPA(TP/(TP+FN))(95% CI)	NPA(TN/(TN+FP))(95% CI)	PPA(TP/(TP+FN))(95% CI)	NPA(TN/(TN+FP))(95% CI)
	ANT	13	100% (1/1)(20.65 - 100)	100% (12/12)(75.75 - 100)	100% (1/1)(20.65 - 100)	100% (12/12)(75.75 - 100)
	CTX-M-1	13	0% (0/1)(0 - 79.35)	100% (12/12)(75.75 - 100)	0% (0/1)(0 - 79.35)	100% (12/12)(75.75 - 100)
	CTX-M-2	13	-	100% (13/13)(77.19 - 100)	-	100% (13/13)(77.19 - 100)
	P.aeruginosagyrAc	12 d	100% (7/7)(64.57 - 100)	100% (5/5)(56.55 - 100)	85.7% (6/7)(48.69 -97.43)	100% (5/5)(56.55 - 100)
	KPC	13	-	100% (13/13)(77.19 - 100)	-	100% (13/13)(77.19 - 100)
	NDM	13	-	100% (13/13)(77.19 - 100)	-	100% (13/13)(77.19 - 100)
	OXA-1	13	50% (1/2)(9.45 - 90.55)	100% (11/11)(74.12 - 100)	50% (1/2)(9.45 - 90.55)	100% (11/11)(74.12 - 100)
	PER	13	-	100% (13/13)(77.19 - 100)	-	100% (13/13)(77.19 - 100)
	SHV	13	-	100% (13/13)(77.19 - 100)	-	100% (13/13)(77.19 - 100)
	TEM	13	0% (0/1)(0 - 79.35)	100% (12/12)(75.75 - 100)	0% (0/1)(0 - 79.35)	100% (12/12)(75.75 - 100)
	VEBd	13	-	92.3% (12/13)(66.69 -98.63)	-	92.3% (12/13)(66.69 -98.63)
	VIM	13	100% (4/4)(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	13	80% (4/5)(37.55 -96.38)	75% (6/8)(40.93 -92.85)	100% (5/5)(56.55 - 100)	100% (8/8)(67.56 - 100)
	ANT	13	100% (1/1)(20.65 - 100)	100% (12/12)(75.75 - 100)	100% (1/1)(20.65 - 100)	100% (12/12)(75.75 - 100)
	APH	13	100% (2/2)(34.24 - 100)	100% (11/11)(74.12 - 100)	100% (2/2)(34.24 - 100)	100% (11/11)(74.12 - 100)
	armAg	13	100% (1/1)(20.65 - 100)	91.7% (11/12)(64.61 -98.51)	100% (1/1)(20.65 - 100)	91.7% (11/12)(64.61 -98.51)
	CMY	13	100% (2/2)(34.24 - 100)	100% (11/11)(74.12 - 100)	100% (2/2)(34.24 - 100)	100% (11/11)(74.12 - 100)
	CTX-M-1h	13	-	92.3% (12/13)(66.69 -98.63)	-	100% (13/13)(77.19 - 100)
Organism	ResistanceMarker	Numberof TestingEvents	Blood Agar		MacConkey Agar
			PPA(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	13	0% (0/1)(0 - 79.35)	91.7% (11/12)(64.61 -98.51)	100% (1/1)(20.65 - 100)	100% (12/12)(75.75 - 100)
	CTX-M-9	13	100% (2/2)(34.24 - 100)	100% (11/11)(74.12 - 100)	100% (2/2)(34.24 - 100)	100% (11/11)(74.12 - 100)
	DFR	13	100% (2/2)(34.24 - 100)	100% (11/11)(74.12 - 100)	100% (2/2)(34.24 - 100)	100% (11/11)(74.12 - 100)
	KPC	13	-	100% (13/13)(77.19 - 100)	-	100% (13/13)(77.19 - 100)
	NDM	13	100% (1/1)(20.65 - 100)	100% (12/12)(75.75 - 100)	100% (1/1)(20.65 - 100)	100% (12/12)(75.75 - 100)
	OXA-1 h	13	100% (2/2)(34.24 - 100)	90.9% (10/11)(62.26 -98.38)	100% (2/2)(34.24 - 100)	100% (11/11)(74.12 - 100)
	OXA-9 f	13	0% (0/1)(0 - 79.35)	100% (12/12)(75.75 - 100)	100% (1/1)(20.65 - 100)	100% (12/12)(75.75 - 100)
	OXA-48 h	13	-	92.3% (12/13)(66.69 -98.63)	-	100% (13/13)(77.19 - 100)
	Sul2 f	13	83.3% (5/6)(43.65 -96.99)	100% (7/7)(64.57 - 100)	100% (6/6)(60.97 - 100)	100% (7/7)(64.57 - 100)
	TEM f	13	83.3% (5/6)(43.65 -96.99)	100% (7/7)(64.57 - 100)	100% (6/6)(60.97 - 100)	100% (7/7)(64.57 - 100)
	VEB	13	-	100% (13/13)(77.19 - 100)	-	100% (13/13)(77.19 - 100)
	VIM	13	-	100% (13/13)(77.19 - 100)	-	100% (13/13)(77.19 - 100)

Table 21 - Performance from MacConkey and Blood Agar

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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.

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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 LDWNumber/External ID	Study LDWNumber	Organism	AMR Genes Detected by Whole GenomeSequencing	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.

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Sample NumberFor Study	#Results	# Positive Results/# Expected PositiveResults (%)	# Negative Results/# Expected NegativeResults (%)
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 GeneTarget	PPA			NPA			Total	Total	Positive for AMRTargets by WGS		Negative for AMRTarget by WGS
	TP/(TP+FN)	%	95% CI	TN/(TN+FP)	%	95% CI	UniqueStrains(n)	Replicates(n)	UniqueStrains(n)	Replicates(n)	UniqueStrains(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 gyrAMutant	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. aeruginosagyrA 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.

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).