Acuitas AMR Gene Panel
Device Facts
| Record ID | K191288 |
|---|---|
| Device Name | Acuitas AMR Gene Panel |
| Applicant | Opgen, Inc. |
| Product Code | PMY · Microbiology |
| Decision Date | Sep 30, 2021 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.1640 |
| Device Class | Class 2 |
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, Enterococcus 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.
Device Story
The Acuitas AMR Gene Panel is a multiplex nucleic acid-based in vitro diagnostic test; it detects genetic resistance markers in pure bacterial colonies. The device uses automated DNA extraction on the QIAGEN EZ1 Advanced XL System and multiplex real-time PCR on the OpGen Qualified QuantStudio 5 Real-Time PCR System. The system processes DNA from bacterial isolates; it uses dried primers and probes in 96-well plates. The Acuitas AMR Gene Analysis Software imports PCR data to generate reports. The device is used in clinical laboratories; it is operated by laboratory technicians. Healthcare providers use the output to identify potential antimicrobial resistance; it serves as an aid to clinical decision-making for patients with suspected resistant infections. The device provides rapid detection of resistance genes, which can help guide infection control and management, though it does not replace phenotypic susceptibility testing.
Clinical Evidence
Multi-site clinical study (1,307 isolates: 1,224 stock, 83 prospective) compared Acuitas AMR Gene Panel to WGS, MALDI-TOF MS, and broth microdilution AST. PPA for AMR genes ranged from 94.4% to 100%; NPA ranged from 96.5% to 100%.
Technological Characteristics
The device uses multiplex real-time PCR with TaqMan probes. DNA extraction is performed on the QIAGEN EZ1 Advanced XL System. Amplification and detection occur on the OpGen Qualified QuantStudio 5 Real-Time PCR System. The assay uses a 96-well plate format with dried primers and probes. It is a qualitative, nucleic acid-based test. Software is used for data analysis and report generation. The device is not networked/cloud-based for primary function.
Indications for Use
Indicated for qualitative detection and differentiation of antibiotic resistance markers in pure bacterial colonies of Pseudomonas aeruginosa, Enterococcus faecalis, or Enterobacterales (Citrobacter, Enterobacter, Escherichia, Klebsiella, Morganella, Proteus, Providencia, Raoultella, Serratia) grown on blood or MacConkey agar. Used as an aid to clinicians in managing patients with known or suspected antibiotic-resistant infections. Not for use in place of phenotypic antimicrobial susceptibility testing.
Regulatory Classification
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.
Predicate Devices
- Xpert Carba-R (K152614)
Submission Summary (Full Text)
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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION
DECISION SUMMARY
ASSAY AND INSTRUMENT
# I Background Information:
A 510(k) Number
K191288
B Applicant
OpGen, Inc.
C Proprietary and Established Names
Acuitas AMR Gene Panel
D Regulatory Information
| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| PMY | Class II | 21 CFR 866.1640 - Antimicrobial Susceptibility Test Powder | MI - Microbiology |
| OOI | Class II | 21 CFR 862.2570 - Instrumentation for clinical multiplex test systems | CH - Clinical Chemistry |
# II Submission/Device Overview:
A Purpose for Submission:
The purpose of the submission is to obtain a substantial equivalence determination for the Acuitas AMR Gene Panel run on the OpGen Qualified QuantStudio 5 Real-time PCR System for the qualitative detection of gene sequences for resistance markers from bacterial colonies.
B Measurand:
The following markers, associated with resistance, are claimed for reporting in the appropriate species:
AAC, AAD, ANT, APH, armA, CMY, CTX-M-1, CTX-M-2, CTX-M-9, DFR, DHA, IMP, KPC, MCR-1, NDM, OXA-1, OXA-48, OXA-9, PER, RMT, SHV, SUL1, SUL2, TEM, vanA, VEB, VIM, P. aeruginosa gyrA mutant, E. coli gyrA mutant
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### C Type of Test:
A multiplexed nucleic acid-based test intended for use with the OpGen Qualified QuantStudio 5 Real-Time PCR System for the qualitative detection and differentiation of select genetic determinants of antimicrobial resistance. The Acuitas AMR Gene Panel is performed with pure bacterial colonies.
### III Intended Use/Indications for Use:
#### A Intended Use(s):
See Indications for Use below.
#### B Indication(s) 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, Enterococcus 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.
Table 1 - Antimicrobial Resistance Gene Markers (Genetic Determinants) Associated with Bacterial Species
| Organism | Reported AMR Gene Marker |
| --- | --- |
| Citrobacter freundii complex\( ^{a} \) | CTX-M-1, CTX-M-9, KPC, NDM, OXA-48 |
| Citrobacter koseri | KPC, OXA-48 |
| Enterobacter cloacae complex\( ^{b} \) | CTX-M-1, CTX-M-9, KPC, TEM\( ^{d} \) |
| Enterococcus faecalis | vanA |
| Escherichia coli | AAC, ANT, CMY, CTX-M-1, CTX-M-2, CTX-M-9, DFR, gyrA Mutant\( ^{c} \), KPC, MCR-1\( ^{c} \), OXA-1, OXA-9, SHV\( ^{d} \), Sul1, Sul2, TEM\( ^{d} \) |
| Klebsiella aerogenes | CTX-M-1, CTX-M-9, KPC, NDM, OXA-48 |
| Klebsiella michiganensis | CTX-M-1, CTX-M-9, KPC, NDM, OXA-48 |
| Klebsiella oxytoca | CTX-M-1, CTX-M-9, KPC, NDM, OXA-48 |
| Klebsiella pneumoniae | AAC, AAD, APH, CMY, CTX-M-1, CTX-M-9, DFR, DHA, IMP, KPC, NDM, OXA-1, OXA-9, OXA-48, RMT, Sul1, Sul2, TEM\( ^{d} \) |
| Klebsiella quasipneumoniae | CTX-M-1, CTX-M-9, KPC, NDM, OXA-48 |
| Klebsiella variicola | CTX-M-1, CTX-M-9, KPC, NDM, OXA-48 |
| Morganella morganii | CTX-M-1, KPC, NDM, OXA-48 |
| Proteus mirabilis | AAC, ANT, APH, armA, CMY, CTX-M-1, CTX-M-2, CTX-M-9, DFR, KPC, NDM, OXA-1, OXA-9, OXA-48, Sul2, TEM\( ^{d} \), VEB, VIM |
| Providencia rettgeri | NDM |
| Providencia stuartii | NDM |
| Pseudomonas aeruginosa | AAC, ANT, CTX-M-1, CTX-M-2, gyrA Mutant\( ^{c} \), KPC, NDM, OXA-1, PER, SHV\( ^{d} \), TEM\( ^{d} \), VEB, VIM |
| Raoultella ornithinolytica | KPC, NDM, OXA-48 |
| Raoultella planticola | KPC |
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| Organism | Reported AMR Gene Marker |
| --- | --- |
| Serratia marcescens | CTX-M-1, CTX-M-9, KPC, NDM, OXA-48 |
aCitrobacter 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.
cPCR assays associated with fluoroquinolone resistance detect and differentiate wild type and mutant variants of gyraseA at amino acid position 87 for E. coli and position 83 for P. aeruginosa.
dPCR assays for SHV and TEM detect several 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.
eThe panel includes an assay for the detection of the mobilized colistin genetic determinant MCR-1 in E. coli.
The Acuitas AMR Gene Panel includes assays for the detection and reporting of genetic resistance markers associated with resistance to select drugs in the following antibiotic groups: aminoglycosides, carbapenems, cephalosporins, fluoroquinolones, penicillins, sulfonamides, trimethoprim, and vancomycin, to aid in the identification of potentially antimicrobial-resistant organisms. The panel includes an assay for the detection of the mobilized colistin genetic determinant MCR-1, a marker of public health importance associated with reduced inhibitory activity of polymyxins.
The results of the Acuitas AMR Gene Panel for detection and identification of genetic determinants associated with antimicrobial resistance are used along with the Acuitas AMR Gene Panel Electronic User Guide (EUG). In certain cases, this information may be used as an aid to clinicians in the management of patients with known or suspected antibiotic non-susceptible or resistant bacterial infections. The EUG contains information on the appropriateness of reporting resistance markers detected by the Acuitas AMR Gene Panel for claimed organisms based on the strength of the collective, totality of scientific evidence delineating the level of association between molecular marker detection with phenotypic, clinical resistance. Test results are not conclusive or prescriptive for labeled use of any specific antimicrobial drug product, and therefore, this test cannot be used in place of or to postpone or delay phenotypic antimicrobial susceptibility testing.
A “Detected” or “Not Detected” result does not rule out the presence of other antimicrobial resistance markers not detected by the Acuitas AMR Gene Panel. A "Not Detected" result for a genetic marker of antimicrobial resistance does not indicate susceptibility to associated antimicrobial drugs or drug classes, as multiple mechanisms of resistance may exist.
### C Special Conditions for Use Statement(s):
Rx - For Prescription Use Only
### D Special Instrument Requirements:
For use with the OpGen Qualified QuantStudio 5 Real-Time PCR System and the QIAGEN EZ1 Advanced XL System
### IV Device/System Characteristics:
### A Device Description:
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The Acuitas AMR Gene Panel is a qualitative nucleic acid-based in vitro diagnostic PCR test that is capable of simultaneous detection and identification of select genetic determinants of antimicrobial resistance. The test is performed on pure colonies of select Enterobacterales and Pseudomonas aeruginosa grown on blood agar or MacConkey agar and Enterococcus faecalis grown on blood agar obtained from clinical specimens in order to detect genetic determinants associated with resistance to drugs in the following antibiotic classes: Aminoglycosides, Carbapenems, Cephalosporins, Fluoroquinolones, Penicillins, Sulfonamides, Trimethoprim, and Vancomycin. In addition, the test detects an emerging genetic marker of public health importance associated with reduced inhibitory activity of polymyxins. An assessment of the levels of evidence, obtained through clinical studies, analytical studies, literature, databases, and in silico analyses was used to assess the association between the molecular (genotypic) marker detected and the resistance phenotype for certain bacterial pathogens.
### B Principle of Operation:
The Acuitas AMR Gene Panel assay is performed on the OpGen Qualified QuantStudio 5 Real-Time PCR System (OpGen Qualified QS5 instrument) using the Acuitas AMR Gene Panel Kit. The Acuitas AMR Gene Panel employs automated DNA extraction on the QIAGEN EZ1 Advanced XL and multiplex real-time PCR on the OpGen Qualified QS5 instrument. The test kit includes PCR plates (96-well) with dried primers and probes for analysis of DNA from four (4) isolates (24 wells per isolate). Each Acuitas AMR Gene Panel test kit includes two PCR plates for analysis of eight isolate samples along with tubes of Master Mix containing reagents and enzymes for PCR.
From bacterial isolates, the DNA is extracted on the QIAGEN EZ1 Advanced XL system according to manufacturer instructions, which includes incorporating the Assay Control within the extraction process. A sample of extracted DNA eluate is transferred to a Reagent Reservoir Trough to which Master Mix is added. Extracted DNA/Master Mix is then transferred to each of 24 wells on the PCR plate per test sample. The contents of each well are mixed, and the plate is sealed and transferred to the OpGen Qualified QS5 instrument for real-time multiplex reaction and detection. Data are exported from the PCR instrument 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 genes as “Detected,” “Not Detected,” or “NA/NR.” A one-page report will be generated if the results are invalid, with a summary of the error(s) listed.
The Acuitas AMR Gene Analysis Software generates a report for each sample on the PCR plate. For a valid sample, there are two (2) report pages:
1. Page 1 of the report contains the following information:
o Run/Report Information: the top of the report provides information on the date the test was performed and the date that the report was generated. The Sample ID and the associated Plate ID are reported, as well as any errors detected during the analysis of the data. Control Results list the status of the Assay Control for a “Valid” Control.
o Class/Gene Table: the results for each antimicrobial resistance gene detected by the Acuitas AMR Gene Panel are reported in this section. Antimicrobial resistance genes are grouped by antibiotic class. Table 1 below summarizes the possible results reported for each resistance gene:
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Table 1 - Results Reported for Antimicrobial Resistance Genes
| Reported Result | Interpretation |
| --- | --- |
| “Detected” | Resistance gene target is detected. There is PCR amplification of the target above the threshold at a Ct value below the assay cut-off. |
| “Not Detected” | Resistance gene target is not detected. There is no PCR amplification of the target above the threshold at a Ct value below the assay cutoff. |
| “NA/NR” | Resistance gene is either Not Associated (NA) with the organism or Non-Reportable (NR) for the organism. |
| “Invalid” | Resistance gene results are not reported if the Assay Control is invalid. A mutant gyrase gene result is also reported “Invalid” if the gyrase Mutant result is not consistent with the user-selected organism. |
2. Page 2 of the assay report contains Supplemental Information, which includes two (2) tables:
○ Table A: table lists the reportable antimicrobial resistance genes on the Acuitas AMR Gene Panel associated for a given species or group. This table only lists corresponding antibiotics for organism/marker combinations with an established combined clinical device performance PPV (Positive Predictive Value) near or above 80%, i.e., for which the detected presence of the AMR Gene in the combined clinical device performance evaluation associated with phenotypic resistance against the antibiotic at/near 80% of those isolates with gene detection.
▪ PPV: Rate of positive genotypic test results for an AMR Gene associated with an isolate in agreement with Antimicrobial Susceptibility Testing (AST) result indicating phenotypic resistance of the isolate against a specific antibiotic.
▪ Actual PPVs for the reported AMR Gene – isolate combinations of the Acuitas AMR Gene Panel observed during the performance evaluation can be queried in the Electronic User Guide (EUG) by selecting a specific AMR gene, organism, and antibiotic combination from the drop-down menu.
○ Table B: table lists the antimicrobial resistance genes on the Acuitas AMR Gene Panel that are Not Associated (NA) or Non-Reportable (NR) for a given species. NA (Not Associated) = Gene not associated with the organism. NR (Non-Reportable) = Minimum performance not demonstrated or not evaluated.
Additional information can be found in the Acuitas AMR Gene Panel EUG at the company website. The Acuitas AMR Gene Panel EUG is an extension of the Acuitas AMR Gene Panel package insert, and it provides additional supporting information about the performance characteristics and interpretation of results for the Acuitas AMR Gene Panel
### C Instrument Description Information:
# 1. Instrument Name:
The following instruments are needed to generate results for the Acuitas AMR Gene Panel:
• OpGen Qualified QuantStudio 5 Real-Time PCR System. Also referred to as “OpGen Qualified QS5 instrument.”
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• QIAGEN EZ1 Advanced XL System
# 2. Sample Identification:
Prior to loading the Acuitas AMR Gene Panel PCR Plate into the instrument, the sample information must be entered. A barcode scanner is available for automatic entry of the PCR plate. After launching the Acuitas AMR Gene Analysis Software, if a sample is present, use the drop-down menu to select the correct species for the isolate that was tested for generating the appropriate report per isolate.
# 3. Specimen Sampling and Handling:
Extracted DNA from pure colonies is added to the Acuitas AMR Gene Panel PCR Plate and inserted into the instrument manually.
# 4. Calibration:
The OpGen Qualified QuantStudio 5 Real-Time PCR System is factory calibrated and does not require calibration at installation. To ensure optimal performance, the Operator's manual recommends performing calibrations at the specified frequency.
# 5. Quality Control:
External controls are not provided with the Acuitas AMR Gene Panel, but quality control strains are recommended in the package insert. External Controls should be used in accordance with laboratory protocols and the appropriate accrediting organization requirements, as applicable.
This medical device product has functions subject to FDA premarket review as well as functions that are not subject to FDA premarket review. For this application, if the product has functions that are not subject to FDA premarket review, FDA assessed those functions only to the extent that they either could adversely impact the safety and effectiveness of the functions subject to FDA premarket review or they are included as a labeled positive impact that was considered in the assessment of the functions subject to FDA premarket review.
# V Substantial Equivalence Information:
# A Predicate Device Name(s):
Xpert Carba-R
# B Predicate 510(k) Number(s):
K152614
# C Comparison with Predicate(s):
| Device & Predicate Device(s): | Device: K191288 | Predicate: K152614 |
| --- | --- | --- |
| General Device Characteristic Similarities | | |
| Device Trade Name | Acuitas AMR Gene Panel | Xpert Carba-R |
| Intended Use/Indications For Use | The Acuitas AMR Gene Panel, performed on the QIAGEN EZ1 Advanced XL | The Xpert Carba-R Assay, performed on the GeneXpert Instrument Systems, is a |
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| | 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, Enterococcus 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 below.Organism (Reported AMR Gene Marker)Citrobacter freundii complex (CTX-M-1, CTX-M-9, KPC, NDM, OXA-48); Citrobacter koseri (KPC, OXA-48); Enterobacter cloacae complex (CTX-M-1, CTX-M-9, KPC, TEM); Enterococcus faecalis (vanA); Escherichia coli (AAC, ANT, CMY, CTX-M-1, CTX-M-2, CTX-M-9, DFR, gyrA Mutant c, KPC, MCR-1, OXA-1, OXA-9, SHV, Sul1, Sul2, TEM); Klebsiella aerogenes (CTX-M-1, CTX-M-9, KPC, NDM, OXA-48); Klebsiella michiganensis (CTX-M-1, CTX-M-9, KPC, NDM, OXA-48); Klebsiella oxytoca (CTX-M-1, CTX-M-9, KPC, NDM, OXA-48); Klebsiella pneumoniae (AAC, AAD, APH, CMY, CTX-M-1, CTX-M-9, DFR, DHA, IMP, KPC, NDM, OXA-1, OXA-9, OXA-48, RMT, Sul1, Sul2, TEM); Klebsiella quasipneumoniae (CTX-M-1, CTX-M-9, KPC, NDM, OXA-48); Klebsiella variicola (CTX-M-1, CTX-M-9, KPC, NDM, OXA-48); Morganella morganii (CTX-M-1, KPC, NDM, OXA-48); Proteus mirabilis (AAC, ANT, APH, armA, CMY, CTX-M-1, CTX-M-2, CTX-M-9, DFR, KPC, NDM, OXA-1, OXA-9, OXA-48, Sul2, TEM, VEB, VIM); Providencia rettgeri (NDM); Providencia stuartii (NDM); Pseudomonas aeruginosa (AAC, ANT, CTX-M-1, CTX-M-2, gyrA Mutant, KPC, NDM, OXA-1, PER, SHV, TEM, VEB, VIM); Raoultella ornithinolytica (KPC, NDM, OXA-48); Raoultella planticola (KPC); Serratia marcescens (CTX-M-1, CTX-M-9, KPC, NDM, OXA-48).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 | qualitative in vitro diagnostic test for the detection and differentiation of the \( bla_{KPC} \), \( bla_{NDM} \), \( bla_{VIM} \), \( bla_{OXA-48} \), and \( bla_{IMP} \) gene sequences associated with carbapenem-non-susceptible pure colonies of Enterobacteriaceae, Acinetobacter baumannii, or Pseudomonas aeruginosa grown on blood agar or MacConkey agar. The test utilizes automated real-time polymerase chain reaction (PCR).A negative Xpert Carba-R Assay result does not preclude the presence of other resistance mechanisms. The Xpert Carba-R Assay should be used in conjunction with other laboratory tests including phenotypic antimicrobial susceptibility testing. The Xpert Carba-R Assay is intended as an aid for infection control in detecting and differentiating genetic markers of resistance to monitor the spread of carbapenem-non-susceptible organisms in healthcare settings. The Xpert Carba-R Assay is not intended to guide or monitor treatment for carbapenem-non-susceptible bacterial infections. |
| --- | --- | --- |
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| | 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. | |
| --- | --- | --- |
| Technological Principles | Qualitative, real-time PCR | Same |
| Sample Type | Bacterial Isolates | Same |
| Detection Probes | TaqMan Probes | Same |
| Interpretation of Test Results | Diagnostic Software | Same |
| **General Device Characteristic Differences** | | |
| Targets | Twenty-eight (28) gene sequences are reported, spanning different antibiotic classes AAC, AAD, ANT, APH, armA CMY, CTX-M-1, CTX-M-2, CTX-M-9, DFR, DHA, IMP, KPC, MCR-1, NDM, OXA-1, OXA-48, OXA-9, PER, RMT, SHV, SUL1, SUL2, TEM, vanA, VEB, VIM, P. | Detects *bla*_{KPC}, *bla*_{NDM}, *bla*_{VIM}, *bla*_{OXA-48}, and *bla*_{IMP} gene sequences 5 gene targets associated with resistance to one antibiotic class |
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| | aeruginosa gyrA Mutant (83-M), E. coli gyrA Mutant (87-M1, 87-M2, 87-M3) | |
| --- | --- | --- |
| Technological Principles | Extraction with the QIAGEN EZ1 Advanced XL System and nucleic acid amplification and target detection with the OpGen Qualified QS5 instrument with the Acuitas AMR Gene Panel Kit. | Fully-automated DNA extraction, nucleic acid amplification (DNA), and target detection |
| Reaction platform | Disposable 96-well plates | Disposable, single-use multi-chambered fluidic cartridge |
| Controls | One Assay Control | Internal sample processing control (SPC) and probe check control (PCC) External controls available |
| Time to obtain Test Results | < 3 hours | Approximately 50 minutes to results |
| Organisms | Select Enterobacterales, Pseudomonas aeruginosa, Enterococcus faecalis | Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter baumannii |
| Instrument System | Data generated with the OpGen Qualified QS5 instrument and analyzed with the Acuitas AMR Gene Analysis Software | GeneXpert Instrument System (includes GeneXpert Dx, Infinity-48, Infinity-48s, and Infinity-80) |
## VI Standards/Guidance Documents Referenced:
- CLSI EP05-A3 Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline - Third Edition; FDA Recognition Number 7-251
- CLSI EP24-A2 Assessment of the Diagnostic Accuracy of Laboratory Tests Using Receiver Operating Characteristic Curves; Approved Guideline – Second Edition; FDA Recognition Number 7-234
- CLSI M100-S28. Performance Standard for Antimicrobial Susceptibility Testing; Approved standard—Twenty-eighth Informational Supplement, 2018; FDA Recognition Number 7-281
- CLSI EP25-A, Evaluation of Stability of In Vitro Diagnostic Reagents; Approved Guideline - Volume 29, 2009; FDA Recognition Number 7-235 IEC 62304:2006/A1:2016 Medical Device Software - Software Lifecycle Processes; FDA Recognition Number 13-79
- EMC-60601-1-2:2014, Medical electrical equipment -- Part 1-2: General requirements for basic safety and essential performance--Collateral Standard: Electromagnetic disturbances--Requirements and tests
## VII Performance Characteristics (if/when applicable):
### A Analytical Performance:
#### 1. Precision/Reproducibility:
The Acuitas AMR Gene Panel Reproducibility study was performed at three sites—two external and one internal site. The study utilized two operators per site, alternating testing over twenty days. Each site performed testing on two QS5 instruments and one EZ1 Advanced XL instrument. At least three lots of all reagents/materials required to perform the Acuitas AMR Gene Panel were used throughout the reproducibility study. Reproducibility panels included bacterial suspensions prepared at an 0.5 McFarland concentration, blinded by OpGen personnel, frozen at -20°C, and shipped to external testing sites. In total, each site tested 300 samples [composed of unique panel isolates, positive controls, negative controls, and no template controls]. During the execution of the Reproducibility Study, a total of (62)
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positive external controls, (62) negative external controls, and (182) no template controls were tested. In all cases the controls passed by producing the expected results. The results are summarized in Table 2.
Overall results for each isolate tested in the reproducibility study (Table 3) are summarized in Table 4 to Table 13 below which show the number of tests and the percentage of correct results obtained (compared to expected). Reproducibility study results were acceptable.
Table 14 summarizes the total false negatives and false positives observed during the Reproducibility Study, as well as a breakdown by organisms exhibiting these results. The Reproducibility Study also included a quantitative analysis of variance components, i.e., average Ct values and calculated SD and %CV, which were determined to be acceptable for the assay.
Table 2. Daily QC Panelᵃ
| Control ID | Organism (Resistance Gene Markers) | Total | % Agreement with Expected Result | Lot Number |
| --- | --- | --- | --- | --- |
| POS Control B | *P. mirabilis* (AAC, , armA, CMY, NDM, Sul2,TEM) | 30 | 100% (30/30) | MN70061 |
| POS Control C | *K. pneumoniae* (AAC, AAD, CTX-M-1, Sul1, Sul2, TEM) | 32 | 100% (32/32) | MN70062 |
| Neg Control | *S. aureus* (No gene targets detected) | 62 | 100% (62/62) | MN90133 |
| NTC (no template control) | None (Negative for all targets)ᵇ | 182 | 100% (182/182) | N/A |
ᵃAll external positive and negative controls gave the expected Acuitas AMR Gene Panel results on all days of testing at each testing site.
ᵇContains PCR Certified Water added at PCR Set-Up
Table 3. Isolates Tested in Reproducibility Panel
| Sample Number | Organism | AMR Markers Present |
| --- | --- | --- |
| 1 | *E. coli* | ANT, DFR, gyraseA-MT, KPC, Sul1, Sul2, TEM |
| 2 | *E. coli* | Sul2, TEM |
| 3 | *E. coli* | DFR, DHA, Sul1, Sul2, TEM |
| 4 | *K. pneumoniae* | AAC, AAD, CMY, CTX-M-1, NDM, OXA-48, Sul1, Sul2, TEM |
| 5 | *K. pneumoniae* | AAC, CTX-M-1, IMP, OXA-1, Sul1, Sul2, TEM |
| 6 | *P. aeruginosa* | gyraseA-MT, PER, VIM |
| 7 | *P. mirabilis* | AAC, ANT, CMY, Sul2, TEM, VEB |
| 8 | *P. mirabilis* | AAC, APH, CTX-M-9, DFR, OXA-1, Sul2, TEM |
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| 9 | E. faecalis | vanA |
| --- | --- | --- |
| 10 | S. aureus | NONE |
Table 4. Reproducibility Results-E. coli (Sample 1)
| E. coli (Sample 1) | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Target^{a} | Expected Result | Site 1 | | | Site 2 | | | Site 3 | | | All Sites |
| | | Op1 | Op2 | Site 1 | Op1 | Op2 | Site 2 | Op1 | Op2 | Site 3 | |
| ANT | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 14/15 (93%) | 15/15 (100%) | 29/30 (97%) | 15/15 100% | 14/15 (93%) | 29/30 (97%) | 88/90 (98%) |
| DFR | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 100% | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
| E. coli gyrA MT | Detected | 14/15 (93%) | 15/15 (100%) | 29/30 (97%) | 15/15 (100%) | 14/15 (93%) | 29/30 (97%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 88/90 (98%) |
| KPC | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 100% | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
| Sul1 | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 100% | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
| Sul2 | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
| TEM | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
$^{a}$No false positive results were observed for other Acuitas AMR Gene Targets to be reported with this organism.
Table 5. Reproducibility Results-E. coli (Sample 2)
| E. coli (Sample 2) | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Target^{a,b} | Expected Result | Site 1 | | | Site 2 | | | Site 3 | | | All Sites |
| | | Op1 | Op2 | Site 1 | Op1 | Op2 | Site 2 | Op1 | Op2 | Site 3 | |
| Sul2 | Detected | 14/14 (100%) | 15/15 (100%) | 29/29 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 89/89 (100%) |
| TEM | Detected | 14/14 (100%) | 15/15 (100%) | 29/29 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 89/89 (100%) |
| CMY | Not Detected | 13/14 (93%) | 15/15 (100%) | 28/29 (97%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 88/89 (99%) |
$^{a}$Except for CMY, all other Acuitas AMR Gene Panel Targets were not detected (did not generate FP results).
$^{b}$One replicate 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.
Table 6. Reproducibility Results-E. coli (Sample 3)
| E. coli (Sample 3) | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Target^{a} | Expected Result | Site 1 | | | Site 2 | | | Site 3 | | | All Sites |
| | | Op1 | Op2 | Site 1 | Op1 | Op2 | Site 2 | Op1 | Op2 | Site 3 | |
| DFR | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
| DHA | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
| Sul1 | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
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| Sul2 | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| TEM | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
| ANT | Not Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 14/15 (93%) | 29/30 (97%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 89/90 (99%) |
| *aExcept for ANT, all other Acuitas AMR Gene Panel Markers did not generate any false positive results.* | | | | | | | | | | | |
Table 7. Reproducibility Results-K. pneumoniae (Sample 4)
| **K. pneumoniae (Sample 4)** | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| **Target^{a}** | **Expected Result** | **Site 1** | | | **Site 2** | | | **Site 3** | | | **All Sites** |
| | | **Op1** | **Op2** | **Site 1** | **Op1** | **Op2** | **Site 2** | **Op1** | **Op2** | **Site 3** | |
| AAC | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
| AAD | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
| CMY | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
| CTX-M-1 | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
| NDM | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
| OXA-48 | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
| Sul1 | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
| Sul2 | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
| TEM | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 14/15 (93%) | 29/30 (97%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 89/90 (99%) |
| IMP | Not Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 14/15 (93%) | 15/15 (100%) | 29/30 (97%) | 89/90 (99%) |
| *aExcept for IMP, all other Acuitas AMR Gene Panel Markers did not generate any false positive results.* | | | | | | | | | | | |
Table 8. Reproducibility Results-K. pneumoniae (Sample 5)
| **K. pneumoniae (Sample 5)** | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| **Target^{a}** | **Expected Result** | **Site 1** | | | **Site 2** | | | **Site 3** | | | **All Sites** |
| | | **Op1** | **Op2** | **Site 1** | **Op1** | **Op2** | **Site 2** | **Op1** | **Op2** | **Site 3** | |
| AAC | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
| CTX-M-1 | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
| IMP | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
| OXA-1 | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
| Sul1 | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
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| Sul2 | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| TEM | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
| ANT | Not Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 14/15 (93%) | 15/15 (100%) | 29/30 (97%) | 89/90 (99%) |
| CTX-M-9 | Not Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 14/15 (93%) | 29/30 (97%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 89/90 (99%) |
aExcept for ANT and CTX-M-9, all other Acuitas AMR Gene Panel Markers did not generate any false positive results.
Table 9. Reproducibility Results-P.aeruginosa (Sample 6)
| P. aeruginosa (Sample 6) | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Target^{a} | Expected Result | Site 1 | | | Site 2 | | | Site 3 | | | All Sites |
| | | Op1 | Op2 | Site 1 | Op1 | Op2 | Site 2 | Op1 | Op2 | Site 3 | |
| P. aeruginosa gyrA MT | Detected | 15/15 (100%) | 14/15 (93%) | 29/30 (97%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 14/15 (93%) | 29/30 (97%) | 88/90 (98%) |
| PER | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
| VIM | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
| ANT | Not Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 14/15 (93%) | 15/15 (100%) | 29/30 (97%) | 89/90 (99%) |
| ^{a}Except for ANT, all other Acuitas AMR Gene Panel Markers did not generate any false positive results. | | | | | | | | | | | |
Table 10. Reproducibility Results-P.mirabilis (Sample 7)
| P. mirabilis (Sample 7) | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Target^{a} | Expected Result | Site 1 | | | Site 2 | | | Site 3 | | | All Sites |
| | | Op1 | Op2 | Site 1 | Op1 | Op2 | Site 2 | Op1 | Op2 | Site 3 | |
| AAC | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
| ANT | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 14/15 (93%) | 29/30 (97%) | 89/90 (99%) |
| CMY | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
| Sul2 | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 14/15 (93%) | 29/30 (97%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 89/90 (99%) |
| TEM | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
| VEB | Detected | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 15/15 (100%) | 15/15 (100%) | 30/30 (100%) | 90/90 (100%) |
| ^{a}All other Acuitas AMR Gene Panel Markers did not generate any false positive results. | | | | | | | | | | | |
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Table 11. Reproducibility Results-P.mirabilis (Sample 8)
| P. mirabilis (Sample 8) | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Target\( ^{a,b} \) | Expected Result | Site 1 | | | Site 2 | | | Site 3 | | | All Sites |
| | | Op1 | Op2 | Site 1 | Op1 | Op2\( ^b \) | Site 2 | Op1 | Op2 | Site 3 | |
| AAC | Detected | 15/15(100%) | 15/15(100%) | 30/30(100%) | 15/15(100%) | 14/14(100%) | 29/29(100%) | 15/15(100%) | 15/15(100%) | 30/30(100%) | 89/89(100%) |
| APH | Detected | 15/15(100%) | 15/15(100%) | 30/30(100%) | 15/15(100%) | 14/14(100%) | 29/29(100%) | 15/15(100%) | 15/15(100%) | 30/30(100%) | 89/89(100%) |
Table 12. Reproducibility Results-P.mirabilis (Sample 9)
| E. faecalis (Sample 9) | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Target\( ^{a} \) | Expected Result | Site 1 | | | Site 2 | | | Site 3 | | All Sites |
| | | Op1 | Op2 | Site 1 | Op1 | Op2 | Site 2 | Op1 | Op2 | |
| vanA | Detected | 15/15(100%) | 15/15(100%) | 30/30(100%) | 15/15(100%) | 15/15(100%) | 30/30(100%) | 15/15(100%) | 15/15(100%) | 30/30(100%) |
| TEM | Detected | 15/15(100%) | 15/15(100%) | 30/30(100%) | 15/15(100%) | 14/14(100%) | 29/29(100%) | 15/15(100%) | 15/15(100%) | 30/30(100%) |
| OXA-48 | Not Detected | 15/15(100%) | 15/15(100%) | 30/30(100%) | 14/15(93%) | 14/14(100%) | 28/29(97%) | 15/15(100%) | 15/15(100%) | 30/30(100%) |
Table 13. Reproducibility Results-S. aureus (Sample 10)
| S. aureus (Sample 10) | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Target\( ^{a} \) | Expected Result | Site 1 | | | Site 2 | | | Site 3 | | All Sites |
| | | Op1 | Op2 | Site 1 | Op1 | Op2 | Site 2 | Op1 | Op2 | |
| AAC | Not Detected | 15/15(100%) | 15/15(100%) | 30/30(100%) | 15/15(100%) | 15/15(100%) | 30/30(100%) | 15/15(100%) | 14/15(93%) | 29/30(97%) |
| \( ^{a} \)Except for AAC, all other Acuitas AMR Gene Panel were not detected in this strain. This strain serves as a negative control for the assay. | | | | | | | | | | |
Table 14. Summary of all Acuitas AMR Gene Panel Reproducibility False Positive (FP) and False Negative (FN) results
| AMR Gene | False Positives | | | False Negatives | | |
| --- | --- | --- | --- | --- | --- | --- |
| | Total FP | Species w/ FP | # FP per Species | Total FN | Species w/ FN | # FN per Species |
| AAC | 1 | S. aureus | 1 | 0 | N/A | N/A |
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| AAD | 1 | E. coli | 1 | 0 | N/A | N/A |
| --- | --- | --- | --- | --- | --- | --- |
| ANT | 3 | E. coli | 1 | 3 | E. coli | 2 |
| | | K. pneumoniae | 1 | | | |
| | | P. aeruginosa | 1 | | P. mirabilis | 1 |
| CMY | 1 | E. coli | 1 | 0 | N/A | N/A |
| CTX-M-9 | 1 | K. pneumoniae | 1 | 0 | N/A | N/A |
| IMP | 1 | K. pneumoniae | 1 | 0 | N/A | N/A |
| OXA-48 | 1 | P. mirabilis | 1 | 0 | N/A | N/A |
| Sul2 | 0 | N/A | N/A | 1 | P. mirabilis | 1 |
| TEM | 0 | N/A | N/A | 1 | K. pneumoniae | 1 |
## 2. Linearity:
N/A
## 3. Analytical Specificity/Interference:
a. Cross-Reactivity
Potential cross-reactivity was investigated for the Acuitas AMR Gene Panel with a total of four hundred and twenty-three (423) isolates. Cross-reactivity was also evaluated for the test's species identification assays for E. coli and P. aeruginosa, which are used in conjunction with the mutant gyrase assays in reporting the mutant gyrase gene for only these two organisms. The Acuitas AMR Gene Panel results were compared with species identification by well-established automated species identification methods and for AMR gene detection by Whole Genome Sequencing (WGS).
Each isolate was tested in duplicate from a single 0.5 McFarland bacterial suspension. All testing results were compared to corresponding WGS results in order to assess cross-reactivity for those resistance genes harbored by a given isolate. In the event of any observed cross-reactivity, serial dilutions were performed, and the isolate was retested until cross-reactivity was no longer observed. Organisms evaluated during the Cross-Reactivity Study are shown in Table 15 below. Appropriate controls were performed during the execution of the Cross-Reactivity Study. A total of 41 Positive External Controls and 40 Negative External Controls were tested. In total, there were six External Control failures: Three (3) Positive Control A failures, one (1) Positive Control C failure, and two (2) Negative Control failures. One Positive Control C failure was not repeated, but all other failures were re-tested and produced expected results.
Table 15. Cross-Reactivity Panel
| Bacterial Species | | |
| --- | --- | --- |
| Achromobacter xylosoxidans | Enterococcus gallinarum | Raoultella planticola |
| Acinetobacter baumannii complex | Enterococcus gilvus | Salmonella enterica |
| Acinetobacter ursingii | Enterococcus hirae\( ^{c} \) | Salmonella species |
| Aeromonas hydrophila | Enterococcus italicus | Serratia marcescens |
| Candida albicans | Enterococcus pseudoavium | Serratia plymuthica |
| Citrobacter braakii | Enterococcus raffinosus | Shigella boydii\( ^{d} \) |
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| Bacterial Species | | |
| --- | --- | --- |
| Citrobacter freundii complex | Hafnia alvei | Shigella dysenteriae^{d} |
| Citrobacter koseri | Klebsiella oxytoca | Sphingomonas paucimobilis |
| Citrobacter youngae | Klebsiella oxytoca ESBL | Staphylococcus aureus |
| Clostridioides (Clostridium) difficile | Leclercia adecarboxylata | Staphylococcus capitis |
| Corynebacterium diphtheriae | Moraxella catarrhalis^{c} | Staphylococcus epidermidis |
| Escherichia fergusonii^{a} | Morganella morganii ssp morganii | Staphylococcus haemolyticus |
| Escherichia hermannii^{b} | Proteus vulgaris | Staphylococcus hominis |
| Enterobacter aerogenes | Providencia rettgeri | Staphylococcus lugdunensis |
| Enterobacter cloacae | Providencia stuartii | Staphylococcus saprophyticus |
| Enterobacter cloacae complex | Pseudomonas fluorescens | Staphylococcus warneri |
| Enterobacter hormaechei^{c} | Pseudomonas luteola | Streptococcus agalactiae |
| Enterococcus dispar^{c} | Pseudomonas oryzihabitans | Streptococcus pyogenes |
| Enterococcus durans | Pseudomonas putida | Yersinia pseudotuberculosis |
| Enterococcus faecium | Pseudomonas stutzeri | |
$^{a}$ One isolate was identified as Escherichia fergusonii by ATCC and by WGS, whereas automated species identification methods identified 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 automated species identification methods designation as E. coli. This isolate is included as E. fergusonii based on ATCC and WGS designation.
$^{b}$ One isolate was identified as Escherichia hermannii by ATCC and by WGS. Automated species identification methods identified the isolate 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 Automated species identification methods designation as E. coli. This isolate is included as E. hermannii based on ATCC and WGS designation.
$^{c}$ These isolates are included based on species designations by ATCC and/or WGS.
$^{d}$ Some but not all isolates of Shigella boydii and Shigella dysenteriae cross-react with the E. coli species ID assay of the Acuitas AMR Gene Panel.
Table 16 below shows false positive (FP) results for organisms tested in the Cross-Reactivity Study without inclusion of results from the Analytical Reactivity (Inclusivity) Study.
Table 16. False Positives Observed in Cross-Reactivity Study for Acuitas AMR Gene Targets
| Marker | Cross Reactivity Study Isolates | | Species Where FP Observed |
| --- | --- | --- | --- |
| | # Times Positive Reported / Total Samples where Target was not Expected | % FP | |
| ANT | 1/227 | 0.4 | C. koseri |
| CMY | 2/229 | 0.9 | C. braakii |
| OXA-9 | 1/227 | 0.4 | S. marcescens |
| OXA-48 | 1/221 | 0.5 | A. hydrophila |
| SUL1 | 4/207 | 1.9 | P. vulgaris |
| VIM | 1/229 | 0.4 | S. marcescens |
Organisms were also evaluated for cross-reacting with E. coli and P. aeruginosa species identification assays (used only in algorithm for gyrase mutant identification) of the Acuitas AMR Gene Panel. No FP were observed, except for Shigella boydii, Shigella
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dysenteriae, Shigella flexneri and Shigella sonnei, which cross-reacted with the E. coli ID assay on the Acuitas AMR Gene Panel as expected from in silico analysis. One isolate of Shigella boydii (ATCC 9207) and one isolate of Shigella dysenteriae (ATCC 49347) did not cross-react with the E. coli ID assay. All Shigella strains demonstrating cross-reactivity with the E. coli ID assay had wild-type gyrase sequences. Shigella species are not claimed for the assay and will not be reported for the test system. This information was included as a limitation in the package insert.
Cross-reactivity with other genes associated with antimicrobial resistance was not observed for any AMR gene assays on the Acuitas AMR Gene Panel.
# 4. Assay Reportable Range:
N/A
# 5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):
a. External Controls
External processing controls consisting of external positive control and negative control organisms were included during analytical and clinical studies evaluating the Acuitas AMR Gene Panel. On each testing day, one negative control and one individual bacterial positive control were tested. Table 17 below shows a list of the external controls prepared and tested during the clinical study (stock and prospective study).
Table 17. Performance of External Controls (Clinical Study)
| Strain | Organism | Target Genes | Observed/Expected |
| --- | --- | --- | --- |
| Positive Control A | E. coli | AAD, CMY | 15/16 |
| Positive Control B | P. mirabilis | AAC, AAD, armA, CMY, NDM, SUL-1, SUL-2, TEM | 16/16 |
| Positive Control C | K. pneumoniae | AAC, AAD, CTX-M-1, SHV, SUL-1, SUL-2,TEM | 14/15 |
| Negative Control | S. aureus | Negative for all targets | 61/63 |
For the analytical performance evaluation, sets of positive external controls representing a variety of organisms and AR Gene targets and one negative control (negative for all targets) were utilized. Individual analytical performance protocols specified the use of these daily external controls, requiring at least one positive external and one negative external control be tested per testing event with valid results in order for data to be included in a given study. Of the 431 external control samples run, 99.1% (427/431) gave a valid result on the first attempt. Only sample test results with valid external controls were included in the analysis of assay performance.
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# b. Assay Controls
The kit also includes eight (8) tubes of dried internal amplification control (Assay Control). The Assay Control for the Acuitas AMR Gene Panel test is Bacillus globigii (ATCC 31028), and it monitors DNA extraction efficiency and PCR inhibition for all pathogens and resistance genes. Since the Bacillus Assay Control test is in a PCR well by itself (one-plex assay), it should be positive independent of the other PCR assays. The Assay Control provided with the Acuitas AMR Gene Panel Test is added to each sample at the start of the extraction process.
# c. Stability
# (i) Sample DNA Stability
A study was conducted to evaluate the stability of DNA extracted from pure bacterial colonies using the QIAGEN EZ1 Advanced XL System with the Acuitas AMR Gene Panel after storage at either 15-25°C or 2-8°C. Testing was performed by one operator across two QS 5 instruments. Three replicates of each isolate (5 isolates) were evaluated. Test results demonstrated that extracted DNA from a 0.5 McFarland suspension for use with Acuitas AMR Gene Panel is stable for up to 6 hours at 15-25°C (room temperature), up to 7 days at 4 °C (2-8°C), and up to 6 hours at room temperature followed by up to 7 days at 2-8 °C. The study panel consisted of the following organisms and target gene panel members as shown in Table 18 below.
Table 18. Organism and Resistance Marker Panel for Sample DNA Stability Study
| Organism | Gene Marker |
| --- | --- |
| E. coli | AAC, CTX-M-9, DFR, EC-gyrA-MT, SUL1, SUL2, TEM |
| K. pneumoniae | AAC, AAD, armA, DHA, KP-gyrA-MT, NDM, SHV, SUL1, SUL2 |
| P. aeruginosa | PA-gyr83-WT^{a} |
| P. mirabilis | AAC, CTX-M-2, SUL1, SUL2, TEM |
| E. faecalis | APH^{b} |
$^{a}$gyr83-WT not reported for P. aeruginosa.
$^{b}$APH not reported for E. faecalis.
During the execution of the Sample Stability Study, a total of four (4) positive external controls, four (4) negative external controls, and twelve (12) no template controls were tested. In all cases the controls passed by producing the expected results.
# (ii) Prepared Acuitas AMR Gene Panel Assay Plate ( with Extracted DNA)
A study was conducted to determine the stability of extracted DNA after being added to the Acuitas AMR Gene Panel Assay plates along with Acuitas AMR Gene Panel Master Mix. This was designated as a "prepared plate." The prepared plate stability protocol evaluated the performance of the prepared assay plate after storage at 2-8°C for 6 hrs with a set of five (5) isolates containing a diverse subset of antimicrobial resistance genes detected by the Acuitas AMR Gene Panel assay (See Table 18 above for list of the organism/marker panel tested). Results from prepared plates were
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compared to plates without storage. Three (3) replicates of each isolate were evaluated for each testing event. Overall, this study showed that prepared plates with extracted DNA were stable for up to 6 hours at 2-8°C with all targets detected.
During the execution of the Sample Stability-Prepared Plate Study, a total of two (2) positive external controls, two (2) negative external controls, and six (6) no template controls were tested. In all cases, the controls passed as the produced the expected results.
### (iii) Fresh vs Frozen Bacterial Suspension Study
To evaluate the equivalence of fresh bacterial suspensions and frozen bacterial suspensions, a Fresh vs. Frozen Study was conducted to determine the performance of the Acuitas AMR Gene Panel with fresh 0.5 McFarland bacterial suspensions vs. frozen 0.5 McFarland bacterial suspensions. Nine (9) isolates [(3) E. coli, (2) K. pneumoniae, (1) P. aeruginosa, (2) P. mirabilis, and (1) E. faecalis] were evaluated. Ten replicates of each isolate were tested by three operators before and 56 days after freezing at -20°C. All resistance markers exhibited 100% agreement when comparing detection before and 56 days after freezing, except for ANT, DFR, and DHA. For ANT, a single FP result was observed for the P. aeruginosa isolate at day 56. This isolate was retested ten times and demonstrated only the expected results during repeat testing. For DFR and DHA, a single FP result for each marker was obtained for a single replicate with a P. mirabilis isolate before freezing. Retesting of ten replicates of this isolate demonstrated only the expected results. The results of this study support that 0.5 McFarland bacterial suspensions can be stored for up to 56 days at -20°C before testing with the Acuitas AMR Gene Panel.
During the execution of the Fresh vs. Frozen Isolate Study, a total of (27) positive external controls, (27) negative external controls, and (12) no template controls were tested. In all but one case, the controls passed by producing the expected results. One Positive Control A failed initial testing, but upon repeat testing produced expected results. The study panel consisted of the following organisms and target gene panel members as shown in Table 19 below. These results support the claim that 0.5 McFarland suspensions stored at -20°C are stable up to 56 days and are acceptable for use in conducting the Reproducibility Study.
Table 19. Organism and Resistance Gene Marker Panel
| Organism | Resistance Gene Markers |
| --- | --- |
| E. coli | EC-gyrA-MT, AAD^{a}, ANT, DFR, KPC, SUL1, SUL2, TEM |
| E. coli | SUL2, TEM |
| E. coli | EC-gyr87-WT^{a}, DFR, DHA, SUL1, SUL2, TEM |
| K. pneumoniae | KP-gyrA-MT^{b}, AAC, armA^{b}, CMY, AAD, NDM, OXA-48, SHV^{b}, CTX-M-1, TEM |
| K. pneumoniae | KP-gyr83-WT^{b}, SUL1, SUL2, AAC, SHV^{b}, TEM, OXA-1, IMP |
| P. aeruginosa | PA-gyrA-MT, SUL1^{c}, PER, VIM |
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| P. mirabilis | SUL1^{d}, SUL2, AAC, ANT, rmt, TEM, VEB, CMY |
| --- | --- |
| P. mirabilis | SUL1^{d}, SUL2, AAC, AAD^{d}, APH, CTX-M-9, DFR, OXA-1, TEM |
| E. faecalis | vanA |
$^{a}$AAD and gyr87-WT are not reported for E. coli.
$^{b}$gyrA-MT, gyr83-WT, armA, and SHV not reported for K. pneumoniae.
$^{c}$SUL1 not reported for P. aeruginosa.
$^{d}$AAD and SUL1 not reported for P. mirabilis.
# (iv) Frozen DNA Stability
A study was conducted to evaluate the stability of DNA extracted from pure bacterial colonies using the QIAGEN EZ1 Advanced XL instrument with the Acuitas AMR Gene Panel after storage at -15°C to -25°C for 30 days. The same organism panel was used as shown in Table 18 above under Sample DNA Stability. Three replicates of each isolate exhibiting the majority of antimicrobial resistance genes detected by the Acuitas AMR Gene Panel assay were evaluated during the Frozen DNA Stability study. Extracted DNA from 0.5 McFarland suspensions was stored under the following conditions: -15°C to -25°C for 14 days and for 30 days. The study results in the summary report showed that there was 100% agreement between T=0 days, T=14, and T=30 days for extracted DNA stored at -15 to -25°C. Based on the results provided, frozen DNA is stable for use with the Acuitas AMR Gene Panel assay for up to 30 days.
During the execution of the Sample Stability Frozen DNA, a total of (5) positive external controls, (5) negative external controls, and (14) no template controls were tested. The controls passed by producing the expected results.
# (v) Acuitas AMR Gene Panel Stability
Three independent lots of the Acuitas AMR Gene Panel will be evaluated for stability in a Real-Time Study at time points established for the respective material after storage under the conditions specified for the plate kit (20-25°C) and reagent kit (2-8°C). Stability studies support two months plate kit storage at 20-25°C and reagent kit storage at 2-8°C. Real-time stability studies for determining shelf-life and expiration dating are ongoing.
# 6. Detection Limit:
a. LoD
N/A. The extracted DNA from a 0.5 McFarland bacterial suspension is used for the assay.
b. Analytical Reactivity
Two hundred ninety-eight (298) isolates covering all resistance genes detected by the Acuitas AMR Gene Panel test were assessed for reactivity in this study. Each isolate was
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tested once, except for isolates harboring rare resistance genes, which were tested in replicates to achieve at least 18 positive data points per gene target assessed. Table 20 shows the detection of various AMR genes during wet testing. The Acuitas AMR Gene Panel results were compared with species identification by well-established automated species identification methods and AMR gene detection by WGS. The genes predicted to be detected by in silico analysis are listed in the last column according to the following categories: “Detectable,” “Likely Detectable,” and “Potentially Detectable.”
During the execution of the Analytical Reactivity study, a total of 36 positive external controls and 37 negative external controls were tested. There were a total of two (2) External Control failures—one (1) Positive Control A failure and one (1) Negative Control failure. All failures were retested and produced expected results.
Table 20. Summary of AMR Gene Variants Tested for Inclusivity/Reactivity on the Acuitas AMR Gene Panel a,c
| AMR Gene | Acuitas AMR Gene Panel | | | | Other AMR Gene Variants Predicted to be Detected by Acuitas AMR Gene Panel Based on In Silico Analysis^{d} |
| --- | --- | --- | --- | --- | --- |
| | Number of Samples Positive for Gene by WGS | Number of Unique Isolates Positive for Gene by WGS | AMR Gene(s) Detected | AMR Gene(s) Not Detected | |
| AAC | 198 | 162 | aac(3)-IIa, aac(3)-IId, aac(3)-IVa, aac(6')-Ib, aac(6')Ib-cr, aacA4-8, aacA4 | | Detectable: aac(3)-IIc, aac(3)-IIe, aac(3)-Ib-aac(6')-Ib, ant(3')-Ih-aac(6')-IId |
| AAD | 70 | 62 | aadA1, aadA2 | | Detectable: aadA13, aadA3, aadA8, aadA8b, aadA7^{e}, aadA17 Likely Detectable: aadA12, aadA21, aadA22, aadA23 |
| ANT | 63 | 40 | aadB | | - |
| APH | 15 | 14 | aph(4)-Ia | aph(4)-Ia_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, blaCMY-99, blaLAT-1, blaCMY-103, blaCMY-117, blaCMY-79 Likely Detectable: blaCMY-13, blaCMY-26, blaCMY-37, blaCMY-49, blaCMY-73, blaCMY-79, blaCMY-116, blaCMY-117, Potentially Detectable: blaCMY-40, blaCMY-53 |
| CTX-M-1 | 72 | 62 | blaCTX-M-1, blaCTX-M-15, blaCTX-M-55, blaCTX-M-64 | blaCTX-M-15_DQ302097 | Detectable: blaCTX-M, blaCTX-M-10, blaCTX-M-101, blaCTX-M-103, blaCTX-M-107, blaCTX-M-108, blaCTX-M-109, blaCTX-M-11, blaCTX-M-114, blaCTX-M-117, blaCTX-M-12, blaCTX-M-123, blaCTX-M-132, blaCTX-M-136, blaCTX-M-139, blaCTX-M-144, blaCTX-M-22, blaCTX-M-28, blaCTX-M-29, blaCTX-M-3, blaCTX-M-30, blaCTX-M-32, blaCTX-M-33, blaCTX-M-34, blaCTX-M-36, blaCTX-M-37, blaCTX-M-38, blaCTX-M-42, blaCTX-M-52, blaCTX-M-53, blaCTX-M-54, blaCTX-M-58, blaCTX-M-60, blaCTX-M-61, blaCTX-M-62, blaCTX-M-66, blaCTX-M-68, blaCTX-M-69, blaCTX-M-71, blaCTX-M-72, blaCTX-M-79, blaCTX-M-82, blaCTX-M-88, blaCTX-M-89, blaCTX-M-96 |
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| AMR Gene | Acuitas AMR Gene Panel | | | | Other AMR Gene Variants Predicted to be Detected by Acuitas AMR Gene Panel Based on In Silico Analysis\( ^{d} \) |
| --- | --- | --- | --- | --- | --- |
| | Number of Samples Positive for Gene by WGS | Number of Unique Isolates Positive for Gene by WGS | AMR Gene(s) Detected | AMR Gene(s) Not Detected | |
| | | | | | Likely Detectable: blaCTX-M-116, blaCTX-M-142, blaCTX-M-23, blaCTX-M-80 |
| 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 |
| 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, |
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| AMR Gene | Acuitas AMR Gene Panel | | | | Other AMR Gene Variants Predicted to be Detected by Acuitas AMR Gene Panel Based on In Silico Analysis\( ^{d} \) |
| --- | --- | --- | --- | --- | --- |
| | Number of Samples Positive for Gene by WGS | Number of Unique Isolates Positive for Gene by WGS | AMR Gene(s) Detected | AMR Gene(s) Not Detected | |
| | | | | | 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)\( ^{a} \), Sul3 (AB281183)\( ^{b} \)Likely Detectable: Sul1 (AM746675)Potentially Detectable: Sul1 (AY260546) |
| Sul2 | 133 | 99 | Sul2 | Sul2_GQ421466 | - |
| TEM | 173 | 132 | blaTEM-1, blaTEM-117, blaTEM-143, blaTEM-176, blaTEM-192, blaTEM-1A, blaTEM-1B, blaTEM-1C, blaTEM-1D, blaTEM-2, blaTEM-33 | | 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-163, blaTEM-164, blaTEM-166, blaTEM-168, 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 |
| vanA | 21 | 4 | VanA-A | | - |
| 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 |
\( ^{a} \) Results include combinations of AMR gene variants across various organisms tested in the study.
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b 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 detected.
c 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.
d 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).
e The test panel for this study was composed of (5) Enterococcus faecalis, (69) Escherichia coli, (3) Klebsiella pneumoniae ssp ozaenae, (89) Klebsiella pneumoniae ssp pneumoniae, (34) Proteus mirabilis, and (98) Pseudomonas aeruginosa strains.
Table 21 shows a summary of false positives and false negatives observed during assessment of reactivity on the Acuitas AMR Gene Panel reported along with a breakdown of species in which these results were observed.
Table 21. Summary of False Positives and False Negatives Observed During Assessment of Reactivity of the Acuitas AMR Gene Panel
| AMR Gene^{a} | False Positives | | | False Negatives | | |
| --- | --- | --- | --- | --- | --- | --- |
| | Total FP | Species w/ FP | # FP per Species | Total FN | Species w/ FN | # FN per Species |
| AAC | 8 | *E. coli* | 1 | 3 | *K. pneumoniae* | 3 |
| | | *K. pneumoniae* | 5 | | | |
| | | *P. aeruginosa* | 1 | | | |
| | | *P. mirabilis* | 1 | | | |
| AAD | 2 | *K. pneumoniae* | 2 | 0 | N/A | N/A |
| ANT | 2 | *P. aeruginosa* | 1 | 0 | N/A | N/A |
| | | *P. mirabilis* | 1 | | | |
| APH | 1 | *K. pneumoniae* | 1 | 1 | *K. pneumoniae* | 1 |
| CMY | 10 | *E. coli* | 2 | 1 | *K. pneumoniae* | 1 |
| | | *K. pneumoniae* | 8 | | | |
| CTX-M-1 | 3 | *E. coli* | 2 | 1 | *K. pneumoniae* | 1 |
| | | *K. pneumoniae* | 1 | | | |
| DFR | 1 | *E. coli* | 1 | 0 | N/A | N/A |
| KPC | 2 | *E. coli* | 1 | 1 | *K. pneumoniae* | 1 |
| | | *K. pneumoniae* | 1 | | | |
| OXA-1 | 0 | N/A | N/A | 1 | *E. coli* | 1 |
| OXA-48 | 1 | *K. pneumoniae* | 1 | 1 | *K. pneumoniae* | 1 |
| OXA-9 | 3 | *E. coli* | 1 | 1 | *K. pneumoniae* | 1 |
| | | *K. pneumoniae* | 2 | | | |
| Sul1 | 6 | *E. coli* | 1 | 1 | *K. pneumoniae* | 1 |
| | | *K. pneumoniae* | 5 | | | |
| Sul2 | 2 | *K. pneumoniae* | 2 | 2 | *K. pneumoniae* | 2 |
| TEM | 1 | *K. pneumoniae* | 1 | 0 | N/A | N/A |
### 7. Assay Cut-Off:
The assay cut-offs for the Acuitas AMR Gene Panel were initially established and validated using pre-clinical data. The results from the four hundred thirty-nine (439) isolates were evaluated. For each assay target, Ct, Cq, Conf, and Delta Rn values were determined from
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which ROC curves were generated to show sensitivity and specificity over a range of Ct values for each assay target.
### 8. Accuracy (Instrument):
N/A
### 9. Carry-Over:
A panel of twelve isolates harboring a variety of targeted resistance genes was utilized for the study (See Table 22 below). Two operators performed testing, where a panel of six positive samples and six negative samples (S. aureus isolate negative for all resistance markers) were prepared and blinded to the other operator. Samples were sub-cultured onto blood agar and used to prepare a 0.5 McFarland suspension. DNA was extracted and the twelve samples were tested in alternating fashion with twelve negative controls to determine the rate of carry-over/cross contamination across multiple plates. During the execution of the Carry Over Cross Contamination Study, no positive external controls were evaluated. The negative external controls passed by producing the expected results (420/420). Isolates included in the test panel also generated expected results (171/171 results).
Table 22. Organism/Markers Included in the Carry-Over Study
| Organism ID | Resistance Genes |
| --- | --- |
| E. coli | AAC, , E. coli gyrA-MT, OXA-1, SHV, Sul1 |
| E. coli | AAC, , Sul1, TEM |
| K. pneumoniae | AAC, AAD, APH, , KPC, Sul1, TEM |
| P. aeruginosa | AAC, ANT, CTX-M-2, P. aeruginosa gyrA-MT |
| P. mirabilis | AAC, APH, CTX-M-9, DFR, OXA-1, Sul2, TEM |
| E. faecalis (2) | None |
| E. coli | AAC, CMY, E. coli gyrA-MT, SHV, Sul1, Sul2, TEM |
| K. pneumoniae | AAC, AAD, CTX-M-1, DFR, OXA-9, , Sul1, Sul2, TEM |
| K. pneumoniae | AAC, AAD, CTX-M-1, , NDM, RMT, Sul2, TEM, |
| P. aeruginosa | P. aeruginosa gyrA-MT, VIM |
| P. mirabilis | AAC, , armA, CTX-M-1, Sul2, TEM |
No cross-contamination was observed indicating that the open-plate format of the Acuitas AMR Gene Panel did not produce carry-over/cross-contamination between test samples.
### 10. Media Equivalency Study
Fifty-two isolates [(13) E. coli, (13) K. pneumoniae, (13) P. aeruginosa, and (13) P. mirabilis), each harboring at least one indicated resistance marker, were tested in a Media Equivalency Study to assess growth of isolates on blood agar and MacConkey agar. The Acuitas AMR Gene Panel results were evaluated for percent agreement with the two comparator methods (automated species identification methods and WGS) for each combination of organism, AMR gene, and agar media. Table 23 summarizes the results below, which includes original results without repeat results. All indicated resistance markers were included in the study, except for DHA, MCR-1, and vanA. E. faecalis was not included as it does not grow on MacConkey agar.
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Isolates were sub-cultured onto blood agar and MacConkey agar, and pure colonies were selected and used to generate 0.5 MacFarland suspensions for testing with the Acuitas AMR Gene Panel. Each isolate was tested once during the study, except for the following organisms, which were tested in duplicate: E. coli, K. pneumoniae, and P. mirabilis. Study results were considered acceptable if there was ≥98% agreement (i.e., PPA and NPA) between Acuitas AMR Gene Panel results for the two medias. All organism/resistant marker combinations met the above outlined criteria, except for those highlighted in grey cells within Table 23 below. These isolates were subjected to retesting. Upon retesting, all isolates yielded the expected results as described in footnotes to the table. This study did not uncover evidence of a media effect between blood and MacConkey agar—both of which are suitable for the Acuitas AMR Gene Panel. During the execution of the Media Equivalency Study, a total of (11) positive external controls, (11) negative external controls, and (19) no template controls were tested. In all cases the controls produced the expected results.
Table 23. Acuitas AMR Gene Panel Media Equivalency Study Performance
| Organism | Resistance Marker | Number of Testing Events | 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) |
| *E. coli* | 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 | -^{(1)} | 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* gyrA-MT | 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) |
| | 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) |
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| Organism | Resistance Marker | Number of Testing Events | Blood Agar | | Mac…
