(871 days)
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.
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.
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.
§ 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).