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The LIAISON PLEX® Gram‐Negative Blood Culture (BCN) Assay, performed using the automated, sample‐to‐result LIAISON PLEX® System, is a qualitative multiplexed in vitro diagnostic test for the simultaneous detection and identification of selected gram‐negative pathogens and/or selected genetic determinants associated with antimicrobial resistance in positive blood culture bottles. LIAISON PLEX® BCN Assay is performed directly on blood culture media using blood culture bottles identified as positive by a continuous monitoring blood culture system which contain gram‐negative bacteria as determined by Gram stain.

The LIAISON PLEX® BCN Assay detects and identifies the following:

Resistance Markers:

• CTX‐M (blaCTX‐M)
• IMP (blaIMP)
• KPC (blaKPC)
• NDM (blaNDM)
• OXA (blaOXA)
• VIM (blaVIM)
• MCR
• SME (blaSME)

Gram Negative Genera and Species:

• Enterobacteriaceae / Morganellaceae
• Acinetobacter baumannii
• Acinetobacter spp.
• Citrobacter spp.
• Enterobacter spp. (1)
• Escherichia coli (2)
• Haemophilus influenzae
• Klebsiella oxytoca
• Klebsiella pneumoniae
• Klebsiella variicola
• Morganella morganii
• Neisseria meningitidis
• Proteus spp.
• Pseudomonas aeruginosa
• Pseudomonas spp.
• Salmonella spp.
• Serratia marcescens
• Stenotrophomonas maltophilia

(1) Due to reclassification, Klebsiella aerogenes will be reported Enterobacter spp.
(2) LIAISON PLEX® BCN Assay will not distinguish between Escherichia coli and Shigella spp. (S. dysenteriae, S. boydii, S. flexneri and S. sonnei)

LIAISON PLEX® BCN Assay contains targets for the detection of genetic determinants associated with resistance to carbapenems (blaCTX‐M, blaIMP, blaKPC, blaNDM, blaOXA48‐like, blaVIM, blaSME) to aid in the identification of potentially antimicrobial‐resistant organisms in positive blood culture samples. In addition, the panel includes an assay for the detection of the mobilized genetic determinant MCR, an emerging marker of public health importance. The antimicrobial resistance gene or marker detected may or may not be associated with the agent responsible for disease. Negative results for these select antimicrobial resistance gene and marker assays do not indicate susceptibility, as multiple mechanisms of resistance to ß‐lactams and colistin exist.

LIAISON PLEX® BCN Assay is indicated for use in conjunction with other clinical and laboratory findings to aid in the diagnosis of bacterial bloodstream infections (BSI). LIAISON PLEX® BCN Assay is not intended to monitor treatment of these infections. Sub‐culturing of positive blood cultures is necessary to recover organisms for antimicrobial susceptibility testing (AST), for identification of organisms not detected by LIAISON PLEX® BCN Assay, to detect mixed infections that may not be detected by LIAISON PLEX® BCN Assay, for association of antimicrobial resistance marker genes to a specific organism, or for epidemiological typing.

The LIAISON PLEX® Gram‐Negative Blood Culture (BCN) Assay is an automated test for the detection and identification of nucleic acid from gram‐negative bacteria in a positive blood culture media sample. The BCN Assay is performed directly on blood culture media using blood culture bottles identified as positive by a continuous monitoring blood culture system, and which contain gram‐negative bacteria, as determined by a Gram stain.

The LIAISON PLEX® System is a fully automated, bench‐top "sample‐to‐answer" device that performs sample preparation, polymerase chain reaction (PCR) and microarray‐based hybridization for the detection of target‐specific nucleic acids. The test reagents are supplied as a single, disposable test cartridge. PCR is not performed on the LIAISON PLEX® BCN Assay, as it is a non‐amplified, direct detection test performed on the LIAISON PLEX® System.

Here's a breakdown of the acceptance criteria and study details for the LIAISON PLEX Gram-Negative Blood Culture Assay, based on the provided FDA 510(k) clearance letter:

1. Table of Acceptance Criteria and Reported Device Performance

Note: The reported performance for individual targets (Sensitivity/PPA and Specificity/NPA) is explicitly given for the "Combined" data set (Prospective + Pre-selected). For Resistance Markers, some N/A entries indicate insufficient positive reference cases in the clinical study.

2. Sample Size and Data Provenance

• Test Set (Clinical Study):
  • Prospective Samples: 381 unique specimens enrolled, 351 included in analysis (30 excluded due to duplicates or incomplete reference testing).
  • Pre-selected Samples: 231 left-over, de-identified specimens.
  • Contrived Samples: 746 specimens.
  • Total Clinical Samples (Prospective + Pre-selected): 582 specimens included in analysis.
  • Total Contrived Samples: 746 specimens included in analysis.
  • Data Provenance:
    • Prospective: Collected from four geographically diverse clinical sites within the United States between March 2024 and July 2024.
    • Pre-selected: Sourced from seven vendors in the United States and one site in Italy.
    • Contrived: Tested at four testing sites during April 2024 – August 2024.
  • Retrospective/Prospective: Prospective data was collected prospectively. Pre-selected and Contrived data were essentially retrospective/prepared, then tested blindly and randomized.

3. Number of Experts and Qualifications for Ground Truth

The document does not mention the use of "experts" in the sense of clinical specialists (e.g., radiologists) establishing ground truth for the test set. Instead, the ground truth was established by laboratory methods.

4. Adjudication Method for the Test Set

The document describes the "Reference Method Algorithm" (Table 19) where the LIAISON PLEX BCN Assay results were compared to:

• Culture followed by Automated microbiological/biochemical identification using VITEK 2 for most organism targets.
• Culture followed by Automated microbiological/biochemical identification using VITEK 2 with positive confirmation of the clinical isolate by PCR/BDS for Acinetobacter baumannii, Klebsiella oxytoca, Klebsiella pneumoniae, and Klebsiella variicola.
• PCR followed by bi-directional sequencing (BDS) for all resistance markers (CTX-M, IMP, KPC, NDM, OXA, VIM, MCR, SME).

This is a hierarchical or sequential method rather than an "adjudication" by multiple human readers for discrepancies. For pre-selected specimens, initial identification was by Standard of Care (SoC) and/or PCR followed by BDS, then confirmed.

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

• No, this was not an MRMC comparative effectiveness study where human readers' improvement with AI vs. without AI assistance was measured. This device is an in vitro diagnostic test for direct detection of nucleic acids, not an AI software intended to assist human readers.

6. Standalone Performance (Algorithm Only)

• Yes, this was a standalone performance study. The LIAISON PLEX BCN Assay is an automated, qualitative multiplexed in vitro diagnostic test performed on the LIAISON PLEX System. The performance metrics (Sensitivity/PPA and Specificity/NPA) described in the clinical study (Table 20 and 21) represent the standalone performance of the device without human interpretation of its results.

7. Type of Ground Truth Used

The ground truth for the clinical study (test set) was established using a combination of:

• Culture followed by Automated microbiological/biochemical identification (VITEK 2).
• PCR followed by bi-directional sequencing (BDS).
• In some cases, a combination of VITEK 2 and PCR/BDS for confirmation.
• For resistance markers, ground truth was solely by PCR followed by bi-directional sequencing (BDS).

8. Sample Size for the Training Set

The document does not explicitly state the sample size used for the training set of the LIAISON PLEX BCN Assay. The provided information focuses on the analytical and clinical validation of the final device. For IVD products like this, internal development and optimization (which would involve data that could be considered a "training set" in a broad sense for assay design) are typically not detailed in the same way as machine learning model training sets in FDA submissions. The inclusivity and exclusivity studies (Analytical Reactivity and Specificity) use laboratory-tested strains and in silico analysis respectively, which are key parts of ensuring the assay's design covers the intended targets and avoids false positives.

9. How the Ground Truth for the Training Set Was Established

Since an explicit "training set" sample size isn't provided, the method for establishing its ground truth isn't detailed. However, the comprehensive Analytical Reactivity (Inclusivity) and Analytical Specificity (Exclusivity) studies describe how the assay's design was validated:

• Analytical Reactivity (Inclusivity): Laboratory testing of 246 on-panel organisms (in triplicate) and in silico analysis using sequences from GenBank and WGS databases (February to April 2024). The ground truth for these would be the known identity of the characterized strains and the genetic sequences from public databases.
• Analytical Specificity (Exclusivity): Laboratory testing of 113 off-panel species (in triplicate) and in silico exclusivity assessment against on-panel and off-panel organisms from GenBank (June 7, 2024). The ground truth here is the known identity of these off-panel organisms and their genetic sequences.

These analytical studies effectively serve as the validation of the assay's "knowledge" or "training" on what to detect and what not to detect.

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The BIOFIRE Blood Culture Identification 2 (BCID2) Panel is a multiplexed nucleic acid test intended for use with BIOFIRE FILMARRAY 2.0 or BIOFIRE FILMARRAY TORCH Systems for the simultaneous qualitative detection and identification of multiple bacterial and yeast nucleic acids and select genetic determinants associated with antimicrobial resistance. The BIOFIRE BCID2 Panel test is performed directly on blood culture samples identified as positive by a continuous monitoring blood culture system. Results are intended to be interpreted in conjunction with Gram stain results. The following organism types and subtypes are identified using the BIOFIRE BCID2 Panel: Gram Positive Bacteria Enterococcus faecalis Staphylococcus spp. Streptococcus spp. Enterococcus faecium Staphylococcus aureus Streptococcus agalactiae (Group B) Listeria monocytogenes Staphylococcus epidermidis Streptococcus pneumoniae Staphylococcus lugdunensis Streptococcus pyogenes (Group A) Gram Negative Bacteria Acinetobacter calcoaceticus-baumannii complex Enterobacterales Bacteroides fragilis Enterobacter cloacae complex Haemophilus influenzae Escherichia coli Neisseria meningitidis (encapsulated) Klebsiella aerogenes Pseudomonas aeruginosa Klebsiella oxytoca Stenotrophomonas maltophilia Klebsiella pneumoniae group Proteus spp. Salmonella spp. Serratia marcescens Yeast Candida albicans Candida krusei Cryptococcus neoformans/gattii Candida auris Candida parapsilosis Candida glabrata Candida tropicalis The BIOFIRE BCID2 Panel contains assays for the detection of genetic determinants associated with resistance to methicillin (mecA/C and mecA/C in conjunction with MREJ, vancomycin (vanA and vanB), ß-lactams including penicillins, cephalosporins, monobactams, and carbapenems (blaCTX-M, blaIMP, blaKPC, blaNDM, blaOXA48-like, blaVIM) to aid in the identification of potentially antimicrobial-resistant organisms in positive blood culture samples. In addition, the panel includes an assay for the mobilized genetic determinant mor-1, an emerging marker of public health importance. The antimicrobial resistance gene or may not be associated with the agent responsible for disease. Negative results for these select antimicrobial resistance gene and marker assays do not indicate susceptibility, as multiple mechanisms of resistance to methicillin, vancomycin, b-lactams, and colistin exist. Antimicrobial Resistance Genes CTX-M KPC mecA/C NDM vanA/B IMP mcr-1 mecA/C and MREJ (MRSA) OXA-48-like VIM The BIOFIRE BCID2 Panel is indicated as an aid in the diagnosis of bloodstream infection and results should be used in conjunction with other clinical and laboratory findings. Positive results do not rule out co-infection with organisms not included in the BIOFIRE BCID2 Panel. The BIOFIRE BCID2 Panel is not intended to monitor treatment for bloodstream infection. Subculturing of positive blood cultures is necessary to recover organisms for susceptibility testing and epidemiological typing, to identify organisms in the blood culture that are not detected by the BIOFIRE BCID2 Panel, and for determination of species detected but not identified within complexes, groups, or genera by the BIOFIRE BCID2 Panel assays.

The BIOFIRE Blood Culture Identification 2 (BCID2) Panel is designed to simultaneously identify 43 bacteria and yeast responsible for bloodstream infections, as well as select genetic determinants of antimicrobial resistance (see Table 1), in a timeframe (about an hour) that allows the test results to be used in determining appropriate patient treatment and management. The BIOFIRE BCID2 Panel is performed directly on positive blood culture samples. The BIOFIRE BCID2 Panel is compatible with BioFire's PCR-based in vitro diagnostic BIOFIRE FILMARRAY 2.0 and FILMARRAY TORCH systems for infectious disease testing. A specific software module (i.e., BIOFIRE BCID2 Panel pouch module) is used to perform BIOFIRE BCID2 Panel testing on these systems. A test is initiated by loading Hydration Solution into one port of the BIOFIRE pouch and positive blood culture specimen mixed with the provided Sample Buffer into the other port of the BIOFIRE BCID2 Panel pouch and placing it in a BIOFIRE System. The pouch contains all the reagents required for specimen testing and analysis in a freeze-dried format; the addition of Hydration Solution and Sample/Buffer Mix rehydrates the reagents. After the pouch is prepared, the BIOFIRE Software guides the user though the steps of placing the the instrument, scanning the pouch barcode, entering the sample identification, and initiating the run. The BIOFIRE System contains a coordinated system of inflatable bladders and seal points, which act on the pouch to control the movement of liquid between the pouch blisters. When a bladder is inflated over a reagent blister, it forces liquid from the blister into connecting channels. Alternatively, when a seal is placed over a connecting channel it acts as a valve to open or close a channel. In addition, electronically-controlled pneumatic pistons are positioned over multiple plungers in order to deliver the rehydrated reagents into the blisters at the appropriate times. Two Peltier devices control heating and cooling of the pouch to drive the PCR reactions and the melt curve analysis. Nucleic acid extraction occurs within the BIOFIRE pouch using mechanical and chemical lysis followed by purification using standard magnetic bead technology. After extracting and purifying nucleic acids from the unprocessed sample, the BIOFIRE system performs a nested multiplex PCR that is executed in two stages. During the first stage, the BIOFIRE System performs a single, large volume, highly multiplexed reverse transcription PCR (rt-PCR) reaction. The products from first stage PCR are then diluted with a fresh, primer-free master mix and a fluorescent double stranded DNA binding dye (LC Green® Plus, BioFire Diagnostics). The solution is then distributed to each well of the array. Array wells contain sets of primers designed specifically to amplify sequences internal to the PCR products generated during the first stage PCR reaction. The 2nd stage PCR, or nested PCR, is performed in single plex fashion in each well of the array. At the end of the 2nd stage PCR, the array is interrogated by melt curve analysis for the detection of signature amplicons denoting the presence of specific targets. A digital camera placed in front of the 2nd stage PCR captures fluorescent images of the PCR reactions and software interprets the data. The BIOFIRE Software automatically interprets the results of each DNA melt curve analysis and combines the data with the results of the internal pouch controls to provide a test result for each organism on the panel.

Here's a breakdown of the acceptance criteria and the study information based on the provided FDA 510(k) summary for the BioFire Blood Culture Identification 2 (BCID2) Panel:

1. Table of Acceptance Criteria and Reported Device Performance:

The document primarily focuses on a software update to mitigate a risk of false negative results for C. tropicalis specifically. It states that the re-analysis with the modified software led to a "very minor adjustment to clinical sensitivity for S. epidermidis from 96.5% [93.0-98.2%] to 96.9%".

The summary does not provide a comprehensive table of acceptance criteria and the overall device performance for all targets. Instead, it highlights the impact of the software update on C. tropicalis detection and the resulting minor change in S. epidermidis sensitivity.

Acceptance Criteria (Implied / Pre-established for Original Device):

While not explicitly stated as "acceptance criteria" in this specific document, the overall context of a 510(k) clearance implies that the device must demonstrate comparable performance to its predicate. The key focus of this Special 510(k) is to show that the software update does not adversely affect, and in fact, improves, the device's performance, particularly concerning the false negative C. tropicalis issue. The acceptance criterion for this update seems to be that the corrected software resolves the C. tropicalis issue and does not significantly degrade performance on other targets.

2. Sample Size Used for the Test Set and Data Provenance:

• Test Set Sample Size: The document refers to "reanalysis of the performance data (clinical and non-clinical studies) with the modified pouch module software." It explicitly mentions that the C. tropicalis issue was identified during testing of Maine Molecular Quality Controls, Inc. (MMQC) FilmArray BCID2 Control Panel M416. While the specific number of tests performed on this control or other test sets with the modified software is not given in this summary, the "reanalysis of the performance data" implies that the original clinical and non-clinical study data from the predicate device (K193519) was re-evaluated using the new software.
• Data Provenance: The issue was identified through testing with an external control material (C. tropicalis synthetic control material). The "reanalysis of the performance data (clinical and non-clinical studies)" implies that data from the original (K193519) approval was used, which would have primarily involved prospective clinical samples from various geographic locations. However, this specific document does not detail the provenance of the original clinical data. The new data generated to address the C. tropicalis issue was from synthetic control material.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and the Qualifications of Those Experts:

• This information is not provided in the given FDA summary. The document focuses on a software update to an already cleared device, addressing a technical issue related to melting temperature analysis. The ground truth for the original clinical studies would have been established through a combination of clinical methods (e.g., standard microbiological culture and identification, susceptibility testing), but those details are not part of this specific Special 510(k) summary.

4. Adjudication Method for the Test Set:

• This information is not provided in the given FDA summary. For the original clinical studies related to the predicate device, an adjudication method (e.g., expert panel review of discrepancies) would likely have been employed, but this specific document does not detail it.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done, If So, What Was the Effect Size of How Much Human Readers Improve with AI vs Without AI Assistance:

• This is not applicable. The device (BIOFIRE Blood Culture Identification 2 Panel) is a fully automated multiplex nucleic acid test. It does not involve human readers interpreting results in the same way an imaging AI device would. Therefore, an MRMC study with human readers and AI assistance is not relevant to this device.

6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done:

• Yes. The BIOFIRE BCID2 Panel operates as a standalone diagnostic system. The "pouch module software" automatically interprets the melt curve analysis and combines data with internal controls to generate a test result. The description explicitly states, "The BIOFIRE Software automatically interprets the results of each DNA melt curve analysis and combines the data with the results of the internal pouch controls to provide a test result for each organism on the panel." There is no "human-in-the-loop" performance component for result generation, although clinical interpretation of results in conjunction with Gram stain and other findings is required.

7. The Type of Ground Truth Used (expert consensus, pathology, outcomes data, etc.):

• For the specific issue addressed in this Special 510(k) regarding C. tropicalis, the ground truth was known composition of synthetic control material.
• For the broader re-analysis of "clinical and non-clinical studies" (referring to the original predicate data), the ground truth for microbial identification in blood cultures typically involves standard microbiological culture (subculturing) and definitive identification methods (e.g., biochemical tests, mass spectrometry, sequencing), often supplemented by clinical context. The document emphasizes that "Subculturing of positive blood cultures is necessary to recover organisms for susceptibility testing and epidemiological typing, to identify organisms in the blood culture that are not detected by the BIOFIRE BCID2 Panel, and for determination of species detected but not identified within complexes, groups, or genera by the BIOFIRE BCID2 Panel assays," highlighting the role of traditional microbiology as a gold standard.

8. The Sample Size for the Training Set:

• This information is not provided in the given FDA summary, as the nature of this submission is a software update to an existing device, not a de novo algorithm development. The "pouch module software" utilizes pre-defined melting temperature (Tm) ranges and analysis parameters. The "reanalysis to include data generated from synthetic control materials" effectively served to refine or "retrain" specific parameters (Tm ranges) within the existing analytical framework, but the original training set size for the entire panel's algorithm is not detailed here.

9. How the Ground Truth for the Training Set Was Established:

• This information is not provided in the given FDA summary. For the original development of the BIOFIRE BCID2 Panel, the ground truth for establishing the algorithms' detection parameters and Tm ranges would have been based on extensive analytical studies using well-characterized microbial isolates (strains with confirmed identities) at various concentrations, likely identified by definitive culture-based methods and sequencing. The update specifically states that "Data from the synthetic control material(s) had not been included in the establishment and validation of the assay Tm ranges used for analysis by the pouch module software," indicating that the "training" for the C. tropicalis Tm range was incomplete previously and was corrected by incorporating this synthetic control material data.

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The GenMark ePlex® Blood Culture Identification Gram-Negative (BCID-GN) Panel is a qualitative nucleic acid multiplex in vitro diagnostic test intended for use on GenMark's ePlex Instrument for simultaneous qualitative detection and identification of multiple potentially pathogenic gram-negative bacterial organisms and select determinants associated with antimicrobial resistance in positive blood culture. In addition, the ePlex BCID-GN Panel is capable of detecting several gram-positive bacteria (Pan Gram-Positive assay) and several Candida species (Pan Candida assay). The ePlex BCID-GN Panel is performed directly on blood culture samples identified as positive by a continuous monitoring blood culture system and which contain gram-negative organism.

The following bacterial organisms and genes associated with antibiotic resistance are identified using the ePlex BCID-GN Panel: Acinetobacter baumannii, Bacteroides fragilis, Citrobacter sakazakii, Enterobacter cloacae complex, Enterobacter (non-cloacae complex), Escherichia coli, Fusobacterium necrophorum, Fusobacterium nucleatum, Haemophilus influenzae, Klebsiella oxytoca, Klebsiella pneumoniae group, Morganii, Neisseria meningitidis, Proteus, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella, Serratia marcescens, Stenotrophomonas maltophilia, CTX-M (blaCTX-M), IMP (blaMP) , KPC (blaKPC) , NDM (blaNDM), OXA (blaOXA) (OXA-23 and OXA-48 groups only), and VIM (blaVIM).

The ePlex BCID-GN Panel contains assays for the detection of genetic determinants associated with resistance to antimicrobial agents including CTX-M(blaCTX-M), which is associated with resistance to extended spectrum betalactamase (ESBL)-mediated resistance to penicillins, cephalosporins, and monobactams, as well as OXA (blaOXA) (OXA-23 and OXA-48 groups only), KPC (blaKPC), and metallo-beta-lactamases IMP (blaIMP), and NDM (blaNDM), which is associated with carbapenemase-mediated resistance. The antimicrobial resistance gene detected may or may not be associated with the agent responsible for disease. Negative results for these select antimicrobial resistance assays do not indicate susceptibility, as there are multiple mechanisms of resistance in gramnegative bacteria.

The ePlex BCID-GN Panel also contains targets designed to detect a broad range of organisms with a potentially misleading Gram stain result or organisms that may be missed by Gram staining altogether, for example in the case of coinfections. These include a broad Pan Gram-Positive assay (which is designed to detect Bacillus cereus group, Bacillus subtilis group, Enterococcus, Staphylococus, and Streptococcus), as well as a Pan Candida assay, which is designed to detect four Candida species: Candida albicans, Candida krusei, and Candida parapsilosis.

The detection and identification of specific bacterial and fungal nucleic acids from individuals exhibiting signs and/or symptoms of bloodstream infection aids in the diagnosis of bloodstream infection when used in conjunction with other clinical information. The results from the ePlex BCID-GN Panel are intended to be interpreted in conjunction with Gram stain results and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.

Negative results in the setting of a suspected bloodstream infection with pathogens that are not detected by this test. Positive results do not rule out co-infection with other organisms; the organism(s) detected by the ePlex BCID-GN Panel may not be the definite cause of disease. Additional laboratory testing (e.g. sub-culturing of positive blood cultures for identification of organisms not detected by ePlex BCID-GN Panel and for susceptibility testing, differentiation of mixed growth, and association of antimicrobial resistance marker genes to a specific organism) and clinical presentation must be taken into consideration in the final diagnosis of bloodstream infection.

The ePlex Blood Culture Identification Gram-Negative (BCID-GN) Panel is based on the principles of competitive nucleic acid hybridization using a sandwich assay format, wherein a single-stranded target binds concurrently to a sequence-specific solution-phase signal probe and a solid-phase electrode-bound capture probe. The test employs nucleic acid extraction, target amplification via polymerase chain reaction (PCR) or reverse transcription PCR (RT-PCR) and hybridization of target DNA. In the process, the double-stranded PCR amplicons are digested with exonuclease to generate single-stranded DNA suitable for hybridization.

Nucleic acid extraction from biological samples occurs within the cartridge via cell lysis, nucleic acid capture onto magnetic beads, and release for amplification. The nucleic acid extraction is processed through microfluidic liquid handling. Once the nucleic acid targets are captured and inhibitors are washed away, the magnetic particles are delivered to the electrowetting environment on the printed circuit board (PCB) and the targets are eluted from the particles and amplified.

During hybridization, the single-stranded target DNA binds to a complementary, single-stranded capture probe immobilized on the working gold electrode surface. Single-stranded signal probes (labeled with electrochemically active ferrocenes) bind to specific target sequence / region adjacent to the capture probe. Simultaneous hybridization of target to signal probes and capture probe is detected by alternating current voltammetry (ACV). Each working electrode on the array contains specific capture probes, and sequential analysis of each electrode allows detection of multiple analyte targets.

The presented document is a 510(k) summary for the GenMark ePlex Blood Culture Identification Gram-Negative (BCID-GN) Panel, a qualitative nucleic acid multiplex in vitro diagnostic test. The study aims to demonstrate that the updated device (Subject Device) is substantially equivalent to its predicate device (original GenMark ePlex BCID-GN Panel, K182619). The data focuses on analytical and clinical performance.

Here's an analysis based on the provided text, addressing your specific points:

1. A table of acceptance criteria and the reported device performance:

The document doesn't explicitly state a single "acceptance criteria" table with pre-defined thresholds for all metrics (like sensitivity, specificity) against which the reported performance is directly compared in a summary table. However, it implicitly demonstrates acceptance by presenting individual performance metrics (Sensitivity/PPA and Specificity/NPA) for each target organism and resistance gene across different sample types (Prospective, Retrospective, Contrived, and Overall). The consistent high percentages for these metrics indicate that the device met the required performance for regulatory acceptance, even if the precise numerical cut-offs aren't explicitly stated in a singular table for all parameters.

Instead of a single "acceptance criteria" table, the document functions as a detailed report of performance against implicit acceptance criteria for in vitro diagnostic devices, which typically demand high sensitivity and specificity. The data tables already present the "reported device performance."

Example of reported device performance for a few key targets (extracted from Tables 7-34):

(Note: "Overall" for Pan targets combines Prospective, Retrospective, and Retrospective (Non-Intended Use), but excludes Contrived. The overall figures for other targets combine Prospective/Retrospective and Contrived samples as a whole.)

2. Sample sized used for the test set and the data provenance:

• Test Set Sample Size:

  • Clinical Samples: 349 prospective samples (167 fresh, 182 frozen) and 577 retrospective samples. Total clinical samples: 926.
  • Contrived Samples: 777 samples.
  • Additional Retrospective (Non-Intended Use) for Pan targets: 741 samples.
  • Total evaluable samples across studies: 349 (prospective) + 577 (retrospective) + 777 (contrived) + 741 (non-intended use for pan targets) = 2444 samples in total tested across various evaluations. The overall performance tables combine various subsets of these.

• Data Provenance:

  • Country of Origin: Not explicitly stated, but 7 clinical sites were involved in prospective collection (suggests multi-center, likely within the US given FDA submission).
  • Retrospective or Prospective: Both.
    • Prospective: 349 samples collected from June 2014 through July 2016 (frozen) and June through July 2018 (fresh).
    • Retrospective: 577 samples collected.
    • Contrived: Laboratory-generated samples.

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts:

The document does not explicitly state the "number of experts" or their specific "qualifications" involved in establishing the ground truth. It refers to "standard laboratory procedures for identification of blood culture isolates, including traditional and automated identification methods, MALDI-TOF IVD, and microbiological and biochemical techniques" (Table 4). For antibiotic resistance genes, it uses "analytically validated qPCR amplification assays followed by bi-directional sequencing." This implies laboratory professionals with expertise in microbiology and molecular diagnostics perform these comparator methods, but specific numbers or individual qualifications are not detailed.

4. Adjudication method (e.g. 2+1, 3+1, none) for the test set:

The document does not describe an explicit "adjudication method" involving multiple human readers (e.g., 2+1 or 3+1). The ground truth is established through comparator methods as described above (standard laboratory procedures, PCR/sequencing). Any discrepancies between the device and these comparator methods are analyzed and explained (e.g., the detailed footnotes in the performance tables and the discussion regarding CTX-M false negatives). This is typical for in vitro diagnostic (IVD) device studies, where ground truth is often determined by a reference laboratory standard or follow-up confirmatory testing, rather than human expert consensus on image interpretation.

5. If a multi reader multi case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance:

No, an MRMC comparative effectiveness study was not done. This device is an in vitro diagnostic (IVD) test for direct detection of pathogens and resistance genes from blood cultures, not an "AI-assisted image interpretation" device to be used by human readers. Therefore, the concept of human readers improving with AI assistance is not applicable here.

6. If a standalone (i.e. algorithm only without human-in-the-loop performance) was done:

Yes, this study represents a standalone technical performance evaluation of the ePlex BCID-GN Panel device. The device itself performs the detection and identification, and its results are compared directly to the gold standard comparator methods. There is no "human-in-the-loop" performance element in the operation of this specific diagnostic test.

7. The type of ground truth used (expert consensus, pathology, outcomes data, etc):

The ground truth was established by a combination of:

• Standard Laboratory Procedures: including traditional and automated identification methods, MALDI-TOF IVD, and microbiological and biochemical techniques for organism identification.
• Molecular Confirmation: Analytically validated PCR assays followed by bi-directional sequencing for specific organism identifications (e.g., Acinetobacter baumannii, Candida parapsilosis) and for all antibiotic resistance genes (qPCR amplification followed by bi-directional sequencing).
• Additional Testing for Discrepancies: Further investigations (e.g., repeat extractions, qPCR testing from isolates, testing with FDA-cleared multiplex assays) were used to resolve discrepancies and confirm the true status of samples, as detailed in the footnotes for several performance tables (e.g., Table 28 for CTX-M).

This ground truth method is based on a hierarchy of established laboratory and molecular techniques rather than human expert consensus on interpretation.

8. The sample size for the training set:

The document does not explicitly mention a "training set" in the context of machine learning, as this is a molecular diagnostic device, not an AI/ML product. However, the development of such a device involves extensive analytical studies related to inclusivity (reactivity), exclusivity (specificity), and limit of detection (LoD), which are analogous to data used in the development or "training" phase.

• Analytical Reactivity (Inclusivity): Evaluated with a panel of 336 strains/isolates.
• Limit of Detection (LoD): Determined using quantified reference strains for each target.
• In silico analysis: Used for predicted reactivity of genus/group assays and resistance markers, involving evaluation of sequence data.

While not a "training set" in the AI sense, these analytical studies inform the design and performance characteristics of the diagnostic assays.

9. How the ground truth for the training set was established:

As noted above, there isn't a "training set" in the AI/ML sense. For the analytical studies that are foundational to the device's design (e.g., inclusivity, LoD):

• Ground truth for inclusivity (analytical reactivity): Established by using characterized strains/isolates with known identity. The strains' identities are determined by standard microbiological and molecular methods.
• Ground truth for LoD: Established by using quantified reference strains where the concentration (CFU/mL) of the organism is precisely known.
• Ground truth for in silico analysis: Based on existing genetic sequence data and bioinformatic analysis to predict reactivity, relying on established genetic databases and characterizations.

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The BioFire® Blood Culture Identification 2 (BCID2) Panel is a multiplexed nucleic acid test intended for use with FilmArray® 2.0 or FilmArray® Torch systems for the simultaneous qualitative detection and identification of multiple bacterial and yeast nucleic acids and select genetic determinants associated with antimicrobial resistance. The BioFire BCID2 Panel test is performed directly on blood culture samples identified as positive by a continuous monitoring blood culture system. Results are intended to be interpreted in conjunction with Gram stain results. The following organism types and subtypes are identified using the BioFire BCID2 Panel:

Gram Positive Bacteria

• Enterococcus faecalis
• · Staphylococcus spp.
• · Streptococcus spp.
• Enterococcus faecium
• Staphylococcus aureus
• · Streptococcus agalactiae (Group B)
• Listeria monocytogenes
• Staphylococcus epidermidis
• Streptococcus pneumoniae
• Staphylococcus lugdunensis
• · Streptococcus pyogenes (Group A)

Gram Negative Bacteria

• Acinetobacter calcoaceticus-baumannii complex
• · Enterobacterales
• · Bacteroides fragilis
• Enterobacter cloacae complex
• Haemophilus influenza
• · Escherichia coli
• · Neisseria meningitidis (encapsulated)
• · Klebsiella aerogenes
• · Pseudomonas aeruginosa
• · Klebsiella oxytoca
• · Stenotrophomonas maltophilia
• · Klebsiella pneumoniae group
• · Proteus spp.
• · Salmonella spp.
• · Serratia marcescens

Yeast

• · Candida albicans
• Candida krusei
• · Cryptococcus neoformans/gattii
• Candida auris
• · Candida parapsilosis
• · Candida tropicalis
• Candida glabrata

The BioFire BCID2 Panel contains assays for the detection of genetic determinants associated with resistance to methicillin (mecA/C and mecA/C in conjunction with MREJ, vancomycin (vanA and vanB), 0-lactams including penicillins, cephalosporins, monobactams, and carbapenems (blaCTX-M, blaKPC, blaNDM, blaOXA48-like, bla VIM) to aid in the identification of potentially antimicrobial-resistant organisms in positive blood culture samples. In addition, the panel includes an assay for the mobilized genetic determinant mcr-1, an emerging marker of public health importance. The animicrobial resistance gene or may not be associated with the agent responsible for disease. Negative results for these select antimicrobial resistance gene and marker assays do not indicate susceptibility, as multiple mechanisms of resistance to methicillin, vancomycin, B-lactams, and colistin exist.

Antimicrobial Resistance Genes

• CTX-M
• КРС
• · mecA/C
• NDM
• vanA/B
• · IMP
• mcr-1
• · mecA/C and MREJ (MRSA)
• OXA-48-like
• VIM

The BioFire BCID2 Panel is indicated as an aid in the diagnosis of bloodstream infection and results should be used in conjunction with other clinical and laboratory findings. Positive results do not rule out co-infection with organisms not included in the BioFire BCID2 Panel is not intended to monitor treatment for bloodstream infection.

Subculturing of positive blood cultures is necessary to recover organisms for susceptibility testing and epidemiological typing, to identify organisms in the blood culture that are not detected by the BioFire BCID2 Panel, and for determination of species detected but not identified within complexes, groups, or genera by the BioFire BCID2 Panel assays.

The BioFire Blood Culture Identification 2 (BCID2) Panel is designed to simultaneously identify 43 bacteria and yeast responsible for bloodstream infections, as well as select genetic determinants of antimicrobial resistance (see Table 1), in a timeframe(about an hour) that allows the test results to be used in determining appropriate patient treatment and management. The BioFire BCID2 Panel is performed directly on positive blood culture samples.

The BioFire BCID2 Panel is compatible with BioFire's PCR-based in vitro diagnostic FilmArray Torch systems for infectious disease testing, A specific software module (i.e., BioFire BCID2 Panel pouch module) is used to perform BioFire BCID2 Panel testing on these systems.

A test is initiated by loading Hydration Solution into one port of the FilmArray pouch and positive blood culture specimen mixed with the provided Sample Buffer into the other port of the BioFire BCID2 Panel pouch and placing it in a FilmArray instrument. The pouch contains all of the reagents required for specimen testing and analysis in a freeze-dried format: the addition of Hydration and Sample/Buffer Mix rehydrates the reagents. After the pouch is prepared, the FilmArray Software quides the user through the pouch into the instrument, scanning the pouch barcode, entering the sample identification, and initiating the run.

The FilmArray instruments contain coordinated systems of inflatable bladders and seal points, which act on the pouch to control the movement of liquid between the pouch blisters. When a bladder is inflated over a reagent blister, it forces liquid from the blister into connecting channels. Alternatively, when a seal is placed over a connecting channel it acts as a valve to open or close a channel. In addition, electronically-controlled pneumatic pistons are positioned over multiple plungers in order to deliver the rehydrated reagents into the blisters at the appropriate times. Two Peltier devices control heating and cooling of the PCR reactions and the melt curve analysis.

Nucleic acid extraction occurs within the FilmArray pouch using mechanical lysis followed by purification using standard magnetic bead technology. After extracting and purifying nucleic acids from the unprocessed sample, the FilmArray performs a nested multiplex PCR that is executed in two stages. During the first stage, the FilmArray performs a single, large volume, highly multiplexed reverse transcription PCR (rt-PCR) reaction. The products from first stage PCR are then diluted and combined with a fresh, primer-free master mix and a fluorescent double-stranded DNA binding dye (LC Green® Plus, BioFire Diagnostics). The solution is then distributed to each well of the array. Array wells contain sets of primers designed specifically to amplify sequences internal to the PCR products generated during the first stage PCR reaction. The 2nd stage PCR, is is performed in singleplex fashion in each well of the conclusion of the 2nd stage PCR, the array is interrogated by melt curve analysis for the detection of signature amplicons denoting the presence of specific targets. A digital camera placed in front of the 2nd stage PCR captures fluorescent images of the PCR reactions and software interprets the data.

The FilmArray Software automatically interprets the results of each DNA melt curve analysis and combines the data with the results of the internal pouch controls to provide a test result for each organism on the panel.

Here is a summary of the acceptance criteria and the study that proves the device meets them, based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are generally implied to be high sensitivity and specificity for each analyte. Rather than explicit criteria, the document presents the achieved performance.

2. Sample Sizes Used for the Test Set and Data Provenance

The "test set" for clinical performance was comprised of:

• Prospective Multi-center Study: 1093 residual PBC specimens, with 1074 evaluable for final analysis.
  • Data Provenance: Nine geographically distinct study sites (seven in the EU) participated. Data collected from October 2018 to May 2019. Specimens were a mix of fresh (1005) and frozen (69) for later testing.
• Archived Specimens: 427 frozen archived PBC specimens, with 395 evaluable. 370 contained confirmed analytes of interest.
  • Data Provenance: Collected from 12 external laboratories (countries not specified, but likely a mix given the mention of EU sites for prospective study). Retrospective.
• Seeded Blood Culture Specimens: 552 seeded specimens.
  • Data Provenance: Laboratory-generated.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Their Qualifications

The document does not explicitly state the "number of experts" or their specific qualifications for establishing ground truth. However, it indicates the following methods were used for reference:

• Bacteria and Cryptococcus: Standard manual and automated microbiological/biochemical identification methods (performed by SOC labs and abstracted from medical charts).
• Candida species: SOC identification for genus level, followed by PCR & sequencing of isolates for species identification.
• AMR Genes:
  • Method 1: One PCR assay performed direct from PBC followed by sequencing of PCR amplicon.
  • Method 2: Commercially available FDA-cleared and CE-marked molecular IVD assays performed on PBC.
  • Method 3: PCR & sequencing for specific resistance gene from applicable cultured isolates.
  • Method 4: Phenotypic antimicrobial susceptibility testing (AST) methods on cultured isolates.

It can be inferred that trained laboratory personnel and potentially infectious disease or microbiology experts interpreted these results, consistent with standard clinical laboratory practices.

4. Adjudication Method for the Test Set

The document details investigations for discrepant results. For example:

• For Enterococcus faecalis FN specimens, additional molecular methods were used for re-testing.
• For Staphylococcus epidermidis FN specimens, additional molecular testing and sequencing were performed.
• For mecA/C and MREJ (MRSA) discrepancies, isolates were tested by PCR/sequencing and had phenotypic AST. Original Cepheid Xpert® MRSA/SA BC tests were also re-tested.

This suggests an internal adjudication process involving further molecular testing and phenotypic analysis to confirm the true status of discrepant samples, rather than a formal blinded 2+1 or 3+1 expert adjudication. Discrepant results were not simply resolved by majority vote but by further in-depth laboratory investigation.

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

No multi-reader multi-case (MRMC) comparative effectiveness study comparing human readers with AI assistance versus without AI assistance was mentioned or conducted. The BioFire BCID2 Panel is a standalone diagnostic device that provides qualitative detection and identification of microbial nucleic acids and resistance genes, not an assistive AI tool for human interpretation.

6. Standalone Performance

Yes, extensive standalone performance studies were conducted. The clinical performance tables (Tables 12-24) and the analytical performance sections (Limit of Detection, Analytical Reactivity (Inclusivity), Analytical Specificity (Cross-Reactivity and Exclusivity), Reproducibility, Interference) directly demonstrate the algorithm's performance without human intervention in result interpretation. The system automatically interprets results and generates reports.

7. Type of Ground Truth Used

The ground truth for the clinical studies was established using a combination of:

• Culture-based microbiology: Standard manual and automated microbiological/biochemical identification methods, followed by species identification where necessary (e.g., Candida species by PCR & sequencing).
• Molecular methods: PCR and sequencing of isolates or direct from positive blood cultures, and commercially available FDA-cleared/CE-marked molecular IVD assays for AMR genes.
• Phenotypic Antimicrobial Susceptibility Testing (AST): Used for AMR gene concordance, particularly for cases like mecA/C and MREJ (MRSA).

For seeded specimens, the "known analyte composition" served as ground truth.

8. Sample Size for the Training Set

The document does not explicitly state the sample size for a "training set" in the context of machine learning. The BioFire BCID2 Panel is a nucleic acid amplification test (PCR-based), where the "training" analogous to model development for AI would involve the design and optimization of primers and probes, and the setting of fluorescence thresholds for detection. This process is primarily based on analytical studies and known genetic sequences, not a labeled dataset like in typical AI development.

However, a large number of isolates were used in the Analytical Reactivity (Inclusivity) study (over 450 isolates) and Analytical Specificity (Cross-Reactivity and Exclusivity) study (many on-panel and off-panel organisms tested, some at high concentrations). These studies would contribute to the robust development and validation of the oligonucleotide sequences and parameters used in the panel tests.

9. How the Ground Truth for the Training Set Was Established

As explained above, for a nucleic acid amplification test like the BioFire BCID2 Panel, the "training set" doesn't strictly align with AI/ML terminology. The development of the assays (e.g., primer and probe design) relied on:

• In silico analysis of sequences: Public databases of genetic sequences were used to design assays that are inclusive of target strains and exclusive of non-target organisms.
• Testing of characterized isolates: Over 450 isolates (representing genetic, geographic, and temporal diversity) were tested at varying concentrations to ensure reliable detection. The "ground truth" for these isolates would have been their known identity (species/subspecies) and genetic markers (e.g., AMR genes) determined by established microbial identification techniques and sequencing (e.g., ATCC strains, clinical isolates with known characteristics).

This rigorous analytical evaluation forms the basis of the device's "knowledge" or "training" on targets and non-targets.

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The iCubate, Inc. iC-GN Assay™ for use on the iC-System™ is a qualitative, multiplexed, in vitro diagnostic test for the detection and identification of potentially pathogenic gram negative bacteria, which may cause bloodstream infection (BSI). The iC-GN Assay™ is performed directly on positive blood cultures, confirmed by Gram stain to contain gram negative bacilli. Cultures demonstrating mixed Gram stain results should not be tested on the assay. The iC-GN Assay™ is validated for use with select BACTEC™, BacT/ALERT® and VersaTREK® blood culture bottles. The iC-GN Assay™ is indicated for use in conjunction with other clinical and laboratory findings, such as culture, to aid in the diagnosis of bacterial bloodstream infections; however, it is not used to monitor bloodstream infections.

The iC-GN Assay™ detects target DNA and identifies the following:

Bacterial Genera and Species: Acinetobacter baumannii complex, Enterobacter cloacae complex, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus species, Serratia marcescens

Resistance Markers: KPC (blaKPC)- associated with resistance to carbapenems, NDM (blaNDM)- associated with resistance to carbapenems, CTX-M group 1(blaCTX-M group 1)- associated with resistance to extended spectrum beta-lactams

In mixed growth, the iC-GN Assay™ does not specifically attribute detection of KPC, NDM, or CTX-M group 1 to a specific genera or species.

Sub-culturing of positive blood cultures is necessary to recover organisms for susceptibility testing, identification of organisms not detected by the iC-GN Assay™, differentiation of mixed growth, association of antimicrobial resistance marker genes to a specific organism, or for epidemiological typing.

The iC-GN Assay™ utilizes polymerase chain reaction (PCR) for the multiplex amplification of specific targets and detects the amplified targets with microarray hybridization. Targets are detected directly from patient positive blood cultures confirmed by Gram stain to contain gram negative bacilli. The iC-GN Assay utilizes proprietary ARM-PCR (Amplicon Rescued Multiplex PCR) technology allowing for multiple targets to be amplified in one reaction. Testing is done in a self-contained, automated, disposable cassette using the iCubate™ processor (iC-Processor™). After the reaction is complete, the cassette is read on the iCubate® reader (iC-Reader™). Results from the iC-Reader™ are interpreted by iC-Report™ software and a final report is displayed on the iMac® computer.

To operate, the user opens the iC-Cassette™ cap and pipettes an aliquot of the diluted positive blood culture sample into the sample/PCR well in the bottom well plate of the cassette. Once inoculated, the cassette cap is closed, and all extraction, amplification and detection processes are completed in the cassette, a closed system. Extraction, amplification and detection sequences are defined by an assay script controlled by the iC-Processor™.

The processing script is defined within a barcode label positioned on the top of each iC-Cassette™ which communicates with the iC-Processor™. To access and pierce the foilsealed reagent wells located in the bottom well plate of the cassette, the processor manipulates the cassette to move the cassette pipette horizontally and vertically. The script directs the transfer of reagents between the wells in the bottom well plate and finally to the array within the cassette. The iC-Processor™ is capable of processing four (4) iC-Cassettes™ with random access.

Once processing is complete, the cassette is manually transferred from the iC-Processor™ to the iC-Reader™ where the microarray within the cassette is read. The iC-Reader™ is capable of reading up to four (4) iC-Cassettes™ at one time. The results are interpreted via the iC-Report™ software and displayed for the user on the iMac®. Raw data and result interpretations are stored within the iMac®; raw data is accessible to iCubate® service personnel only and not to the end user.

When finished with a loaded iC-GN Cassette™, it should be disposed as biohazardous waste.

Here's a breakdown of the acceptance criteria and the study that proves the device meets them, based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are generally implied by the "Percent Agreement" values (positive and negative) to reference methods. While specific numeric acceptance thresholds are not explicitly stated as "acceptance criteria," performance metrics of 95% or higher with tight confidence intervals are typically considered strong evidence of meeting performance expectations for such devices. For reproducibility and equivalency, "≥ 95% performance" is explicitly stated as the acceptance criteria.

2. Sample size used for the test set and the data provenance

• Method Comparison Study (Clinical Study):

  • Total specimens enrolled: 1107
  • Specimens included in performance analysis: 1002
    • Fresh prospective specimens: 976
    • Frozen prospective specimens (retrospectively tested): 26 (2.6%)
  • Contrived samples: 170
  • Data Provenance: Five geographically dispersed clinical sites in the U.S. (NY, WI, NM, FL, IN). The specimens were "leftover de-identified specimens from anaerobic blood culture bottles flagged as positive." This indicates a retrospective collection of clinical samples taken from patients, which were then de-identified and used for the study.

• Reproducibility Study:

  • Eighteen-organism panel tested in triplicate across five, non-consecutive days by two independent operators at each of three sites. This implies approximately 18 organisms * 3 replicates * 5 days * 2 operators * 3 sites = 1620 tests, but the table breaks down performance by target with denominators of ~90, suggesting a more specific number per target/concentration.

• Limit of Detection (LoD) Study:

  • Twenty-seven representative strains, a minimum of three per target. Tested in a minimum of twenty replicates (for confirmation phase) on each of three unique cassette lots.

• Inclusivity Study:

  • Eighty-two (82) representative strains, a minimum of ten strains for each target analyte. Each strain tested in triplicate.

• Exclusivity Study:

  • A total of 114 strains. Each strain tested in triplicate (retested in replicates of 3 or 10 for discordant results).

• Microbial Interference Study:

  • Sixty (60) gram negative exclusivity strains in combination with eight (8) iC-GN target organisms. Each combination tested in triplicate (retested in replicates of 3 or 10 for discordant results).

• Competitive Inhibition Study:

  • One target organism (low concentration) combined with a second target organism (high concentration). Each combination tested in triplicate (retested in replicates of 10 for false negatives).

• Interfering Substances Study:

  • Eight representative target organisms plus one non-target organism. Organism/interferent combination tested in triplicate (retested in replicates of 10 or 3 for discordant results).

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts

The document does not specify the number or qualifications of experts used to establish the ground truth. It states that the ground truth for the clinical method comparison was based on:

• "reference culture followed by MALDI identification" for organism targets.
• "PCR amplification followed by confirmatory bidirectional sequencing" for resistance markers.
• "Phenotypic antimicrobial susceptibility testing (AST)" was also performed to identify samples requiring sequencing.

While these are standard laboratory methods, the involvement of specific "experts" (e.g., microbiologists, infectious disease specialists) beyond performing these standard laboratory procedures is not detailed.

4. Adjudication method for the test set

The document does not describe a formal adjudication method (like 2+1 or 3+1). For the method comparison study, discordant samples were "sequenced" (for resistance markers) or further analyzed by PCR/bi-directional sequencing for organism targets. For other performance studies (inclusivity, exclusivity, microbial interference), discordant results led to retesting in replicates (3 or 10). This indicates a re-testing approach for discrepancies rather than a multi-expert consensus.

5. If a multi reader multi case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance

No, a multi-reader multi-case (MRMC) comparative effectiveness study was not done. The iC-GN Assay is an in vitro diagnostic (IVD) device, specifically a molecular diagnostic test for identifying microorganisms and resistance markers from positive blood cultures. It does not involve "human readers" interpreting images or data in a way that would typically be evaluated in an MRMC study for AI assistance. Its performance is compared against laboratory reference methods.

6. If a standalone (i.e. algorithm only without human-in-the loop performance) was done

Yes, the studies presented evaluate the standalone performance of the iC-GN Assay. The assay is an automated system (iC-System) that detects and identifies targets directly from positive blood cultures using PCR and microarray hybridization. The results are interpreted by software (iC-Report™) and displayed for the user. The performance data (reproducibility, LoD, inclusivity, exclusivity, microbial interference, competitive inhibition, and method comparison) all reflect the device's inherent analytical and clinical performance without a human interpreting the primary result, but rather the system's output. The human role is operational (loading samples, reading the final report), not interpretive of the raw data.

7. The type of ground truth used (expert consensus, pathology, outcomes data, etc.)

The ground truth used for the clinical method comparison study was established using:

• Reference culture followed by MALDI identification: This is a combination of microbiological culture methods and advanced biochemical analysis to identify bacterial species.
• PCR amplification followed by confirmatory bidirectional sequencing: This is a molecular method used to confirm the presence and identity of specific genes (e.g., resistance markers) or bacterial species through DNA sequencing.
• Phenotypic antimicrobial susceptibility testing (AST): Used to guide which resistance markers might need further sequencing for confirmation.

For other analytical studies (Inclusivity, Exclusivity, LoD, etc.), the ground truth was based on the known identity and concentration of the characterized bacterial strains used.

8. The sample size for the training set

The document does not provide details about a "training set" in the context of an algorithm or AI development. This device is a molecular diagnostic test, not an AI/ML-based diagnostic that would typically involve a separate training, validation, and test set for algorithm development. The studies described are performance validation studies for the finished medical device.

9. How the ground truth for the training set was established

As there's no explicit mention of a "training set" for an algorithm, the method for establishing its ground truth is not described. The ground truth for the verification and validation studies (as detailed in point 7) was established through standard microbiological and molecular laboratory techniques, which served as the comparator for assessing device performance.

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The GenMark ePlex® Blood Culture Identification Gram-Negative (BCID-GN) Panel is a qualitative nucleic acid multiplex in vitro diagnostic test intended for use on GenMark's ePlex Instrument for simultaneous qualitative detection and identification of multiple potentially pathogenic gram-negative bacterial organisms and select determinants associated with antimicrobial resistance in positive blood culture. In addition, the ePlex BCID-GN Panel is capable of detecting several gram-positive bacteria (Pan Gram-Positive assay) and several Candida species (Pan Candida assay). The ePlex BCID-GN Panel is performed directly on blood culture samples identified as positive by a continuous monitoring blood culture system and which contain gram-negative organism.

The following bacterial organisms and genes associated with antibiotic resistance are identified using the ePlex BCID-GN Panel: Acinetobacter baumannii, Bacteroides fragilis, Citrobacter, Cronobacter sakazakii. Enterobacter cloacae complex, Enterobacter (non-cloacae complex), Escherichia coli, Fusobacterium necrophorum, Fusobacterium nucleatum, Haemophilus influenzae, Klebsiella oxytoca, Klebsiella pneumoniae group, Morganella morganii, Neisseria meningitidis, Proteus, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella, Serratia, Serratia marcescens, Stenotrophomas maltophilia, СТХ-М (blactх-м), IMP (blамм) , КРС (blakec) , NDM (bland), OXA (blaoxa) (OXA-23 and OXA-48 groups only), and VIM (blaviм).

The ePlex BCID-GN Panel contains assays for the detection of genetic determinants associated with resistance to antimicrobial agents including CTX-M(blactx.M), which is associated with resistance to extended spectrum beta-lactamase (ESBL)-mediated resistance to penicillins, cephalosporins and monobactams, as well as OXA (blaoxA) (OXA-23 and OXA-48 groups only), KPC (blakpc), and metallo-beta-lactamases IMP (blavM), VIM (blavM), and NDM (blaNDM), which is associated with carbapenemase-mediated resistance. The antimicrobial resistance gene detected may or may not be associated with the agent responsible for disease. Negative results for these select antimicrobial resistance assays do not indicate susceptibility, as there are multiple mechanisms of resistance in gram-negative bacteria.

The ePlex BCID-GN Panel also contains targets designed to detect a broad range of organisms with a potentially misleading Gram stain result or organisms that may be missed by Gram staining altogether, for example in the case of co-infections. These include a broad Pan Gram-Positive assay (which is designed to detect Bacillus cereus group, Bacillus subtilis group, Enterococcus, Staphylococcus, and Streptococcus), as well as a Pan Candida assay, which is designed to detect four Candida species: Candida albicans, Candida glabrata, Candida krusei, and Candida parapsilosis.

The detection and identification of specific bacterial and fungal nucleic acids from individuals exhibiting signs and/or symptoms of bloodstream infection aids in the diagnosis of bloodstream infection when used in conjunction with other clinical information. The results from the ePlex BCID-GN Panel are intended to be interpreted in conjunction with Gram stain results and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.

Negative results in the setting of a suspected bloodstream infection may be due to infection with pathogens that are not detected by this test. Positive results do not rule out co-infection with other organisms; the organism(s) detected by the ePlex BCID-GN Panel may not be the definite cause of disease. Additional laboratory testing (e.g. sub-culturing of positive blood cultures for identification of organisms not detected by ePlex BCID-GN Panel and for susceptibility testing, differentiation of mixed growth, and association of antimicrobial resistance marker genes to a specific organism) and clinical presentation must be taken into consideration in the final diagnosis of bloodstream infection.

The ePlex Blood Culture Identification Gram-Negative (BCID-GN) Panel is based on the principles of competitive nucleic acid hybridization using a sandwich assay format, wherein a single-stranded target binds concurrently to a sequence-specific solution-phase signal probe and a solid-phase electrode-bound capture probe. The test employs nucleic acid extraction, target amplification via polymerase chain reaction (PCR) or reverse transcription PCR (RT-PCR) and hybridization of target DNA. In the process, the double-stranded PCR amplicons are digested with exonuclease to generate single-stranded DNA suitable for hybridization.

Nucleic acid extraction from biological samples occurs within the cartridge via cell lysis, nucleic acid capture onto magnetic beads, and release for amplification. The nucleic acid extraction is processed through microfluidic liquid handling. Once the nucleic acid targets are captured and inhibitors are washed away, the magnetic particles are delivered to the electrowetting environment on the printed circuit board (PCB) and the targets are eluted from the particles and amplified.

During hybridization, the single-stranded target DNA binds to a complementary, single-stranded capture probe immobilized on the working gold electrode surface. Single-stranded signal probes (labeled with electrochemically active ferrocenes) bind to specific target sequence / region adjacent to the capture probe. Simultaneous hybridization of target to signal probes and capture probe is detected by alternating current voltammetry (ACV). Each working electrode on the array contains specific capture probes, and sequential analysis of each electrode allows detection of multiple analyte targets.

Here's a summary of the acceptance criteria and study information for the ePlex Blood Culture Identification Gram Negative (BCID-GN) Panel, extracted from the provided text:

Acceptance Criteria and Device Performance for ePlex Blood Culture Identification Gram Negative (BCID-GN) Panel

The ePlex BCID-GN Panel is a qualitative nucleic acid multiplex in vitro diagnostic test for the simultaneous qualitative detection and identification of multiple potentially pathogenic gram-negative bacterial organisms and select determinants associated with antimicrobial resistance in positive blood cultures. Performance characteristics were evaluated through clinical and analytical studies.

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are generally implied by the achieved performance metrics, which show high Positive Percent Agreement (PPA) for sensitivity and Negative Percent Agreement (NPA) for specificity. While explicit numerical acceptance thresholds are not directly stated as "acceptance criteria" in a dedicated section, the provided tables demonstrate the device's performance against its intended use. For brevity, the "Overall" performance (combining prospective, retrospective, and contrived samples where applicable) is used here to represent the device's reported performance against implied high accuracy criteria.

2. Sample Sizes Used for the Test Set and Data Provenance

The test set comprised a combination of clinical and contrived samples:

• Prospective Clinical Samples: 349 evaluable samples (167 fresh, 182 frozen). Patients were of all ages and genders. Collected at 7 clinical sites.
• Retrospective Clinical Samples: 577 evaluable samples. Collected at 10 clinical sites.
• Contrived Samples: 777 samples.

Data Provenance: The clinical samples (prospective and retrospective) were collected at multiple clinical sites, suggesting data from different countries/regions, but specific countries of origin are not detailed. Both prospective and retrospective collection methods were used.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Those Experts

The document does not explicitly state the number of experts or their specific qualifications (e.g., radiologist with 10 years of experience) used to establish the ground truth. It refers to "standard laboratory procedures for identification of blood culture isolates, including traditional and automated identification methods, MALDI-TOF IVD, and microbiological and biochemical techniques" as the comparator method. For Acinetobacter baumannii and Candida parapsilosis, as well as antibiotic resistance genes, PCR assays followed by bi-directional sequencing were used for confirmation. This implies a reliance on established laboratory protocols and potentially expert interpretation within those processes, but specific expert count or detailed qualifications are not provided.

4. Adjudication Method for the Test Set

The document does not describe an explicit adjudication method (e.g., 2+1, 3+1). The "Comparator Method" served as the reference standard and was used to determine true positive (TP), false negative (FN), true negative (TN), and false positive (FP) results. Discrepancies were investigated using PCR/sequencing (as noted in footnotes for various tables), suggesting a re-evaluation process for conflicting results but not a formal multi-reader adjudication scheme for the initial ground truth establishment.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done

No MRMC comparative effectiveness study involving human readers and AI assistance was performed or described in the provided text. The study focuses on the standalone performance of the ePlex BCID-GN Panel against laboratory reference methods.

6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done

Yes, the study described is a standalone performance study of the ePlex BCID-GN Panel. The performance metrics (PPA and NPA) are calculated by comparing the device's output directly against the "Comparator Method" without human interpretation influencing the device's result.

7. The Type of Ground Truth Used

The ground truth was established using expert consensus methods based on standard laboratory procedures for organism identification and analytically validated molecular assays (qPCR followed by bi-directional sequencing) for confirming specific organisms and resistance genes. This includes:

• Traditional and automated identification methods
• MALDI-TOF IVD
• Microbiological and biochemical techniques
• PCR/sequencing for specific organisms and resistance markers.

8. The Sample Size for the Training Set

The document does not mention a distinct "training set" in the context of the clinical performance evaluation. The clinical studies (prospective, retrospective, and contrived samples) are described as evaluation/test sets. However, the "Analytical Reactivity (Inclusivity)" and "Limit of Detection (LoD)" studies (Tables 56 and 57) describe testing of various strains and isolates to establish the device's analytical performance and broad reactivity. These analytical studies are crucial for the development and "training" (in a broader sense of assay design and validation) of such molecular diagnostic panels.

Specifically:

• Limit of Detection (LoD): At least 20 replicates per target were tested for each condition using quantified reference strains (Table 56 lists 38 organisms/targets with specific strains).
• Analytical Reactivity (Inclusivity): A panel of 336 strains/isolates was evaluated (Table 57 provides a partial list of these validated strains).
• Predicted (in silico) Reactivity: Bioinformatic analysis covered many additional variants for genus/group assays and resistance markers (Tables 58-76).

9. How the Ground Truth for the Training Set Was Established

For the analytical "training" aspects:

• LoD: The ground truth for LoD was established by using quantified reference strains with known concentrations in simulated blood culture sample matrix. The lowest concentration detected in ≥95% of tested replicates was defined as the LoD.
• Analytical Reactivity (Inclusivity): The ground truth was established by testing well-characterized strains/isolates with known identities at specified concentrations, confirming their detection by the panel.
• Predicted (in silico) Reactivity: This involved bioinformatic analysis of existing sequence data for various organisms and resistance gene variants to predict the panel's ability to detect them. This is a computational method for establishing theoretical ground truth based on genetic sequences.

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The FilmArray Blood Culture Identification (BCID) Panel is a qualitative multiplexed nucleic acid-based in vitro diagnostic test intended for use with FilmArray systems. The FilmArray BCID Panel is capable of simultaneous detection and identification of multiple bacterial and yeast nucleic acids and select genetic determinants of antimicrobial resistance. The FilmArray BCID Panel assay is performed directly on blood culture samples identified as positive by a continuous monitoring blood culture system. Results are intended to be interpreted in conjunction with Gram stain results.

The following gram-positive bacteria, gram-negative bacteria, and yeast are identified using the FilmArray BCID Panel: Enterococci, Listeria monocytogenes, Staphylococci (including specific differentiation of Staphylococcus aureus), Streptococci (with specific differentiation of Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyogenes), Acinetobacter baumannii, Enterobacteriaceae (including specific differentiation of the Enterobacter cloacae complex, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus, and Serratia marcescens), Haemophilus influenzae, Neisseria meningitidis (encapsulated), Pseudomonas aeruginosa, Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, and Candida tropicalis.

The FilmArray BCID Panel also contains assays for the detection of genetic determinants of resistance to methicillin (mecA), vancomycin (vanA and vanB), and carbapenems (blaxe) to aid in the identification of potentially antimicrobial resistant organisms in positive blood culture samples. The antimicrobial resistance gene detected may or may not be associated with the agent responsible for disease. Negative results for these select antimicrobial resistance gene assays do not indicate susceptibility, as multiple mechanisms of resistance to methicillin, vancomycin, and carbapenems exist.

FilmArray BCID Panel is indicated as an aid in the diagnosis of specific agents of bacteremia and fungemia and results should be used in conjunction with other clinical and laboratory findings. Positive FilmArray results do not rule out co-infection with organisms not included in the FilmArray BCID Panel. FilmArray BCID Panel is not intended to monitor treatment for bacteremia or fungemia.

Subculturing of positive blood cultures is necessary to recover organisms for susceptibility testing and epidemiological typing, to identify organisms in the blood culture that are not detected by the FilmArray BCID Panel, and for species determination of some Staphylococci, Enterococci, Streptococci, and Enterobacteriaceae that are not specifically identified by the FilmArray BCID Panel assays.

The FilmArray Blood Culture Identification (BCID) Panel is a multiplex nucleic acid test designed to be used with a FilmArray system. The FilmArray BCID pouch contains freeze-dried reagents to perform nucleic acid purification and nested, multiplex PCR with DNA melt analysis. The FilmArray Blood Culture Identification (BCID) Panel simultaneously tests a single positive blood culture sample to provide results for 24 different organisms and organism groups that cause bloodstream infections and three genetic markers that are known to confer antimicrobial resistance (see Table 1).

A test is initiated by loading Hydration Solution and a positive blood culture sample mixed with the provided Sample Buffer into the FilmArray BCID pouch. The pouch contains all of the reagents required for specimen testing and analysis in a freeze-dried format; the addition of Hydration Solution and sample Buffer Mix rehydrates the reagents. After the pouch is prepared, the software guides the user though the steps of placing the pouch into the instrument. scanning the pouch barcode, entering the sample identification, and initiating the run.

The FilmArray instrument contains a coordinated system of inflatable bladders and seal points, which act on the pouch to control the movement of liquid between the pouch blisters. When a bladder is inflated over a reagent blister, it forces liquid from the blister into connecting channels. Alternatively, when a seal is placed over a connecting channel it acts as a valve to open or close a channel. In addition, electronically controlled pneumatic pistons are positioned over multiple plungers in order to deliver the rehydrated reagents into the blisters at the appropriate times. Two Peltier devices control heating and cooling of the pouch to drive the PCR reactions and the melt curve analysis.

Nucleic acid extraction occurs within the FilmArray pouch using mechanical lysis and standard magnetic bead technology. After extracting and purifying nucleic acids from the unprocessed sample, a nested multiplex PCR is executed in two stages. During the first stage, a single, large volume, highly multiplexed PCR reaction which includes all primers of the outer primer sets, is performed. The products from first stage PCR are then diluted and combined with a fresh, primer-free master mix and a fluorescent double stranded DNA binding dye (LC Green® Plus, BioFire Defense, LLC). The solution is then distributed to each well of the array. Array wells contain sets of primers designed specifically to amplify sequences internal to the PCR products generated during the first stage PCR reaction. The 2nd stage PCR, or nested PCR, is performed in singleplex fashion in each well of the array. At the conclusion of the 2nd stage PCR, the array is interrogated by melt curve analysis for the detection of signature amplicons denoting the presence of specific targets. A digital camera placed in front of the array captures fluorescent images of the PCR reactions and software interprets the data.

The FilmArray software automatically interprets the results of each DNA melt curve analysis and combines the data with the results of the internal pouch controls to provide a test result for each organism on the panel.

The provided document is a 510(k) summary for a modification to an existing medical device, the FilmArray Blood Culture Identification (BCID) Panel. This submission primarily focuses on changes to the intended use and labeling, rather than a full re-evaluation of the device's original performance. As such, the document does not contain a typical study outlining comprehensive acceptance criteria and performance against those criteria in the same way an initial submission might.

However, based on the information provided, we can infer some "acceptance criteria" related to the changes being made and how the device meets them.

Here's an attempt to structure the information as requested, with caveats due to the nature of the document:

1. Table of Acceptance Criteria and Reported Device Performance

Since this is a modification submission, the "acceptance criteria" discussed are largely related to ensuring the changes (removal of false positive warnings, additional interference testing, and updated cross-reactivity information) do not negatively impact the device's overall performance as established by the predicate device.

2. Sample Size Used for the Test Set and Data Provenance

Due to the nature of this submission being a modification of use and labeling, the "test set" described is specifically for the interference testing. The document does not detail the full original clinical validation test set.

• Interference Testing (bioMerieux BacT/ALERT FA Plus and BacT/ALERT FN Plus bottles):

  • Sample Size: 7 contrived blood culture samples (6 positive and 1 negative) per media type. As three media types were tested (BD BACTEC Plus Aerobic/F, BacT/ALERT FA Plus, BacT/ALERT FN Plus), this implies a total of 21 runs.
  • Data Provenance: Not explicitly stated, but these were contrived samples prepared in a laboratory setting, likely at BioFire Diagnostics. Therefore, it's a prospective analytical study. Country of origin is implied to be within the US, where BioFire Diagnostics is located.

• Cross-Reactivity Updates:

  • The document notes that some cross-reactivities were observed "infrequently in pre-analytical studies and the clinical evaluation (estimated occurrence of ~0.25% of all Staphylococcus positive patient samples)," and "in contrived samples and/or clinical blood culture specimens."
  • For Klebsiella quasipneumoniae, it's stated as a "new species (with subspecies) that is closely related to K. pneumoniae," implying some form of ongoing surveillance or re-evaluation. Similarly, Raoultella ornithinolytica cross-reactivity was "observed in clinical positive blood cultures."
  • This suggests some data comes from retrospective analysis of previously collected clinical data or ongoing surveillance, as well as new analytical testing (contrived samples). Specific sample sizes for these cross-reactivity discoveries are not provided in this summary.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and the Qualifications of Those Experts

• Interference Testing: Not applicable in the same way as a clinical study. Ground truth for the contrived samples would have been precisely known based on their preparation. For example, a "positive" sample would have been spiked with a known organism at a known concentration.
• Cross-Reactivity: The document does not specify experts involved in establishing ground truth for the discovery of cross-reactivities. However, the identification of microorganisms and their genetic markers typically relies on established microbiology laboratory methods, often overseen by experienced microbiologists. Given the "in silico analysis" mentioned, bioinformatics experts would also be involved.

4. Adjudication Method for the Test Set

• Interference Testing: No formal adjudication method like "2+1" or "3+1" is mentioned or expected for this type of analytical validation using contrived samples. The results (valid runs, no errors, lack of interference) are directly observable from the instrument's output and comparison against expected results based on sample preparation.
• Cross-Reactivity: The document doesn't detail an adjudication process for identifying cross-reactivities, but it references "pre-analytical studies," "clinical evaluation," and "in silico analysis." This implies internal validation and expert review of results from various sources.

5. If a Multi Reader Multi Case (MRMC) Comparative Effectiveness Study Was Done, If So, What Was the Effect Size of How Much Human Readers Improve with AI vs Without AI Assistance

• No, an MRMC comparative effectiveness study was not done. The FilmArray BCID Panel is an in vitro diagnostic (IVD) device that directly detects nucleic acids from blood culture samples and provides an automated interpretation. It is not an imaging AI device that "assists human readers" in the traditional sense. The device generates a definitive result (presence/absence of specific organisms/resistance genes), and while healthcare professionals interpret these results in conjunction with other clinical findings (like Gram stain), there isn't a human-in-the-loop "reading" task that the device is assisting.

6. If a Standalone (i.e. algorithm only without human-in-the loop performance) Was Done

• Yes, the primary performance of the FilmArray BCID Panel is standalone algorithm performance. The device performs nucleic acid extraction, PCR, and melt curve analysis, and its software automatically interprets the results. The "test interpretation" is described as "Automated test interpretation and report generation. User cannot access raw data." This confirms it's an algorithm-only, standalone performance. The document does not present separate standalone performance data for this specific submission as it is about modifications to an existing, already cleared device. The "performance" being demonstrated here is primarily the robustness of the existing system to new conditions (bottle types) and the transparent reporting of newly identified cross-reactivities. The performance of the predicate device (K160457) would have established its standalone performance.

7. The Type of Ground Truth Used

• Interference Testing: The ground truth for the contrived samples was known composition (i.e., specific organisms spiked at known concentrations, or negative controls).
• Cross-Reactivity: The ground truth for identified cross-reactivities likely came from a combination of:
  • Analytical testing (e.g., testing known isolates/species in contrived samples).
  • Clinical culture results (identifying organisms in patient samples that cross-reacted with the panel).
  • In silico analysis (bioinformatics prediction based on sequence homology).

8. The Sample Size for the Training Set

• Not applicable for this modification submission. This document describes modifications to an existing device (K160457). The "training set" (if applicable from a machine learning perspective, which is unlikely for a PCR-based IVD) would have been part of the original development and validation of the predicate device, not detailed in this modification summary. The FilmArray panel design (primers, probes) itself is developed through a sophisticated "training" process involving bioinformatic analysis of target sequences, but the document does not provide details on specific sample sizes for that primer/probe design process.

9. How the Ground Truth for the Training Set Was Established

• Also not applicable for this modification submission as a "training set" for an algorithm in the machine learning sense isn't detailed, nor is its ground truth establishment. For the original development of the PCR panel, the ground truth for probe design and target identification would have been established through extensive genomic sequencing data, validated microbial culture collections, and molecular characterization methods.

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The FilmArray Blood Culture Identification (BCID) Panel is a qualitative multiplexed nucleic acid-based in vitro diagnostic test intended for use with FilmArray BCID Panel is capable of simultaneous detection and identification of multiple bacterial and yeast nucleic acids and select genetic determinants of antimicrobial resistance. The BCID assay is performed directly on blood culture samples identified as positive by a continuous monitoring blood culture system that demonstrate the presence of organisms as determined by Gram stain.

The following gram-positive bacteria, gram-negative bacteria, and yeast are identified using the FilmArray BCD Panel: Enterococci, Listeria monocytogenes, Staphylococci (including specific differentiation of Staphylococcus aureus), Streptococci (with specific differentiation of Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyogenes), Acinetobacter baumannii, Enterobacteriaceae (including specific differentiation of the Enterchacter cloacae complex, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus, and Serratia marcescens), Haemophilus influenzae, Neisseria meningitidis (encapsulated), Pseudomonas aeruginosa, Candida glabrata, Candida krusei, Candida parapsilosis, and Candida tropicalis.

The FilmArray BCID Panel also contains assays for the determinants of resistance to methicillin (mecA), vancomycin (vanA and vanB), and carbapenems (blaKPC) to aid in the identification of potentially antimicrobial resistant organisms in positive blood culture samples. The antimicrobial resistance gene detected may or may not be associated with the agent responsible for disease. Negative results for these select antimicrobial resistance gene assays do not indicate susceptibility, as multiple mechanisms of resistance to methicillin, vancomycin, and carbapenens exist. FilmArray BCID is indicated as an aid in the diagnosis of bacteremia and fungemia and results should be used in conjunction with other clinical and laboratory findings. Positive FilmArray results do not rule out co-infection with organisms not included in the FilmArray BCID Panel. FilmArray BCID is not intended to monitor treatment for bacteremia or fungemia.

Subculturing of positive blood cultures is necessary to recover organisms for susceptibility testing and epidemiological typing, to identify organisms in the blood culture that are not detected by the FilmArray BCID Panel, and for species determination of some Staphylococci, Streptococci, Streptococci, and Enterobacteriaceae that are not specifically identified by the FilmArray BCID Panel assays.

The FilmArray Blood Culture Identification (BCID) Panel is a multiplex nucleic acid test designed to be used with a FilmArray system. The FilmArray BCID pouch contains freeze-dried reagents to perform nucleic acid purification and nested, multiplex PCR with DNA melt analysis. The FilmArray Blood Culture Identification (BCID) Panel simultaneously tests a single positive blood culture sample to provide results for 24 different organisms and organism groups that cause bloodstream infections and three genetic markers that are known to confer antimicrobial resistance (see Table 1).

A test is initiated by loading Hydration Solution and a positive blood culture sample mixed with the provided Sample Buffer into the FilmArray BCID pouch. The pouch contains all of the reagents required for specimen testing and analysis in a freeze-dried format; the addition of Hydration Solution and sample Buffer Mix rehydrates the reagents. After the pouch is prepared, the software guides the user though the steps of placing the pouch into the instrument. scanning the pouch barcode, entering the sample identification, and initiating the run.

The FilmArray instrument contains a coordinated system of inflatable bladders and seal points, which act on the pouch to control the movement of liquid between the pouch blisters. When a bladder is inflated over a reagent blister, it forces liquid from the blister into connecting channels. Alternatively, when a seal is placed over a connecting channel it acts as a valve to open or close a channel. In addition, electronically controlled pneumatic pistons are positioned over multiple plungers in order to deliver the rehydrated reagents into the blisters at the appropriate times. Two Peltier devices control heating and cooling of the pouch to drive the PCR reactions and the melt curve analysis.

Nucleic acid extraction occurs within the FilmArray pouch using mechanical lysis and standard magnetic bead technology. After extracting and purifying nucleic acids from the unprocessed sample, a nested multiplex PCR is executed in two stages. During the first stage, a single, large volume, highly multiplexed PCR reaction which includes all primers of the outer primer sets, is performed. The products from first stage PCR are then diluted and combined with a fresh, primer-free master mix and a fluorescent double stranded DNA binding dye (LC Green® Plus, BioFire Defense, LLC). The solution is then distributed to each well of the array. Array wells contain sets of primers designed specifically to amplify sequences internal to the PCR products generated during the first stage PCR reaction. The 2nd stage PCR, or nested PCR, is performed in singleplex fashion in each well of the array. At the conclusion of the 2nd stage PCR, the array is interrogated by melt curve analysis for the detection of signature amplicons denoting the presence of specific targets. A digital camera placed in front of the array captures fluorescent images of the PCR reactions and software interprets the data.

The FilmArray software automatically interprets the results of each DNA melt curve analysis and combines the data with the results of the internal pouch controls to provide a test result for each organism on the panel.

Here's a breakdown of the acceptance criteria and study information for the FilmArray Blood Culture Identification (BCID) Panel for use with FilmArray Torch, based on the provided document:

Acceptance Criteria and Device Performance

The document doesn't explicitly state numerical acceptance criteria in a dedicated section with thresholds. However, it describes the performance goal and the reported device performance as follows:

Study Information

2. Sample size used for the test set and the data provenance:

• Sample Size: 30 replicates per analyte per system for 90 total replicates per analyte. (The document doesn't specify how many analytes are on the panel, but Table 1 lists 24 organism targets and 4 resistance markers, implying a total of 28 analytes. So, for each of these 28 analytes, 90 samples were tested).
• Data Provenance: The study used "contrived samples" containing analytes at specific concentrations. This indicates a prospective, laboratory-controlled study, rather than retrospective clinical data. No country of origin is explicitly stated, but the submission is to the FDA (USA).

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts:

• This study evaluates an in vitro diagnostic (IVD) device that directly detects nucleic acids. The ground truth for the contrived samples would be established by the known composition and concentration of the analytes spiked into the samples during their preparation, not by human expert interpretation. Therefore, no external human experts were involved in establishing the ground truth for this specific test set.

4. Adjudication method (e.g. 2+1, 3+1, none) for the test set:

• Not applicable. As this is an IVD device testing contrived samples against known concentrations, there is no need for expert adjudication. The result is either "Detected" or "Not Detected" compared to the known content of the contrived sample.

5. If a multi-reader multi-case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance:

• No, a multi-reader multi-case (MRMC) comparative effectiveness study was not done. This device is an automated IVD and does not involve human readers interpreting results in the same way an AI for image analysis would. Its output is an automated interpretation of molecular detection.

6. If a standalone (i.e. algorithm only without human-in-the loop performance) was done:

• Yes, this was effectively a standalone performance evaluation of the FilmArray Torch system running the BCID Panel. The "algorithm" (the automated processes and software interpretation within the FilmArray system) directly processed the samples and generated results without human interpretation of raw data; the user only sees automated test interpretation and report generation.

7. The type of ground truth used (expert consensus, pathology, outcomes data, etc.):

• For the test set, the ground truth was known composition and/or spiking of analytes (nucleic acids) at specific concentrations in the contrived samples.

8. The sample size for the training set:

• The document describes a submission for a modified device (FilmArray Torch) to be used with an existing reagent panel (FilmArray BCID Panel). It states "There have been no changes to the previously cleared FilmArray BCID Panel reagent kit itself." Therefore, the training of the assay (the BCID Panel's detection algorithms) would have occurred during the development and clearance of the original K143171 predicate device. This document does not provide details on the training set for that original assay, as the focus here is on the new instrument's equivalence. The study described (using 90 replicates per analyte) is a validation/test set, not a training set.

9. How the ground truth for the training set was established:

• As noted above, details about the training set for the original BCID Panel assay are not provided in this document. For molecular diagnostic assays like this, training data would typically involve:
  • Known positive samples: Samples containing confirmed target organisms/genes (e.g., pure cultures, reference materials).
  • Known negative samples: Samples confirmed to lack the target organisms/genes.
  • Specificity panels: Samples containing closely related organisms or common co-infecting agents to ensure accurate differentiation and minimize cross-reactivity.
  • The ground truth would be established through gold standard microbiology methods (e.g., culture, sequencing, validated reference PCRs) for organisms, and often sequencing or known genetic constructs for resistance genes.

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The FilmArray Blood Culture Identification (BCID) Panel is a qualitative multiplexed nucleic acid-based in vitro diagnostic test intended for use with FilmArray systems. The FilmArray BCID Panel is capable of simultaneous detection and identification of multiple bacterial and yeast nucleic acids and select genetic determinants of antimicrobial resistance. The BCID assay is performed directly on blood culture samples identified as positive by a continuous monitoring blood culture system that demonstrate the presence of organisms as determined by Gram stain.

The following gram-positive bacteria, gram-negative bacteria, and yeast are identified using the FilmArray BCID Panel: Enterococci, Listeria monocytogenes, Staphylococci (including specific differentiation of Staphylococcus aureus), Streptococci (with specific differentiation of Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyogenes), Acinetobacter baumannii, Enterobacteriaceae (including specific differentiation of the Enterobacter cloacae complex, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus, and Serratia marcescens), Haemophilus influenzae, Neisseria meningitidis (encapsulated), Pseudomonas aeruginosa, Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, and Candida tropicalis.

The FilmArray BCID Panel also contains assays for the detection of genetic determinants of resistance to methicillin (mecA), vancomycin (vanA and vanB), and carbapenems (blakpr) to aid in the identification of potentially antimicrobial resistant organisms in positive blood culture samples. The antimicrobial resistance gene detected may or may not be associated with the agent responsible for disease. Negative results for these select antimicrobial resistance gene assays do not indicate susceptibility, as multiple mechanisms of resistance to methicillin, vancomycin, and carbapenems exist.

FilmArray BCID is indicated as an aid in the diagnosis of specific agents of bacteremia and fungemia and results should be used in conjunction with other clinical and laboratory findings. Positive FilmArray results do not rule out co-infection with organisms not included in the FilmArray BCID Panel. FilmArray BCID is not intended to monitor treatment for bacteremia or fungemia.

Subculturing of positive blood cultures is necessary to recover organisms for susceptibility testing and epidemiological typing, to identify organisms in the blood culture that are not detected by the FilmArray BCID Panel, and for species determination of some Staphylococci, Enterococci, Streptococci, and Enterobacteriaceae that are not specifically identified by the FilmArray BCID Panel assays.

The FilmArray Blood Culture Identification (BCID) Panel is a multiplex nucleic acid test designed to be used with a FilmArray system. The FilmArray BCID pouch contains freeze-dried reagents to perform nucleic acid purification and nested, multiplex PCR with DNA melt analysis. The FilmArray Blood Culture Identification (BCID) Panel simultaneously tests a single positive blood culture sample to provide results for 24 different organisms and organism groups that cause bloodstream infections and three genetic markers that are known to confer antimicrobial resistance (see Table 1).

A test is initiated by loading Hydration Solution and a positive blood culture sample mixed with the provided Sample Buffer into the FilmArray BCID pouch. The pouch contains all of the reagents required for specimen testing and analysis in a freeze-dried format; the addition of Hydration Solution and sample Buffer Mix rehydrates the reagents. After the pouch is prepared, the software guides the user though the steps of placing the pouch into the instrument, scanning the pouch barcode, entering the sample identification, and initiating the run.

The instrument contains a coordinated system of inflatable bladders and seal points, which act on the pouch to control the movement of liquid between the pouch blisters. When a bladder is inflated over a reagent blister, it forces liquid from the blister into connecting channels. Alternatively, when a seal is placed over a connecting channel it acts as a valve to open or close a channel. In addition, electronically controlled pneumatic pistons are positioned over multiple plungers in order to deliver the rehydrated reagents into the blisters at the appropriate times. Two Peltier devices control heating and cooling of the pouch to drive the PCR reactions and the melt curve analysis.

Nucleic acid extraction occurs within the FilmArray pouch using mechanical lysis and standard magnetic bead technology. After extracting and purifying nucleic acids from the unprocessed sample, a nested multiplex PCR is executed in two stages. During the first stage, a single, large volume, highly multiplexed PCR reaction which includes all primers of the outer primer sets, is performed. The products from first stage PCR are then diluted and combined with a fresh, primer-free master mix and a fluorescent double stranded DNA binding dye (LC Green" Plus, BioFire Defense, LLC). The solution is then distributed to each well of the array. Array wells contain sets of primers designed specifically to amplify sequences internal to the PCR products generated during the first stage PCR reaction. The 2nd stage PCR, or nested PCR, is performed in singleplex fashion in each well of the array. At the conclusion of the 21th stage PCR, the array is interrogated by melt curve analysis for the detection of signature amplicons denoting the presence of specific targets. A digital camera placed in front of the array captures fluorescent images of the PCR reactions and software interprets the data.

The software automatically interprets the results of each DNA melt curve analysis and combines the data with the results of the internal pouch controls to provide a test result for each organism on the panel.

Here's an analysis of the provided text regarding the acceptance criteria and study for the FilmArray Blood Culture Identification (BCID) Panel:

The document describes a 510(k) premarket notification for the FilmArray BCID Panel for use with the FilmArray 2.0 system, claiming substantial equivalence to a previously cleared device (K130914). The studies discussed are primarily focused on demonstrating this equivalence rather than establishing wholly new acceptance criteria for the device itself. The acceptance criteria essentially align with the performance of the predicate device.

1. A table of acceptance criteria and the reported device performance

The acceptance criteria are implicitly based on demonstrating comparable performance to the predicate FilmArray BCID Panel on the original FilmArray system. The reported performance for the new device (FilmArray BCID Panel on FilmArray 2.0) is effectively the validation that it meets these implicit acceptance criteria.

2. Sample sized used for the test set and the data provenance

• Clinical Performance Study Test Set:
  • Sample Size: 100 specimens (plus 2 additional runs for the modified system, totaling 102 runs for the current system and 202 for the modified system). These were selected such that each analyte (and antibiotic resistance marker) was represented 3-5 times.
  • Data Provenance: Retrospective. Specimens were "previously obtained during the FilmArray BCID prospective clinical evaluation." Seeded blood cultures (remnant from the prospective BCID clinical study) were used for rare BCID analytes. The country of origin is not explicitly stated, but the FDA submission and company location (Salt Lake City, UT, USA) suggest it is likely US-based data.
• Low Analyte (Titration) Study Test Set:
  • Sample Size: Not explicitly stated as a single number, but involves dilution series of samples for various analytes. Each test level for each organism was tested with 5 replicates (e.g., 5/5 (100%) in Table 4). The total number of tests for each analyte across the dilution series is 20 (5 replicates x 4 dilutions). With ~30 analytes, this implies hundreds of individual tests.
  • Data Provenance: This appears to be an analytical study using contrived samples (dilution series of samples containing a mix of BCID analytes), not directly clinical specimens. Country of origin not specified, but likely internal lab data.
• Reproducibility Study Test Set:
  • Sample Size: For each analyte, at each test level (Positive/Negative), 30 tests were performed at each of 3 sites, for a total of 90 data points per analyte/test level/loading procedure combination. This was then done for both syringe and injection vial loading procedures, effectively 180 data points per analyte/test level. There were 6 organisms with 2 test levels each for the most detailed analysis (positive/negative), and several other analytes with only negative test levels contributing to overall statistics.
  • Data Provenance: Contrived blood culture samples spiked with various concentrations of BCID organisms, tested across three different test sites (likely in the US based on the submission).

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts

The document does not mention the use of experts to establish ground truth for the test sets. For the clinical performance study, the ground truth for previously collected clinical specimens would have been established by standard clinical microbiology methods. For the seeded blood cultures and contrived samples, the ground truth is inherently known by the study design (i.e., what organisms were spiked into the samples). This is a common approach for in vitro diagnostic (IVD) device studies.

4. Adjudication method for the test set

Not applicable, as ground truth was established by microbiological culture or by design (for contrived samples), not by expert opinion requiring adjudication.

5. If a multi reader multi case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance

Not applicable. This is an in vitro diagnostic (IVD) device, not an AI-assisted diagnostic imaging system that uses human readers. The "reader" here is the instrument and its software, and the comparison is between two versions of the instrument/software system, and different loading methods.

6. If a standalone (i.e. algorithm only without human-in-the-loop performance) was done

Yes, the device (FilmArray BCID Panel) performs as a standalone algorithm/instrument system to detect and identify nucleic acids. There is no human interpretation of raw data; "The software automatically interprets the results of each DNA melt curve analysis and combines the data with the results of the internal pouch controls to provide a test result for each organism on the panel."

7. The type of ground truth used (expert consensus, pathology, outcomes data, etc)

• Clinical Performance Study: Ground truth for patient specimens would have been established by conventional microbiology methods (culture, identification, susceptibility testing). For seeded blood cultures, the ground truth was based on the known spiked organisms.
• Low Analyte Study: Ground truth was based on the known composition and concentration of the spiked organisms in the contrived samples.
• Reproducibility Study: Ground truth was based on the known composition and expected detection of the spiked organisms in the contrived samples.

8. The sample size for the training set

The document does not provide information about a separate "training set" in the context of device development or any machine learning approach. This is an IVD device based on molecular biology principles (PCR and melt analysis), not a machine learning algorithm that typically requires explicit training datasets. The development and optimization of the assays would have involved extensive R&D, but not in the "training set" sense of AI/ML.

9. How the ground truth for the training set was established

As noted in point 8, the concept of a "training set" in the AI/ML sense is not directly applicable to this type of IVD device. The ground truth for developing and validating the PCR assays would involve standard molecular biology and microbiology techniques to confirm the presence and identity of target organisms and resistance genes.

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The Verigene® Gram Negative Blood Culture Nucleic Acid Test (BC-GN), performed using the sample-to-result Verigene System, is a qualitative multiplexed in vitro diagnostic test for the simultaneous detection and identification of selected gram-negative bacteria and resistance markers. BC-GN is performed directly on blood culture media using blood culture bottles identified as positive by a continuous monitoring blood culture system and which contain gram-negative bacteria as determined by gram stain.

BC-GN detects and identifies the following:

BC-GN will not distinguish Escherichia coli from Shigella spp. (S. dysenteriae, S. flexneri, S. boydii, and S. sonnei)

BC-GN is indicated for use in conjunction with other clinical and laboratory findings to aid in the diagnosis of bacterial bloodstream infections; however. is not used to monitor these infections. Sub-culturing of positive blood cultures is necessary to recover organisms for antimicrobial susceptibility testing (AST), for identification of organisms not detected by BC-GN, to detect mixed infections that may not be detected by BC-GN, for association of antimicrobial resistance marker genes to a specific organism, or for epidemiological typing.

The Verigene Gram Negative Blood Culture Nucleic Acid Test (BC-GN) is a molecular assay which relies on detection of specific nucleic acid targets in a microarray format. For each of the bacterial nucleic acid sequences detected by the BC-GN test, unique Capture and Mediator oligonucleotides are utilized, with gold nanoparticle probe-based endpoint detection. The Capture oligonucleotides are covalently bound to the microarray substrate and hybridize to a specific portion of the nucleic acid targets. The Mediator oligonucleotides have regions which bind to a different portion of the same nucleic acid targets and also have sequences which allow binding of gold nanoparticle probes. Specific silver enhancement of the bound gold nanoparticle probes at the capture sites results in gold-silver aggregates that scatter light with high efficiency and provide accurate detection of target capture.

The BC-GN test is performed on the Verigene System, a sample-to-result, fully automated. bench-top molecular diagnostics workstation consisting of two components: the Verigene Reader and the Verigene Processor SP. For the BC-GN test, the Verigene System allows automated nucleic acid extraction from positive bacteria-containing blood culture specimens and target detection of bacteria-specific DNA. The BC-GN test utilizes single-use disposable test consumables and a self-contained Verigene Test Cartridge for each sample tested.

The Reader is the Verigene System's central control unit and user interface, and, with a touch-screen control panel and barcode scanner, guides the user through test processing. imaging, and test result generation. The Verigene Processor SP executes the test procedure. automating the steps of (1) Sample Preparation- cell lysis and magnetic bead-based bacterial DNA isolation from blood culture samples, and (2) Hybridization-- detection and identification of bacterial-specific DNA in a microarray format by using gold nanoparticle probe-based technology. Once the specimen is loaded by the operator, all other fluid transfer steps are performed by an automated pipette that transfers reagents between wells of the trays and loads the specimen into the Test Cartridge for hybridization. Single-use disposable test consumables and a self-contained Verigenc Test Cartridge are utilized for each sample tested with the BC-GN test.

To obtain the test results after processing is complete. the user removes the Test Cartridge from the Processor SP, and inserts the substrate holder into the Reader for analysis. Light scatter from the capture spots is imaged by the Reader and intensities from the microarray spots are used to make a determination regarding the presence (Detected) or absence (Not Detected) of a bacterial nucleic acid sequence/analyte. This determination is made by means of software-based decision algorithm resident in the Reader.

The Nanosphere Verigene® Gram Negative Blood Culture Nucleic Acid Test (BC-GN) is a qualitative multiplexed in vitro diagnostic test designed for the simultaneous detection and identification of selected gram-negative bacteria and resistance markers directly from positive blood culture media.

Here's an analysis of its acceptance criteria and the supporting studies:

1. Table of Acceptance Criteria (Performance Goals) and Reported Device Performance (Method Comparison Study)

The document does not explicitly state "acceptance criteria" as a set of predefined thresholds for performance metrics. However, the "Method Comparison" study presents the device's performance against reference methods, which implicitly serve as the comparison points for its effectiveness. The reported performance metrics are Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA), along with their 95% Confidence Intervals.

Note: The document does not explicitly state numerical acceptance criteria. The "implied acceptance criterion" section is a general interpretation based on typical regulatory expectations for diagnostic accuracy.

2. Sample Size Used for the Test Set and Data Provenance

• Sample Size for Test Set: A total of 1412 specimens were analyzed in the method comparison study.
  • 604 prospectively-collected fresh specimens
  • 272 prospectively-collected frozen specimens
  • 239 selected frozen specimens
  • 297 simulated frozen specimens
• Data Provenance: The study was conducted at thirteen (13) investigational sites. The document does not specify the countries of origin for these sites or the data itself, but such clinical trials for FDA submissions are typically conducted in the US or under comparable regulatory frameworks. The inclusion of "prospectively-collected fresh specimens" and "prospectively-collected frozen specimens" indicates prospective data collection, while "selected frozen specimens" and "simulated frozen specimens" indicate retrospective or artificially prepared samples. This suggests a mixed approach to sample collection.

3. Number of Experts Used to Establish Ground Truth for the Test Set and Their Qualifications

The document does not explicitly state the number of experts used to establish ground truth or their specific qualifications (e.g., "radiologist with 10 years of experience"). Instead, it describes the methods used to establish ground truth:

• For bacterial organisms: "standard culture-based automated phenotypic bacterial identification reference methods." This implies that the ground truth was established by clinical microbiology laboratories following established protocols, likely interpreted by qualified clinical microbiologists or medical laboratory scientists.
• For resistance markers: "the combination of PCR amplification and bidirectional sequencing confirmation." This indicates molecular biology techniques, which would also be performed and interpreted by appropriately trained laboratory personnel.

4. Adjudication Method for the Test Set

The document does not mention an explicit "adjudication method" involving multiple human readers for the test set results. The ground truth was established using standard reference laboratory methods, not through an adjudication process of human interpretations of the device's output. The device's results were directly compared to these reference methods.

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

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not reported. This device is an in vitro diagnostic test (an algorithm-only device without human-in-the-loop performance being evaluated in the clinical study presented) rather than an imaging or interpretive AI device where human reader performance is typically assessed and compared to AI-assisted performance. The study focuses on the agreement of the device's output with standard reference methods. Therefore, an effect size of human readers improving with AI vs. without AI assistance is not applicable here.

6. Standalone (Algorithm-Only Without Human-in-the-Loop Performance) Study

Yes, the method comparison study directly assesses the standalone performance of the Verigene BC-GN test (algorithm only). The device's results are automatically generated by the Verigene System (Reader and Processor SP) and then compared to the ground truth established by reference laboratory methods, without human intervention in the interpretation of the device's output that would then be compared to human interpretation.

7. Type of Ground Truth Used

The ground truth used was:

• Culture-based automated phenotypic bacterial identification reference methods for bacterial organisms.
• PCR amplification and bidirectional sequencing confirmation for resistance markers.

8. Sample Size for the Training Set

The document does not explicitly specify a "training set" in the context of machine learning model development. This device appears to be a molecular diagnostic assay using a microarray and a software-based decision algorithm, rather than a system heavily reliant on a continuously learning or adaptable AI model that would typically have a distinct training phase with a specific dataset.

However, analytical studies involved extensive testing that could be considered analogous to developing and refining the device's performance characteristics:

• Analytical Sensitivity (LOD): Tested 12 bacterial strains.
• Analytical Reactivity (Inclusivity): Tested 195 strains of 44 different organisms (including 79 with resistance markers).
• Analytical Specificity (Exclusivity): Tested 172 "non-BC-GN panel" organisms.
• Competitive Inhibition / Mixed Growth: Multiple studies involving combinations of bacterial organisms.
• Precision/Repeatability: 18-member panel tested for 864 replicates.
• Reproducibility: 18-member panel tested for 1620 replicates across 3 external sites.

These studies contribute to the design and validation of the test's targets, probes, and decision algorithm, but a "training set" in the context of statistical machine learning for inferential models is not presented.

9. How the Ground Truth for the Training Set Was Established

As noted in point 8, a distinct "training set" with ground truth in the typical machine learning sense is not explicitly described. For the analytical studies (e.g., LOD, inclusivity, exclusivity), the ground truth for bacterial identification and resistance marker presence would have been established using well-characterized laboratory strains and standard microbiological and molecular identification techniques. These involve:

• Pure cultures: For LOD and inclusivity, known bacterial strains are used.
• Conventional identification methods: Such as cell morphology, growth characteristics, biochemical tests, and potentially 16S rRNA sequencing for difficult or novel strains.
• Molecular techniques for resistance markers: Such as PCR and sequencing to confirm the presence and identity of specific resistance genes.

These methods are the gold standards in microbiology for characterizing bacteria and their genetic elements, forming the basis of the device's design and analytical performance evaluation.

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