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

The iC-GPC Assay detects organism DNA and identifies the following bacterial species and resistance markers:

Bacterial Species Staphylococcus aureus Staphylococcus epidermidis Streptococcus pneumoniae Enterococcus faecalis Enterococcus faecium

Resistance Markers

mecA- associated with methicillin resistance vanA- associated with vancomycin resistance vanB- associated with vancomycin resistance

The iC-GPC Assay detects the mecA resistance marker, inferring mecA-mediated methicillin resistance, and the vanA and vanB resistance markers, inferring vanAlvanB-mediated vancomycin resistance. In mixed growth, the iC-GPC Assay does not specifically attribute van-mediated vancomycin resistance to either E. faecalis or E. faecium, or mecA-mediated methicillin resistance to either S. aureus or S. epidermidis.

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

The iC-GPC Assay utilizes polymerase chain reaction (PCR) for the amplification of specific targets and detects the amplified target DNA with fluorescence-based microarray hybridization. The iC-GPC Assay uses proprietary ARM-PCR (Amplicon Rescued Multiplex PCR) technology allowing for multiple targets to be amplified in one reaction. Targets are detected directly from patient positive blood cultures, confirmed by Gram stain to contain gram positive cocci. Testing is performed within a closed, disposable cassette that contains all the reagents required to complete the iC-GPC Assay, including a universal microarray.

To operate, the user opens the iC-GPC Cassette cap and pipettes an aliquot of the positive blood culture sample into the sample well of the cassette is then inserted into the iC-Processor, which performs the processes of DNA extraction, multiplex amplification, and microarray After processing is complete, the cassette is inserted into the iC-Reader for hybridization. fluorescence-based detection and data analysis. Final results are generated by iC-Report, computer software for data acquisition, analysis, and display.

Extraction, amplification, and hybridization 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-GPC Cassette which communicates with the iC-Processor. To access and pierce the foil-sealed reagent wells located in the bottom well plate of the cassette, the processor manipulates the cassette to move the internal 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 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 4 iC-Cassettes at one time. The results are interpreted via the iC-Report software and displayed for the user on an iMac computer. Raw data and result interpretations are stored within the iMac; raw data is accessible to authorized personnel only and is not available to the end user.

The provided document describes the iC-GPC Assay for use on the iC-System, a qualitative, multiplexed, in vitro diagnostic test for the detection and identification of potentially pathogenic gram-positive bacteria and resistance markers from positive blood cultures. The document details the performance studies conducted to support its substantial equivalence to a legally marketed predicate device (VERIGENE® Gram Positive Blood Culture Nucleic Acid Test (BC-GP)) and its overall analytical and clinical performance.

Here's an breakdown of the acceptance criteria and the studies proving the device meets them:

1. Table of Acceptance Criteria and Reported Device Performance

The document doesn't explicitly state "acceptance criteria" in a single, consolidated table with pre-defined thresholds. Instead, it presents performance metrics in various tests (reproducibility, LoD, inclusivity, exclusivity, method comparison). Acceptance for these tests is implied by the successful outcomes. The core performance is summarized in the method comparison study against an FDA-cleared multiplex assay and against traditional culture and susceptibility methods.

Below, I've compiled a table summarizing the key performance metrics from the Method Comparison section, which represents the device's main performance claim for its intended use. The "Acceptance Criteria" here are implicitly High agreement (e.g., >95%) for both positive and negative agreement, as is common for in vitro diagnostics devices aiming for substantial equivalence.

2. Sample Size and Data Provenance

• Test Set Sample Size:
  • Clinical Method Comparison Study: 966 positive blood culture specimens initially enrolled, reduced to 913 (879 fresh prospective, 34 frozen prospective) after withdrawals.
  • Contrived Samples: An additional 168 contrived samples.
• Data Provenance: The general text of the letter indicates "four geographically dispersed clinical sites." Given the context of a US regulatory submission to the FDA, it is highly likely these sites were in the United States. The "Expected Values" table further specifies "U.S. State" data from NY, WI, NM, and FL. The study collected prospective (fresh and frozen) and contrived samples.

3. Number of Experts and Qualifications for Ground Truth

The document does not explicitly state the "number of experts" or their "qualifications" for establishing the ground truth for the clinical method comparison test set.

However, for the comparison methods that served as ground truth:

• FDA-cleared multiplex assay: This is a pre-existing, validated device, implying its results are considered reliable.
• Conventional reference methods: This includes "subculture, identification of isolates using biochemical methods or MALDI-TOF, and phenotypic antimicrobial susceptibility testing (AST)." These methods are standard clinical laboratory practices performed by trained microbiologists and clinical laboratory scientists. The qualification of these individuals would adhere to standard laboratory accreditation and personnel requirements.

4. Adjudication Method for the Test Set

The document details discordant analysis for the method comparison studies:

• Against FDA-Cleared Multiplex Assay: PCR and bidirectional sequencing were performed for all specimens with positive results for resistance markers by either the iC-GPC Assay or the FDA-cleared multiplex assay. These results were used to analyze and investigate discordant results. This implies a form of 2+1 adjudication or investigation where if the iC-GPC and comparator disagreed, an independent gold standard (PCR/sequencing) was used to resolve.
• Against Conventional Reference Methods: PCR and bi-directional sequencing were not used for discordant analysis between iC-GPC and conventional reference method results. This suggests that the conventional methods served as the direct reference, without further independent adjudication for discrepancies with the iC-GPC assay.

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

This is not a multi-reader multi-case (MRMC) study. The iC-GPC Assay is an in vitro diagnostic device for laboratory use that provides automated results, not a diagnostic imaging AI algorithm that assists human readers. Therefore, there is no human-in-the-loop performance measurement or effect size reported here regarding human reader improvement with AI assistance.

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

Yes, this entire submission revolves around the standalone performance of the iC-GPC Assay, which is an automated system (iC-System) that performs DNA extraction, multiplex amplification, hybridization, and detection to generate results. The reported performance metrics (positive/negative agreement, LoD, inclusivity, exclusivity) are a direct measure of the algorithm's (device's) performance.

7. Type of Ground Truth Used

• For Analytical Studies (LoD, Inclusivity, Exclusivity, Reproducibility, etc.): Ground truth was established by known concentrations/strains of organisms, obtained from characterized sources (e.g., ATCC strains, well-characterized clinical isolates) and confirmed by methods such as colony counts.
• For Clinical Method Comparison Study:
  • Primary Ground Truth 1: An FDA-cleared multiplex assay.
  • Secondary Ground Truth 2: Conventional laboratory reference methods (subculture, biochemical identification, MALDI-TOF, and phenotypic antimicrobial susceptibility testing (AST)).
  • Discordant Resolution (for resistance markers in comparison to FDA-Cleared Multiplex Assay): PCR and bi-directional sequencing as an independent, highly accurate molecular method.

8. Sample Size for the Training Set

The document does not provide information on the training set size for the iC-GPC Assay's development. This FDA 510(k) submission focuses on the validation of the device through performance studies (test sets) rather than detailing the specifics of its development or "training" phase. For in vitro diagnostics that use molecular techniques, the "training" analogous to machine learning models usually involves extensive research and development to optimize primer and probe design, reaction conditions, and analytical algorithms based on large collections of known positive and negative samples, but these are typically internal R&D activities not explicitly reported as "training set size" in the final validation summary.

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

As the document does not detail a "training set," it also does not describe how its ground truth was established. However, for a molecular diagnostic assay like this, the underlying "ground truth" for developing and optimizing the assay's targets and reaction conditions would typically be established through:

• Known, characterized bacterial strains and clinical isolates: Identified and confirmed using a combination of standard microbiological methods (culture, Gram stain, biochemical tests, phenotypic susceptibility testing) and advanced molecular methods (16S rRNA gene sequencing, whole-genome sequencing) to determine precise species and resistance marker presence.
• Synthetic DNA/RNA targets: Used for initial assay design and analytical performance testing.
• Bioinformatics analysis: To select highly specific gene targets (like gseA, nuc, lytA, ddl, fcm, mecA, vanA, vanB) that are unique to the target organisms and resistance mechanisms.

This developmental phase would involve rigorous, iterative testing and refinement against a comprehensive panel of both target and non-target organisms to ensure high sensitivity and specificity before formal validation studies like those presented in the 510(k) summary are conducted.

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