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The Cepheid® Xpert® MRSA/SA Blood Culture Assay, performed on the GeneXpert® Instrument Systems, is a qualitative in vitro diagnostic test intended for the detection of Staphylococcus aureus (SA) and methicillin-resistant Staphylococcus aureus (MRSA) DNA directly from positive blood cultures. The assay utilizes automated real-time polymerase chain reaction (PCR) for the amplification of MRSA/SA specific DNA targets and fluorogenic target-specific hybridization probes for the real-time detection of the amplified DNA. The assay is performed directly on positive blood culture samples from BD BACTEC™ Plus Aerobic/F, BacT/ALERT® SA (Standard Aerobic) or VersaTREK REDOX 1® (aerobic) blood culture bottles that are determined by Gram Stain as Gram Positive Cocci in Clusters (GPCC) or as Gram Positive Cocci in singles (GPC). The Xpert MRSA/SA Blood Culture Assay is indicated for use in conjunction with other laboratory tests, such as culture, and clinical data available to the clinician as an aid in the detection of MRSA/SA from positive blood cultures. Subculturing of positive blood cultures is necessary to recover organisms for susceptibility testing or for epidemiological typing. The Cepheid Xpert MRSA/SA Blood Culture Assay is not intended to monitor treatment for MRSA/SA infections.

The Cepheid Xpert® MRSA/SA Blood Culture Assay (Xpert MRSA/SA Blood Culture Assay) is a rapid, automated DNA test for the simultaneous qualitative detection of MRSA and SA DNA directly from blood culture bottle specimens that are detected as positive for microbial growth and shown to contain Gram Positive Cocci by Gram stain. The primers and probes in the Xpert MRSA/SA Assay detect nucleic acid sequences of the staphylococcal protein A (spa), the gene for methicillin/oxacillin resistance (mecA), and staphylococcal cassette chromosome (SCCmec) inserted in the SA chromosomal attB site.

The test includes a Sample Processing Control (SPC) to control for adequate processing of the target bacteria and to monitor the presence of inhibitor(s) in the PCR assay. The Probe Check Control (PCC) verifies reagent rehydration, PCR tube filling in the cartridge, probe integrity, and dye stability.

The specimen for testing with the Xpert MRSA/SA Blood Culture Assay consists of an aliquot taken from a positive blood culture bottle. Using one of the disposable fixed 50 µL volume transfer pipettes provided with the test kit, an aliquot of the positive blood culture is transferred into a single-use tube of Elution Reagent, also provided with the kit. The Elution Reagent is briefly vortexed and the entire content is transferred to the "S" chamber of the disposable fluidic cartridge (the Xpert MRSA/SA Blood Culture Assay cartridge), after which the cartridge is ready to place on the instrument.

The assay is performed on the Cepheid GeneXpert Instrument Systems, which automate and integrate sample purification, nucleic acid amplification of the target sequences in simple or complex samples using real-time PCR. The systems consist of an instrument, personal computer, and preloaded software for running the tests and viewing the results. The GeneXpert Instrument Systems require the use of single-use disposable cartridges that hold the PCR reagents and host the PCR process. Because the cartridges are self-contained, crosscontamination between samples is minimized. In this platform, additional sample preparation, amplification, and real-time detection are all fully-automated and completely integrated. The Xpert MRSA/SA Blood Culture Assay performed on the GeneXpert Instrument Systems provides results in approximately 60 minutes.

The GeneXpert Instrument Systems, comprised of the GeneXpert Dx Systems and the GeneXpert Infinity Systems, have 1 to 80 randomly accessible modules, depending upon the instrument, that are each capable of performing separate sample preparation and real-time PCR tests. Each module contains a syringe drive for dispensing fluids (i.e., the syringe drive activates the plunger that works in concert with the rotary valve in the cartridge to move fluids between chambers), an ultrasonic horn for lysing cells or spores, and a proprietary I-CORE® thermocycler for performing real-time PCR and detection.

Here's a breakdown of the acceptance criteria and study details for the Cepheid Xpert® MRSA/SA Blood Culture Assay:

Acceptance Criteria and Reported Device Performance

Study Details

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

   • Sample Size: A total of 792 specimens were tested for MRSA and SA.
   • Data Provenance: The data was collected from a multi-site prospective study at eight US institutions.

2. 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 for establishing ground truth within the clinical comparison study.
   • The ground truth was established by reference culture results and susceptibility testing, which are standard laboratory practices and would generally involve trained microbiologists or laboratory technicians rather than individual "experts" in the context of interpretation. Susceptibility testing was performed in accordance with CLSI documents M2-A11 and M100-S22, using cefoxitin disc as a surrogate for methicillin/oxacillin resistance, indicating reliance on established guidelines.

3. Adjudication method for the test set:

   • The document does not specify an explicit adjudication method (like 2+1 or 3+1). The ground truth was determined by comparing the device's results to "reference culture results and susceptibility testing (the current standard of care)." This implies a direct comparison to established laboratory methods, rather than an expert consensus process for adjudication of conflicting results between readers.

4. 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 a Nucleic Acid Amplification Test (NAAT), which is an automated molecular diagnostic test producing a definitive positive/negative result, not an imaging-based AI system that would assist human readers in interpretation. Therefore, the concept of human readers improving with or without AI assistance is not applicable here.

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

   • Yes, a standalone performance study was done. The entire clinical comparison study (792 specimens) directly evaluated the performance of the Xpert MRSA/SA Blood Culture Assay (an automated DNA test) against reference culture methods. The device's output is a definitive positive or negative result for MRSA and SA, generated automatically by the instrument system without human interpretive input for each individual test result.

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

   • The ground truth used was reference culture results and susceptibility testing. This represents established laboratory diagnostic methods considered the "standard of care" for identifying and characterizing Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus.

7. The sample size for the training set:

   • The document focuses on the validation or test set performance rather than detailing a specific "training set" in the context of machine learning. The device is a NAAT, meaning its "learning" or development process involved analytical studies (inclusivity, LoD, specificity, etc.) and wet-lab experiments during its development, rather than observational data-driven machine learning.
   • For analytical inclusivity (reactivity), 250 SA strains (47 MSSA and 203 MRSA) from multiple sources were tested. This could be considered part of the development/training of the assay's target detection capabilities, but it's not a "training set" in the common AI/ML sense.

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

   • As noted above, a "training set" in the machine learning sense is not directly applicable. For the analytical studies that inform the assay's design and performance claims:
     • Analytical Inclusivity (Reactivity): Strains were characterized by methods like pulsed-field gel electrophoresis (PFGE) to confirm their USA types and classifications (e.g., USA100, USA300, USA400). The document also mentions a SA strain (LGA251) with a novel mecA gene (SCCmec type XI) which was incorrectly identified, implying that the ground truth for these strains was established through advanced genomic and microbiological characterization.
     • Limit of Detection (LoD): Ground truth was established by precise quantification of bacterial cells (CFU/mL, then CFU/test) using plate counts in triplicate.
     • Analytical Specificity (Exclusivity): Organisms were identified as Gram positive, Gram negative, or yeast, and specifically classified (e.g., methicillin-sensitive, coagulase negative Staphylococcus) based on standard microbiological techniques and sourced from reputable culture collections (ATCC, CCUG, NCTC, NARSA).

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The Cepheid Xpert™ MRSA/SA Blood Culture Assay performed on the GeneXpert® Dx System™ is a qualitative in vitro diagnostic test intended for the detection of Staphylococcus aureus (SA) and methicillin-resistant Staphylococcus aureus (MRSA) DNA directly from patient positive blood cultures. The assay utilizes automated realtime polymerase chain reaction (PCR) for the amplification of MRSA/SA specific DNA targets and fluorogenic target-specific hybridization probes for the real-time detection of the amplified DNA. The assay is performed directly on positive blood culture specimens using BD BACTEC™ Plus Aerobic/F blood culture bottles that are determined as Gram Positive Cocci in Clusters (GPCC) or as Gram Positive Cocci in singles (GPC) by Gram stain. The Cepheid Xpert™ MRSA/SA Blood Culture Assay is not intended to monitor treatment for MRSA/SA infections. Subculturing of positive blood cultures is necessary to recover organisms for susceptibility testing or for epidemiological typing.

The Cepheid Xpert™ MRSA/SA Blood Culture Assay (Xpert MRSA/SA Blood Culture Assay) is a rapid, automated DNA test for simultaneously detecting MRSA and SA Alber) I a rapids, and culture specimens. The specimens. The specimen consists of an aliquot taken from a positive blood culture bottle for testing with the Xpert MRSA/SA Blood Culture Assay. Using one of the small disposable transfer pipettes provided with the test kit, a single drop of the positive blood culture aliquot (approximately 50 µL) is transferred into the Elution Reagent. The Elution Reagent is vortexed for 10 seconds and the entire contents are transferred to "S" chamber of the disposable fluidic cartridge (the Xpert MRSA/SA Blood Culture Assay cartridge). The two single-use reagents (Reagent 1 and Reagent 2) that are provided with the assay are transferred to different, uniquelylabeled chambers of the Xpert MRSA/SA cartridge. The user initiates a test from the system user interface and places the cartridge into the GeneXpert® Dx System instrument platform, which performs hands-off real-time, multiplex polymerase chain reaction (PCR) for detection of DNA. In this platform, additional sample preparation, amplification, and real-time detection are all fully-automated and completely integrated.

The GeneXpert Dx System consists of a GeneXpert instrument, personal computer, and the multi-chambered fluidic cartridges that are designed to complete sample preparation and real-time PCR for detection of MRSA and SA in approximately 50 minutes. Each system has 2 to 16 randomly accessible modules that are each capable of performing separate sample preparation and real-time PCR tests. Each module contains a syringe drive for dispensing fluids, an ultrasonic horn for lysing cells or spores, and a proprietary I-CORE® thermocycler for performing real-time PCR and detection.

The Xpert MRSA/SA Assay includes reagents for the simultaneous detection of the target organisms, SA and MRSA. The primers and probes in the Xpert MRSA/SA Assay detect nucleic acid sequences of the staphylococcal protein A (spa), the gene for methicillin/oxacillin resistance (mecA), and staphylococcal cassette chromosome (SCCmec) inserted in the SA chromosomal attB site.

The test includes a Sample Processing Control (SPC) to control for adequate processing of the target bacteria and to monitor the presence of inhibitor(s) in the PCR assay. The Probe Check Control (PCC) verifies reagent rehydration, PCR tube filling in the cartridge, probe integrity, and dye stability.

The acceptance criteria and study proving the device meets them are detailed below for the Cepheid Xpert™ MRSA/SA Blood Culture Assay.

1. Table of Acceptance Criteria and Reported Device Performance:

The document implicitly defines acceptance criteria based on the performance observed in the predicate device and the clinical study results. The clinical study results serve as the primary demonstration of meeting these criteria.

Note: The predicate device's performance is listed as "Positive % Agreement" and "Negative % Agreement," which are equivalent to sensitivity and specificity in this context. The implicit acceptance criteria for the new device are to perform at least comparably to the predicate and the "standard of care" (reference culture results and susceptibility testing).

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

• Sample Size for Clinical Test Set: 249 specimens.
• Data Provenance: Multi-site prospective investigation study at three US institutions. The data is prospective, collected from subjects whose routine care included blood culture testing.

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 or their specific qualifications (e.g., years of experience as a radiologist) used to establish the ground truth for the clinical test set. It mentions "reference culture results and susceptibility testing, the current standard of care" and that susceptibility testing was performed "in accordance with the CLSI documents M2-A9 and M100-S17." This implies that qualified laboratory personnel, following established clinical microbiology guidelines, performed these reference methods.

4. Adjudication Method for the Test Set:

The document does not explicitly describe an adjudication method (e.g., 2+1, 3+1). The ground truth was established by "reference culture results and susceptibility testing, the current standard of care." This suggests a direct comparison against the established laboratory gold standard, rather than a consensus among multiple human readers.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done, and Effect Size:

No, a multi-reader multi-case (MRMC) comparative effectiveness study was not explicitly done in the context of comparing human readers with and without AI assistance. The study compares the device's performance directly against "reference culture results and susceptibility testing," which is the established standard of care for identifying bacterial infections. The device is an automated nucleic acid amplification test, not an AI assistance tool for human interpretation.

6. If a Standalone (Algorithm Only Without Human-in-the-Loop Performance) Was Done:

Yes, a standalone performance study was conducted. The Cepheid Xpert™ MRSA/SA Blood Culture Assay is an automated device that performs sample preparation, amplification, and real-time detection without human intervention once the sample is loaded. The "Overall Results" section (Page 12) directly reports the device's performance in identifying MRSA and SA specimens relative to culture. This refers to the algorithm's performance without a human in the loop for interpretation.

7. The Type of Ground Truth Used:

The ground truth used for the clinical study was based on:

• Reference Culture Results: This involves standard microbiological culture techniques to isolate and identify organisms.
• Susceptibility Testing: Performed in accordance with CLSI documents (M2-A9 and M100-S17) to determine methicillin/oxacillin resistance, using cefoxitin as a surrogate.

This represents established microbiological laboratory standards, often considered the "gold standard" for pathogen identification and antimicrobial susceptibility.

8. The Sample Size for the Training Set:

The document does not explicitly state a separate "training set" for the clinical validation studies in the way one might for a machine learning algorithm. For analytical studies, various strains were tested:

• Analytical Inclusivity Study on CDC Staphylococcus aureus Specimens: 25 Staphylococcus aureus strains.
• Analytical Inclusivity Study on Expanded Panel of Staphylococcus aureus Specimens: 121 additional Staphylococcus aureus strains.
• "Empty Cassette Variants" Study: 22 Staphylococcus aureus isolates.
• Analytical Limit of Detection: Replicates of 20 for 6 MRSA isolates and 3 MSSA isolates.
• Cross-reactivity Study: 105 strains (98 ATCC, 7 NARSA).

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

For the analytical "training" (or rather, validation) sets mentioned above, the ground truth was established through:

• Bacterial strain identification, PFGE type, and SCCmec type determined by the CDC (for CDC specimens).
• Catalase, tube coagulase, and Gram stain for characterizing discordants in the expanded panel study.
• Methicillin susceptibility assessed by disk diffusion using a cefoxitin disk.
• Known characteristics and genetic information of the cultured strains (e.g., MRSA, SA, Cfu/test).

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