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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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The Cepheid Xpert MRSA/SA Skin and Soft Tissues Infection Assay (Xpert MRSA/SA SSTI Assay) performed in 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) from skin and soft tissue infection swabs. The test utilizes automated real-time polymerase chain reaction (PCR) to detect MRSA/SA DNA. The Xpert MRSA/SA SSTI Assay is indicated for use in conjunction with other laboratory tests such as microbiology culture, and clinical data available to the clinician as an aid in the detection of MRSA/SA from skin and soft tissue infections. The Xpert MRSA/SA SSTI Assay is not intended to monitor treatment for MRSA/SA infections. Concomitant cultures for SA and MRSA are necessary to recover organisms for susceptibility testing or epidemiological typing. In a mixed culture containing MRSA/SA and other organisms (e.g. Gram negative bacilli, yeast), results can be false negative or variable depending on the concentration of MRSA/SA present, particularly if the concentration of MRSA/SA is close to the LoD of the assay.

The Cepheid Xpert MRSA/SA Skin and Soft Tissue Infection Assay (Xpert MRSA/SA SSTI Assay) is a rapid, automated DNA test for simultaneously detecting MRSA and SA directly from skin and soft tissue specimen is collected on a double swab, which is placed in a tube containing elution reagent. Following brief vortexing, the eluted matcrial and two single-use reagents (Reagent 1 and Reagent 2) that are provided with the assay are transferred to different, uniquely-labeled chambers of the disposable fluidic cartridge (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® 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 1 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 MecA-Mediated 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.

Here's a breakdown of the acceptance criteria and study details for the Cepheid Xpert MRSA/SA SSTI Assay, based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

The document doesn't explicitly state "acceptance criteria" numerical targets in a table format. Instead, it presents performance data (Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA)) in comparison to a reference culture method. These performance metrics implicitly serve as the acceptance criteria for regulatory approval.

Implicit Acceptance Criteria (Performance Targets) and Reported Device Performance

Note: The document states "The test results showed the Xpert MRSA/SA SSTI to be substantially equivalent to the current standard of care" and explicitly lists performance data, which are interpreted as meeting implicit acceptance criteria for regulatory clearance.

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

• Clinical Test Set Sample Size: A total of 848 specimens were collected from patients.
  • No Antibiotic Use (within 3 weeks): 441 subjects
  • Unknown Antibiotic Use (within 3 weeks): 200 subjects
  • Known Antibiotic Use (within 3 weeks): 207 subjects
• Data Provenance: The study was a multi-site prospective investigation conducted at four US institutions. This means the data was collected specifically for this study in a forward-looking manner, and from within the United States.

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

The document states that the reference culture method involved "confirmation of presumptive positive colonies was performed with catalase, tube coagulase, and Gram stain. MecA-Mediated Oxacillin resistance was tested by disk diffusion test using a 30 µg cefoxitin disk and cutoff of 21/22 mm." This implies standard microbiology laboratory procedures were followed by trained laboratory personnel.

• Number of Experts: Not explicitly stated as a specific number of individual "experts."
• Qualifications of Experts: Implied to be trained microbiologists or clinical laboratory technologists experienced in performing and interpreting standard microbiology culture, identification (catalase, coagulase, Gram stain), and susceptibility testing (cefoxitin disk diffusion). The reference culture was performed at a "centralized laboratory," suggesting specialized expertise.

4. Adjudication Method for the Test Set

The document does not describe a formal adjudication method (like 2+1 or 3+1 consensus) for the ground truth of the clinical test set. The "reference culture" is treated as the definitive ground truth, performed at a centralized laboratory following established protocols. Implicitly, any discrepancies would be resolved by the standard operating procedures of that centralized laboratory, but a multi-reader adjudication process is not detailed.

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

No, an MRMC comparative effectiveness study involving human readers with and without AI assistance was not done. This device is an automated in vitro diagnostic test (Nucleic Acid Amplification Test) performed on a GeneXpert Dx System, designed to provide results directly without requiring human interpretation of complex images or data that AI might otherwise augment for human readers. Its comparison is against a reference culture method.

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

Yes, a standalone performance study was done. The Xpert MRSA/SA SSTI Assay is an automated system (algorithm only without human interpretation in the loop beyond sample preparation and loading). The "Performance Characteristics" and "Clinical Performance" sections detail the direct comparison of the Xpert MRSA/SA SSTI Assay's results against the reference culture method, making it a standalone evaluation.

7. The Type of Ground Truth Used

The primary ground truth used for the clinical performance study was reference microbiology culture and susceptibility testing. This involved:

• Enrichment in trypticase soy broth.
• Streaking onto plates with and without cefoxitin.
• Sub-culturing presumptive colonies onto blood agar.
• Confirmation of positive colonies with catalase, tube coagulase, and Gram stain for Staphylococcus aureus (SA) identification.
• MecA-Mediated Oxacillin resistance tested by disk diffusion using a 30 µg cefoxitin disk and a cutoff of 21/22 mm for MRSA identification.

8. The Sample Size for the Training Set

The document does not explicitly state a sample size for a "training set" in the context of machine learning (AI). This device is a real-time PCR assay, which is a molecular diagnostic method based on known biological reactions, not typically developed using machine learning algorithms that require distinct training sets.

However, if "training set" is interpreted more broadly as data used for analytical development and optimization, the document mentions extensive analytical studies:

• Analytical Inclusivity Study on CDC Staphylococcus aureus Specimens: 21 MRSA strains and 3 MSSA strains (total 24 strains).
• Analytical Inclusivity Study on Expanded Panel of Staphylococcus aureus Specimens: 121 additional Staphylococcus aureus strains (78 MRSA, 43 SA).
• Evaluation of Empty Cassette Variants: 22 isolates.
• Limit of Detection Studies: Multiple individual isolates (6 MRSA, 3 SA) each tested with 20 replicates at various concentrations.
• Linearity Studies: SA and MRSA isolates tested across ten-fold serial dilutions.
• Cross-reactivity Study: 105 strains (various bacteria and yeast).
• Evaluation of BORSA Strains: 7 BORSA strains.

These analytical tests provide the data and parameters for the assay's design and demonstrate its performance characteristics, which is analogous to the role of a training set for algorithm development, even though it's a traditional molecular test.

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

For the analytical studies (which can be considered analogous to a training/development phase for a molecular assay), the ground truth was established through:

• Confirmed Identification: Using CDC-reported strains, phylogenetically characterized strains, ATCC cultures, and NARSA strains.
• Phenotypic Testing: Catalase, tube coagulase, Gram stain, and cefoxitin disk diffusion (for oxacillin resistance) were used to characterize discrepant results and confirm the identity and resistance profile of isolates.
• Genetic Characterization: PFGE for USA types and SCCmec typing for MRSA strains provided detailed genetic ground truth.
• Known Concentrations: For LoD and linearity studies, bacterial concentrations were carefully quantified (CFU/sample) to establish precise analytical ground truth.

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