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The Cepheid Xpert Flu-RSV Xpress Assay, performed on the GeneXpert Xpress System, is an automated, multiplex realtime, reverse transcriptase polymerase chain reaction (RT-PCR) assay intended for the in vitro qualitative detection and differentiation of influenza B, and respiratory syncytial virus (RSV) viral RNA. The Xpert Flu+RSV Xpress Assay uses nasopharyngeal swab specimens collected from patients with signs and symptoms of respiratory infection. The Xpert Flu+RSV Xpress Assay is intended as an aid in the diagnosis of influenza and respiratory syncytial virus infections in conjunction with clinical and epidemiological risk factors.

Negative results do not preclude influenza virus or respiratory syncytial virus infection and should not be used as the sole basis for treatment or other patient management decisions.

Performance characteristics for influenza A were established during the 2014-2015 influenza season. When other novel influenza A viruses are emerging, performance characteristics may vary.

If infection with a novel influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to state or local health department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.

The Xpert® Nasopharyngeal Sample Collection Kit is designed to collect, preserve, and transport nasopharyngeal swab specimens and to preserve and transport nasal aspirate/wash specimens containing viruses from patients with signs and symptoms of respiratory infection prior to analysis with the Xpert Flu Assay or the Xpert Flu/RSV XC Assay. The Xpert® Nasopharyngeal Sample Collection Kit is designed to collect, preserve, and transport nasopharyngeal swab specimens containing viruses from patients with signs and symptoms of respiratory infection prior to analysis with the Xpert Flu+RSV Xpress Assay.

The Xpert Flu+RSV Xpress Assay is an automated in vitro diagnostic test for qualitative detection and differentiation of influenza A. influenza B, and respiratory syncytial virus (RSV). The assay is performed on the Cepheid GeneXpert Xpress System (GeneXpert Dx System, GX-I). The GeneXpert Xpress System platform automates and integrates sample extraction, purification, amplification, and detection of the target sequence in simple or complex samples using real-time PCR and reverse transcriptase PCR (RT-PCR) assays. The systems require the use of single-use disposable cartridges (the Xpert Flu+RSV Xpress cartridges) that hold the PCR reagents and host the PCR process. Because the cartridges are self-contained and specimens never come into contact with working parts of the instrument modules, cross-contamination between samples is minimized.

The Xpert Flu+RSV Xpress Assay includes reagents for the detection and differentiation of influenza A, influenza B, and RSV viral RNA directly from nasopharyngeal (NP) swab specimens collected from patients with signs and symptoms of respiratory infection. A Sample Processing Control (SPC) and a Probe Check Control (PCC) are also included in the cartridge. The SPC is present to control for adequate processing of the target viruses and to monitor the presence of inhibitors in the PCR reaction. The Probe Check Control (PCC) verifies reagent rehydration, PCR tube filling in the cartridge, probe integrity, and dye stability.

The single-use, multi-chambered fluidic cartridges are designed to complete sample preparation and real-time RT-PCR for detection and differentiation of influenza B and RSV viral RNA in approximately 60 minutes. The GeneXpert Xpress System, comprised of the GeneXpert Dx System GX-I, has one module that is capable of performing separate sample preparation and real-time PCR and RT-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 realtime PCR and RT-PCR and detection.

Specimens are collected following the instructions for collecting NP swab specimens provided in Xpert Flu+RSV Xpress Assay package insert for influenza and RSV testing. The Cepheid Xpert Nasopharyngeal Sample Collection Kit (Cepheid catalog #SWAB/B-100) is required but not provided for use with the assay. The NP swab specimen is placed in the Xpert viral transport medium and sent to the GeneXpert® Xpress testing area for processing. When stored in the transport medium, the NP swab specimen is stable for up to 24 hours at 2-30 °C or up to seven days at 2-8 °C. When ready to test the specimen, the user briefly mixes the specimen by inverting the tube five times, transfers the eluted material to the sample chamber in the top of the disposable fluidic cartridge. The user initiates a test from the system user interface and places the cartridge into the GeneXpert Xpress instrument platform, which performs hands-off real-time, multiplex polymerase chain reaction (PCR) for detection of RNA. The results are automatically generated at the end of the process in a report that can be viewed and printed.

1. Table of Acceptance Criteria and Reported Device Performance

• Flu A: PPA 100% (98.5-100), NPA 94.8% (93.7-95.7)
• Flu B: PPA 100% (94.3-100), NPA 99.5% (99.1-99.8)
• RSV: PPA 96.9% (92.3-99.1), NPA 99.6% (99.2-99.8)
  Pre-selected Frozen NP Swab Specimens:
• Flu A: NPA 98.5% (96.1-99.6) (PPA not applicable as no positive collected)
• Flu B: PPA 100% (96.4-100), NPA 98.7% (95.5-99.8)
• RSV: PPA 97.6% (87.1-99.9), NPA 99.5% (97.5-100) |
  | Reproducibility (Total Agreement) | High percentage of samples yielding expected results across sites, days, and operators, especially for positive samples. | Negative: 100% (88/88)
  Flu A-Low Pos: 98.9% (89/90)
  Flu A-Mod Pos: 100% (90/90)
  Flu B-Low Pos: 98.9% (89/90)
  Flu B-Mod Pos: 100% (90/90)
  RSV-Low Pos: 97.8% (87/89)
  RSV-Mod Pos: 100% (90/90) (Note: High Neg samples show lower agreement, consistent with near cutoff concentrations). |
  | Reproducibility (Ct Variation) | Low variability (SD, CV) in Ct values across sites, days, and operators for detected targets. | SPC: Total Std Dev 1.5, Total CV 4.8%
  Flu A1 (Low Pos): Total Std Dev 1.6, Total CV 4.5%
  Flu A1 (Mod Pos): Total Std Dev 1.4, Total CV 4.2%
  Flu B (Low Pos): Total Std Dev 1.9, Total CV 5.9%
  Flu B (Mod Pos): Total Std Dev 1.0, Total CV 3.3%
  RSV (Low Pos): Total Std Dev 1.7, Total CV 5.0%
  RSV (Mod Pos): Total Std Dev 1.0, Total CV 3.0% (Similar values for corresponding High Neg samples). |

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

• Clinical Comparison Study (Test Set):
  • Total NP Swab Specimens: 2435
  • Fresh, Prospectively Collected: 2176
  • Pre-selected Frozen, Archived Specimens: 259
  • Data Provenance: United States (12 institutions in the U.S.), retrospective for archived samples and prospective for fresh samples. The study was conducted during the 2014-2015 influenza season.

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 used to establish the ground truth for the test set or their specific qualifications (e.g., radiologist with 10 years of experience).

Instead, the ground truth for the clinical comparison study was established using a FDA-cleared molecular comparator assay. For discrepant results between the Xpert Flu+RSV Xpress Assay and the comparator assay, bidirectional sequencing was performed. While sequencing is a definitive molecular method, it's not described as an "expert" review in the traditional sense of a clinical expert.

4. Adjudication Method for the Test Set

• Initial Comparison: Xpert Flu+RSV Xpress Assay results were compared to an FDA-cleared molecular comparator assay.
• Discrepancy Resolution/Adjudication: For specimens where the Xpert Flu+RSV Xpress Assay and the comparator assay were discrepant, bidirectional sequencing was performed. The results from sequencing were provided for informational purposes only. This implies that while sequencing helped understand discrepancies, the primary comparison was with the FDA-cleared molecular assay.

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

• No, a multi-reader multi-case (MRMC) comparative effectiveness study was not explicitly mentioned or described in the provided text.
• The study focuses on the diagnostic performance of the device itself (algorithm only) against a comparator assay, and its reproducibility under different operator conditions, not on human reader performance with or without AI assistance.
• The statement "The study results showed the Xpert Flu+RSV Xpress Assay is acceptable for its intended use with inexperienced lab users" refers to the device's performance when used by such users, not an improvement in human reader performance aided by AI.

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

• Yes, a standalone study (algorithm-only performance) was effectively done. The clinical comparison study evaluates the performance of the Xpert Flu+RSV Xpress Assay (the device/algorithm) against an FDA-cleared molecular comparator assay.
• While operators load samples and initiate the test, the entire process of sample extraction, purification, amplification, and detection, and interpretation of the fluorescent signals to yield a positive/negative result, is automated by the device ("GeneXpert Xpress System platform automates and integrates sample extraction, purification, amplification, and detection..."). The output is a "report that can be viewed and printed," indicating an algorithm-generated result.

7. The Type of Ground Truth Used

• Clinical Comparison Study: The primary ground truth was established by an FDA-cleared molecular comparator assay. For discordant results, bidirectional sequencing was used as an additional, more definitive form of molecular ground truth for informational purposes.
• Non-Clinical Studies (Analytical Sensitivity, Specificity, Reactivity): The ground truth was based on known concentrations of purified viral cultures (TCID50/mL or pg/µL) or bacterial/yeast cultures (CFU/mL), a laboratory-defined ground truth.

8. The Sample Size for the Training Set

The document does not explicitly specify a sample size for a "training set." This type of in-vitro diagnostic device (PCR assay) typically relies on extensive analytical validation (LoD, specificity, inclusivity, interference) and clinical validation with prospectively and retrospectively collected samples, rather than a distinct "training set" in the machine learning sense. The device's underlying algorithm is based on established molecular biology principles (RT-PCR) calibrated during development, not specifically "trained" on a dataset in the way an AI model would be.

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

As noted above, the concept of a clear "training set" with ground truth in the context of machine learning is not directly applicable here. For the development and establishment of the assay's performance characteristics, ground truth would have been established through:

• Analytical studies: Using known concentrations and strains of target and non-target pathogens to define sensitivity (LoD), specificity, and inclusivity.
• Internal validation/optimization: During the assay development phase, characterized clinical samples or contrived samples with confirmed presence/absence of targets via established reference methods (e.g., PCR, sequencing, culture) would have been used to optimize assay parameters (e.g., primer/probe design, cycle thresholds).

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The Cepheid Xpert Flu/RSV XC Assay is an automated, multiplex real-time, reverse transcriptase polymerase chain reaction (RT-PCR) assay intended for the in vitro qualitative detection and differentiation of influenza B, and respiratory syncytial virus (RSV) viral RNA. The Xpert Flu/RSV XC Assay uses nasopharyngeal swab and nasal aspirate/wash specimens collected from patients with signs and symptoms of respiratory infection. The Xpert Flu/RSV XC Assay is intended as an aid in the diagnosis of influenza and respiratory syncytial virus infections in conjunction with clinical and epidemiological risk factors.

Negative results do not preclude influenza virus or respiratory syncytial virus infection and should not be used as the sole basis for treatment or other patient management decisions.

Performance characteristics for influenza A were established during the 2013-2014 influenza season. When other novel influenza A viruses are emerging, performance characteristics may vary.

If infection with a novel influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to state or local health department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.

The Xpert Flu/RSV XC Assay is a rapid, automated in vitro diagnostic test for qualitative detection and differentiation of influenza A. influenza B, and respiratory syncytial virus (RSV). The assay is performed on the Cepheid GeneXpert Instrument Systems (GeneXpert Dx systems and GeneXpert Infinity Systems). The GeneXpert Instrument System platform automates and integrates sample purification, nucleic acid amplification, and detection of the target sequence in simple or complex samples using real-time PCR and reverse transcriptase PCR (RT-PCR) assays. The systems require the use of singleuse disposable cartridges (the Xpert Flu/RSV XC cartridges) that hold the PCR reagents and host the PCR process. Because the cartridges are self-contained and specimens never come into contact with working parts of the instrument modules, cross-contamination between samples is minimized.

The Xpert Flu/RSV XC Assay includes reagents for the detection and differentiation of influenza A, influenza B, and RSV viral RNA directly from nasopharyngeal (NP) swab and nasal aspirate/wash (NA/W) specimens collected from patients with signs and symptoms of respiratory infection. A Sample Processing Control (SPC) and a Probe Check Control (PCC) are also included in the cartridge. The SPC is present to control for adequate processing of the target viruses and to monitor the presence of inhibitors in the PCR reaction. The Probe Check Control (PCC) verifies reagent rehydration, PCR tube filling in the cartridge, probe integrity and dye stability.

The single-use, multi-chambered fluidic cartridges are designed to complete sample preparation and real-time RT-PCR for detection and differentiation of influenza A, influenza B and RSV viral RNA in approximately 60 minutes or less. 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 and RT-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 RT-PCR and detection.

Specimens are collected following the user's institution standard procedures for collecting NA/W specimens and NP swab specimens for influenza and RSV testing. The ancillary Cepheid Xpert Nasopharyngeal Sample Collection Kit (Cepheid catalog #SWAB/B-100) or Cepheid's Sample Collection Kit (Cepheid catalog #NASL-100N-100) are required but not provided for use with the assay. Both kits contain the identical viral transport medium and sterile nylon flocked swab. The NA/W specimen or the NP swab specimen is placed into the Xpert viral transport medium and sent to the GeneXpert® testing area for processing. When stored in the transport medium, the NA/W specimen or NP swab specimen is stable for up to 24 hours at 2-30 ℃ or up to seven days at 2-8 ℃. When ready to test the specimen, the user briefly mixes the specimen by inverting the tube five times, transfers the eluted material to the sample chamber in the top of the disposable fluidic cartridge. The user initiates a test from the system user interface and places the cartridge into the GeneXpert instrument platform, which performs hands-off real-time, multiplex polymerase chain reaction (PCR) for detection of RNA. The results are automatically generated at the end of the process in a report that can be viewed and printed.

Here's an analysis of the provided text, focusing on acceptance criteria and study details:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state formal "acceptance criteria" for the clinical performance in terms of specific PPA/NPA thresholds that had to be met for clearance. It presents the performance of the device and claims substantial equivalence to predicate devices. However, we can infer the desired performance from the presented data and the implicit expectation for a diagnostic assay to perform well. The analytical studies (LoD, specificity, inclusivity) demonstrate performance against well-defined criteria.

Inferred Clinical Acceptance Criteria (Based on Comparator Performance and FDA Clearance): The device's performance (PPA and NPA) should be demonstrably similar to or better than previously cleared predicate devices, with high positive and negative agreement. While no explicit thresholds like "PPA > X%" are stated for clinical studies, the demonstration of high agreement values and the claim of "substantially equivalent" implies these levels were considered acceptable by the FDA.

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The Cepheid Xpert® Flu Assay, performed on the GeneXpert® Instrument Systems, is an automated, multiplex real-time RT-PCR assay intended for the in vitro qualitative detection and differentiation of influenza A, influenza B and 2009 H1N1 influenza viral RNA. The Xpert Flu Assay uses nasal aspirates/washes and nasopharyngeal swab specimens collected from patients with signs and symptoms of respiratory infection in conjunction with clinical and epidemiological risk factors. The Xpert Flu Assay is intended as an aid in the diagnosis of influenza.

Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions.

Performance characteristics for influenza A were established during the 2009-2010 influenza season when 2009 H1N1 influenza was the predominant influenza A virus in circulation. Performance characteristics for influenza A were confirmed when influenza A/H3 and influenza A/2009 H1N1 were the predominant influenza A viruses in circulation (2009-2010, 2010-2011 and 2011-2012). When other influenza A viruses are emerging, performance characteristics may vary.

If infection with a novel influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to state or local health department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.

The Xpert Flu Assay is a rapid, automated in vitro diagnostic test for qualitative detection and differentiation of influenza A, influenza B and influenza A, subtype 2009 H1N1. The assay is performed on the Cepheid GeneXpert Instrument Systems. The GeneXpert Instrument Systems automate and integrate sample purification, nucleic acid amplification, and detection of the target sequence in simple or complex samples using real-time PCR and RT-PCR assays. The 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 and specimens never come into contact with working parts of the instrument modules, cross-contamination between samples is minimized.

The Xpert Flu Assay includes reagents for the detection and differentiation of influenza A, influenza B and influenza A, subtype 2009 H1N1 directly from nasal aspirates/washes (NA/W) and nasopharyngeal (NP) swab specimens from patients suspected of having influenza. A Sample Processing Control (SPC) and a Probe Check Control (PCC) are also included. The SPC is present to control for adequate processing of the target viruses and to monitor the presence of inhibitors in the PCR reaction. The Probe Check Control (PCC) verifies reagent rehydration, PCR tube filling in the cartridge, probe integrity and dye stability.

The liquid specimen (NA/W) or swab specimen (NP) is collected according to the institution's standard procedures and placed into Universal Transport Medium (3mL UTM tubes). Following a brief mixing by inverting the UTM tube five times, the eluted material and one single-use reagent (Reagent 1), that is provided with the assay, are transferred to different, uniquely-labeled chambers of the disposable fluidic cartridge (the Xpert Flu cartridge). The user initiates a test from the system user interface and places the cartridge into the GeneXpert instrument platform, which performs hands-off realtime, multiplex polymerase chain reaction (PCR) for detection of DNA. In this platform, additional sample preparation, amplification, and real-time detection are all fullyautomated and completely integrated.

The single-use, multi-chambered fluidic cartridges are designed to complete sample preparation and real-time PCR for detection and differentiation of influenza A. influenza B and influenza A, subtype 2009 H1N1 in 75 minutes. The GeneXpert Instrument Systems, which consist of the GeneXpert Dx System, the GeneXpert Infinity-48 System, and the GeneXpert Infinity-80 System, have 1 to 80 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 (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.

The Cepheid Xpert® Flu Assay, a molecular diagnostic device, was evaluated through analytical and clinical performance studies to demonstrate its substantial equivalence to a previously cleared predicate device (K120911).

1. Acceptance Criteria and Reported Device Performance

The acceptance criteria for the Xpert Flu Assay, as demonstrated by its performance against a predicate device, are based on positive and negative agreement rates for the detection of influenza A, influenza A subtype 2009 H1N1, and influenza B in different specimen types (Nasal Aspirates/Washes and Nasopharyngeal Swabs). While explicit numerical acceptance criteria are not presented as thresholds, the observed performance values for the modified Xpert Flu Assay are provided as evidence of its substantial equivalence.

Table of Acceptance Criteria and Reported Device Performance:

The study demonstrates that the modified Xpert Flu Assay exhibits very high positive and negative agreement rates when compared to the predicate Xpert Flu Assay across all targets and specimen types.

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

The clinical performance study used a total of 302 Nasal Aspirate/Wash (NA/W) specimens and 352 Nasopharyngeal (NP) swab specimens for the test set.

The data provenance for these specimens is described as:

• Retrospective/Prospective: The specimens included a mix of "frozen leftover, prospectively collected, unlinked prospectively collected archived and/or pre-selected banked" specimens.
• Country of Origin: Not explicitly stated, but assumed to be from locations where standard clinical influenza testing is conducted, given the context of US FDA submission.

Additionally, ten contrived specimens (5 NP swab and 5 NA/W) were prepared and tested separately. These were not included in the primary dataset analysis of agreement rates.

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

Not applicable. This is a molecular diagnostic device comparing its performance to a predicate molecular diagnostic device. The ground truth for the clinical performance study was established by the predicate device's results, with sequencing used for discrepant results. No human expert interpretation of images or clinical findings for ground truth establishment is mentioned.

4. Adjudication Method for the Test Set

The adjudication method specifically mentioned for the clinical performance study was sequencing for all discrepant specimens. This means if the modified Xpert Flu Assay result differed from the predicate Xpert Flu Assay result, a third, independent method (sequencing) was used to determine the true positive/negative status for those particular samples.

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

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not done. This device is an automated, standalone diagnostic assay, not an AI-assisted human reading system. Therefore, the concept of human readers improving with AI vs. without AI assistance is not applicable.

6. Standalone Performance Study

Yes, a standalone performance study was done. The entire evaluation of the "modified Xpert Flu Assay" against the "current Xpert Flu Assay" (the predicate device) represents its standalone performance. The device provides a qualitative detection and differentiation of influenza A, B, and 2009 H1N1 viral RNA directly from patient specimens without human interpretation of the assay's primary output beyond reading the automated result.

7. Type of Ground Truth Used

For the clinical performance study, the primary ground truth was implicitly the results from the predicate Xpert Flu Assay. For any discrepancies between the modified device and the predicate, sequencing was used as the confirmatory ground truth.

For the analytical studies (Analytical Reactivity and Limit of Detection), the ground truth for positive results was known viral strains at defined concentrations (TCID50/mL or pg/µL). For Analytical Specificity, the ground truth was known non-target viral, bacterial, and yeast strains.

8. Sample Size for the Training Set

Not applicable. This is a molecular diagnostic test. The concept of a "training set" is typically associated with machine learning or AI models. For this type of device, performance is established through analytical validation (known concentrations of specific strains) and clinical validation (comparison to a predicate or established reference method in patient samples). There is no mention of a machine learning component requiring a distinct training set.

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

Not applicable, as there was no explicit training set for a machine learning model. For the analytical and clinical studies, ground truth was established as described in section 7.

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The Cepheid Xpert® Flu Assay is an automated, multiplex real-time RT-PCR assay intended for the in vitro qualitative detection and differentiation of influenza A, influenza B and 2009 H1N1 influenza viral RNA. The Xpert Flu Assay uses nasal aspirates/washes and nasopharyngeal swab specimens collected from patients with signs and symptoms of respiratory infection in conjunction with clinical and epidemiological risk factors. The Xpert Flu Assay is intended as an aid in the diagnosis of influenza.

Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions.

Performance characteristics for influenza A were established during the 2009-2010 influenza season when 2009 H1N1 influenza was the predominant influenza A virus in circulation. When other influenza A viruses are emerging, performance characteristics may vary.

If infection with a novel influenza A virus is suspected based on current clinical and enidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to state or local health department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.

The Cepheid Xpert® Flu Assay is a rapid, automated in vitro diagnostic test for qualitative detection and differentiation of influenza B, and influenza A, subtype 2009 H1N1 from nasal aspirates/washes (NA/W) and nasopharyngeal (NP) swab specimens from patients with signs and symptoms of respiratory infection. The assay is performed on the Cepheid GeneXpert Instrument Systems, which consist of the GeneXpert Dx System and the GeneXpert Infinity-48 System. The Device is being modified with this 510(k), to add the GeneXpert Infinity-80 System as an additional instrument system for use with the Xpert Flu Assay.

The GeneXpert Instrument Systems automate and integrate sample purification, nucleic acid amplification, and detection of the target sequence in simple or complex samples using real-time PCR and rRT-PCR assays. The GeneXpert Instrument System family comprises a GeneXpert (GX) instrument, GX-I, GX-IV, GX-XVI; a GeneXpert XVI, available with 4, 8, 12 or 16 modules, a GeneXpert Infinity-48 available with 16, 24, 32 or 48 modules, or a GeneXpert Infinity-80 available with 16, 24, 32, 40, 48, 56, 64, 72, or 80 modules. The instrument systems also contain a computer, and preloaded software for running tests and viewing the results. The GeneXpert Infinity Systems contain robotic features for cartridge handling. Each module contains a syringe drive for dispensing fluids, an ultrasonic horn for lysing cells or spores, a valve drive for sample movement, and I-CORE® thermocycler for performing real-time PCR and detection.

All systems require the use of the assay-specific single-use disposable cartridges that hold the PCR reagents and host the PCR process. The patented single-use cartridges contain: (1) eleven chambers for holding sample, reagents, or other materials, (2) a valve body composed of a plunger and syringe barrel, (3) a rotary valve system for controlling the movement of fluids between chambers, (4) an area for capturing, concentrating, washing, and lysing cells, (5) dry real-time PCR reagents, (6) an integrated PCR reaction tube that can be automatically filled by the instrument, and (7) liquid reagents. To eliminate testto-test contamination, all fluids including amplicons, are contained within the disposable cartridge. The instrument never comes into contact with any fluids within the cartridge. Each disposable cartridge is intended to test one sample. Cartridges are not re-usable.

A sample processing control (SPC) and a system control (Probe Check Control) are controls utilized by the GeneXpert Instrument System platform. The SPC is pre-loaded into the GeneXpert cartridge provided with the assay. The SPC is an encapsidated RNA made up of recombinant fragments developed so that there is no homology to the influenza genome. The SPC is present to control for adequate processing of the target viruses and to monitor the presence of inhibitors in the PCR reaction to reduce the possibility of false negative results. The SPC also ensures the PCR reaction conditions (temperature and time) are appropriate for the amplification reaction and that the PCR reagents are functional. The Probe Check Control verifies reagent rehydration, PCR tube filling in the cartridge, probe integrity and dye stability.

Commercially-available external controls may also be run in accordance with local, state, and federal accrediting organizations, as applicable.

The Xpert Flu Assay includes reagents for the simultaneous detection and differentiation of the target viruses. The primers and probes in the Xpert Flu Assay detect the presence of nucleic acid sequences for influenza A (Flu A), influenza B (Flu B) and influenza A sub-type 2009 H1N1 (2009 H1N1) directly from nasal aspirates/washes (NA/W) and nasopharyngeal (NP) swab specimens collected from patients suspected of having influenza. The specimens are collected in Universal Transport Medium (UTM) and transported to the GeneXpert area.

The specimen is prepared according to package insert instructions and transferred to the sample chamber (large opening) of the Xpert Flu Assay Cartridge. Reagent 1 (Binding Reagent) is dispensed into the chamber with the small opening of the Xpert Flu Assay Cartridge. The GeneXpert Cartridge is loaded onto the GeneXpert® Instrument System platform, which performs hands-off automated sample processing and real-time PCR for detection of Flu RNA. Summary and detailed test results are obtained in 75 minutes.

The results are interpolated by the GeneXpert Instrument Systems software from measured fluorescent signals and embedded calculation algorithms and are shown in the "View Results" window in tabular and graphic formats. The Xpert Flu Assay provides test results for influenza A, influenza B and influenza A, subtype 2009 H1N1. It also reports if the test is "Invalid," "Error" or "No Result," and instructs the user to repeat the test.

Here's an analysis of the acceptance criteria and study detailed in the provided 510(k) summary:

1. Table of Acceptance Criteria and Reported Device Performance

The document describes a reproducibility/precision study conducted to compare the performance of the GeneXpert Dx and the GeneXpert Infinity-80 Instrument Systems. The acceptance criteria are implicitly good agreement between the two systems, with specific percentage agreement targets for different sample types and concentrations.

Note: The "high negative" samples, particularly for 2009 H1N1 and Flu B, show lower agreement percentages. This is often expected for samples around the limit of detection, where results can fluctuate more. The study's conclusion that "The two Instrument Systems were shown to provide comparable results" indicates these levels were deemed acceptable for the purpose of demonstrating substantial equivalence of the new instrument system.

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

• Test Set Sample Size: The study used a panel of 10 specimens (with varying concentrations of influenza A, B, and 2009 H1N1). Each specimen was tested:
  • 4 times per day
  • for 12 days
  • by 2 operators
  • on 2 instrument systems (GeneXpert Dx and GeneXpert Infinity-80).
  • This results in (4 * 12 * 2 * 2 = 192) individual tests per specimen type, or 192 total tests for each specific sample ID category (e.g., 192 tests for "Negative", 192 tests for "Flu A moderate positive", etc.).
  • For the "2009 H1N1 high negative" sample on the Infinity-80, 3 out of 96 samples yielded indeterminate results on both attempts, reducing the effective sample size to 93 (93/96) for that specific comparison.
• Data Provenance: Not explicitly stated. The study was conducted by Cepheid, indicating it's likely internal analytical data, not from a specific country or clinical setting. It is a prospective analytical study designed for reproducibility/precision testing.

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

• Ground Truth Experts: The document does not mention the use of experts to establish the ground truth for these analytical samples. The samples were prepared as "Negative," "moderate positive," "low positive," and "high negative" concentrations of known viral targets. This suggests the "ground truth" was established based on the known composition and concentration of the prepared panel specimens themselves, rather than clinical adjudication by experts.
• Qualifications: Not applicable, as expert adjudication was not used for this analytical study.

4. Adjudication Method for the Test Set

• Adjudication Method: Not applicable. The "ground truth" for the analytical samples was determined by their known viral content and concentration as prepared by the manufacturer for the study. The study's purpose was to compare agreement between two instrument systems for these known samples, not to
  adjudicate ambiguous clinical results.

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

• MRMC Study: No, an MRMC comparative effectiveness study was not done. This study solely focused on the analytical performance (reproducibility/precision) of the device across different instrument platforms. It did not involve human readers interpreting results, nor did it measure the effect size of human readers with or without AI assistance.

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

• Standalone Performance: Yes, this study effectively represents a standalone performance evaluation of the integrated system (instrument + assay + embedded software). The results are generated automatically by the GeneXpert Instrument Systems software from measured fluorescent signals and embedded calculation algorithms. Human intervention is limited to specimen preparation and loading the cartridge.

7. The Type of Ground Truth Used

• Type of Ground Truth: The ground truth for the test set was based on prepared samples with known concentrations of viral RNA. These were spiked or formulated samples to represent negative, moderate positive, low positive, and high negative (near limit of detection) states for influenza A, B, and 2009 H1N1.

8. The Sample Size for the Training Set

• Training Set Sample Size: The document does not provide information on the training set sample size. This 510(k) submission is for a modification to an existing device (Xpert Flu Assay, predicate device K103766) to add a new instrument system (GeneXpert Infinity-80). It is highly likely that the original predicate device was developed and cleared with extensive training data, but that information is not part of this specific submission's summary for the modification.

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

• Training Set Ground Truth: The document does not provide information on how the ground truth for the training set was established. As with the training set sample size, this information would likely be found in the original 510(k) submission for the predicate device (K103766).

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The Cepheid® Xpert Flu Assay is an automated, multiplex real-time RT-PCR assay intended for the in vitro qualitative detection and differentiation of influenza B and 2009 H1N1 influenza viral RNA. The Xpert Flu Assay uses nasal aspirates/washes and nasopharyngeal swab specimens collected from patients with signs and symptoms of respiratory infection in conjunction with clinical and epidemiological risk factors. The Xpert Flu Assay is intended as an aid in the diagnosis of influenza.

Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions.

Performance characteristics for influenza A were established during the 2009-2010 influenza season when 2009 H1N1 influenza was the predominant influenza A virus in circulation. When other influenza A viruses are emerging, performance characteristics may vary.

If infection with a novel influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to state or local health department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.

The Xpert Flu Assay is a rapid, automated in vitro diagnostic test for qualitative detection and differentiation of influenza A, influenza B and influenza A, subtype 2009 HINI. The assay is performed on the Cepheid GeneXpert Instrument Systems. The GeneXpert Instrument Systems automate and integrate sample purification, nucleic acid amplification, and detection of the target sequence in simple or complex samples using real-time PCR and RT-PCR assays. The 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 and specimens never come into contact with working parts of the instrument modules, cross-contamination between samples is eliminated.

The Xpert Flu Assay includes reagents for the detection and differentiation of influenza A, influenza B and influenza A, subtype 2009 H1N1 directly from nasal aspirates/washes (NA/W) and nasopharyngeal (NP) swab specimens from patients with signs and symptoms of respiratory infection. A Sample Processing Control (SPC) and a Probe Check Control (PCC) are also included. The SPC is present to control for adequate processing of the target viruses and to monitor the presence of inhibitors in the PCR reaction. The Probe Check Control (PCC) verifies reagent rehydration, PCR tube filling in the cartridge, probe integrity and dye stability.

The liquid specimen (NA/W) or swab specimen (NP) is collected according to the institution's standard procedures and placed into Universal Transport Medium (3mL UTM tubes). Following a brief mixing by inverting the UTM tube five times, the eluted material and one single-use reagent (Reagent 1), that is provided with the assay, are transferred to different, uniquely-labeled chambers of the disposable fluidic cartridge (the Xpert Flu cartridge). The user initiates a test from the system user interface and places the cartridge into the GeneXpert instrument platform, which performs hands-off reverse transcription and 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 single-use, multi-chambered fluidic cartridges are designed to complete sample preparation and real-time RT-PCR for detection and differentiation of influenza A, influenza B and influenza A, subtype 2009 H1N1 in 75 minutes. The GeneXpert Instrument Systems have 1 to 48 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 (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.

Acceptance Criteria and Device Performance Study for Xpert Flu Assay

This report details the acceptance criteria and the study proving the Cepheid Xpert Flu Assay meets these criteria, based on the provided 510(k) summary (K103766).

1. Table of Acceptance Criteria and Reported Device Performance

The 510(k) summary does not explicitly state pre-defined quantitative acceptance criteria for sensitivity and specificity. Instead, the clinical study results are presented as the "performance characteristics" and are compared against a reference method. It's implied that achieving high agreement with the reference method across various influenza types and specimen types constitutes acceptable performance for substantial equivalence.

Based on the clinical performance study, the device performance is reported as follows:

Clinical Performance on Prospective Nasal Aspirates/Washes (NA/W)

Clinical Performance on Prospective Nasopharyngeal (NP) Swabs

Clinical Performance on Archived NA/W Specimens (vs. FDA Cleared Molecular Comparator)

Clinical Performance on Archived NP Swabs (vs. Viral Culture + DFA)

Clinical Performance on Archived NP Swabs (vs. FDA Cleared Molecular Comparator)

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

Prospective Specimens:

• NA/W specimens: 342
• NP swab specimens: 297

Archived Specimens:

• NA/W specimens: 425
• NP swab specimens: 150 (compared to viral culture + DFA), 177 (compared to FDA cleared molecular assay)

Data Provenance: The clinical study was conducted at six institutions in the U.S. and Australia. The study included both prospective and archived specimens.

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 X years of experience) used to establish the ground truth. However, the ground truth for "viral culture followed by direct fluorescent assay (DFA)" is a standard laboratory method, implying trained laboratory personnel perform these tests, which typically require specific certifications and experience. Sequencing results for influenza A positive specimens were also used as part of the ground truth.

4. Adjudication Method for the Test Set

The concept of an "adjudication method" (like 2+1, 3+1) is typically associated with studies where multiple human readers interpret results, and disagreement is resolved by an adjudicator. This is not directly applicable to a molecular diagnostic assay where results are objectively determined by instrumentation.

The document describes sequencing being performed for all influenza A positive specimens (identified by viral culture/DFA or the FDA cleared molecular assay) to differentiate subtypes. This acts as a confirmatory "adjudication" step for influenza A subtyping.

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

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not done. This study is for a diagnostic assay, not an AI-assisted human reading task.

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 Flu Assay is an automated, multiplex real-time RT-PCR assay intended for in vitro qualitative detection and differentiation of influenza viral RNA. The performance characteristics described in the "Clinical Performance Study" sections (Pgs. 14-21) are for the device (Xpert Flu Assay) operating independently, generating its own results. There is no human interpretation of imaging or other complex data involved in generating the primary test result from the assay itself.

7. The Type of Ground Truth Used

The ground truth used for the clinical performance study varied based on the specimen type and whether it was prospective or archived:

• Prospective specimens: Viral culture followed by direct fluorescent assay (DFA) was the primary comparator. This is a recognized laboratory standard.
• Archived specimens (where viral culture was not performed prior to freezing): An FDA cleared molecular assay was performed as the comparator assay.
• For all influenza A positive specimens (from both prospective and archived sets): Sequencing was used to differentiate influenza A subtypes (e.g., 2009 H1N1 from other influenza A). This can be considered as a highly specific confirmatory method.

8. The Sample Size for the Training Set

The document describes analytical and clinical performance studies but does not detail a separate "training set" or its size for an algorithm development since this is a molecular diagnostic assay, not a machine learning model in the typical sense. The assay is based on predefined biological reactions and detection thresholds, not trainable parameters derived from a large dataset. The analytical studies (Analytical Sensitivity, LoD, Analytical Specificity) and reproducibility studies define the assay's fundamental performance characteristics.

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

As noted above, there isn't a traditional "training set" as understood in machine learning. The assay's design and operating parameters would have been established through extensive laboratory work and optimization, including:

• Analytical Reactivity (Inclusivity): Testing against known influenza strains at specific concentrations (Table 5.2).
• Limit of Detection (LoD): Empirically determined as the lowest concentration (TCID50/mL) where 19/20 or 20/20 replicates were positive (Tables 5.3-5.6).
• Analytical Specificity (Exclusivity): Testing against potentially interfering viral, bacterial, and yeast strains at specified concentrations (Table 5.7).

These studies use well-defined, characterized strains and concentrations as their "ground truth" to ensure the assay performs as expected.

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