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The Prodesse® ProFAST + Assay is a multiplex Real Time RT-PCR in vitro diagnostic test for the qualitative detection and discrimination of seasonal Influenza A/H1, seasonal Influenza A/H3 and 2009 H1N1 Influenza viral nucleic acids isolated and purified from nasopharyngeal (NP) swab specimens from human patients with signs and symptoms of respiratory infection in conjunction with clinical and epidemiological risk factors. This Assay targets conserved regions of the Hemagglutinin (HA) gene for seasonal Influenza A/H1, seasonal Influenza A/H3 and 2009 H1N1 Influenza Virus, respectively. This Assay is not intended to detect Influenza B or Influenza C Viruses.

A negative ProFAST+ Assay result is a presumptive negative result for Influenza A. These results should be confirmed by an FDA cleared nucleic acid-based test (NAT) detecting Influenza A.

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

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 Prodesse® ProFAST + Assay is a multiplex Real Time RT-PCR in vitro diagnostic test for the qualitative detection and discrimination of seasonal Influenza A/H1, seasonal Influenza A/H3 and 2009 H1N1 Influenza viral nucleic acids isolated and purified from nasopharyngeal (NP) swab specimens from human patients with signs and symptoms of respiratory infection in conjunction with clinical and epidemiological risk factors. This Assay targets conserved regions of the Hemagglutinin (HA) gene for seasonal Influenza A/H1, seasonal Influenza A/H3 and 2009 H1N1 Influenza Virus, respectively. This Assay is not intended to detect Influenza B or Influenza C Viruses.

The ProFAST+ Assay enables detection and discrimination of Influenza A Virus subtypes: seasonal A/H1, seasonal A/H3, and 2009 H1N1 and internal control nucleic acid. Nasopharyngeal swab specimens are collected from patients with signs and symptoms of a respiratory infection using a polyester, rayon or nylon tipped swab and placed into viral transport medium.

A Universal Internal Control (UIC) is added to each sample prior to nucleic acid isolation to monitor for inhibitors present in the specimens. The isolation and purification of the nucleic acids is performed using either a MagNA Pure LC Instrument (Roche) and the MagNA Pure Total Nucleic Acid Isolation Kit (Roche) or a NucliSENS® easyMAG™ System (bioMérieux) and the Automated Magnetic Extraction Reagents (bioMérieux).

The purified nucleic acids are added to ProFAST+ Supermix along with enzymes included in the ProFAST+ Assay Kit. The ProFAST+ Supermix contains oligonucleotide primers and targetspecific oligonucleotide probes. The primers are complementary to highly conserved regions of the Hemagglutinin (HA) gene for seasonal influenza A/H1, seasonal influenza A/H3 and 2009 H1N1 Influenza Virus. The probes are dual-labeled with a reporter dye attached to the 5'-end and a quencher dye attached to the 3'-end.

Reverse transcription of the RNA in the sample into complementary DNA (cDNA) and subsequent amplification of DNA is performed in a Cepheid SmartCycler® II instrument. In this process, the probe anneals specifically to the template followed by primer extension and amplification. The ProFAST+ Assay is based on Tagman chemistry, which utilizes the 5' - 3' exonuclease activity of the Taq polymerase to cleave the probe thus separating the reporter dye from the quencher. This generates an increase in fluorescent signal upon excitation from a light source. With each cycle, additional reporter dye molecules are cleaved from their respective probes, further increasing fluorescent signal. The amount of fluorescence at any given cycle is dependent on the amount of amplification products present at that time. Fluorescent intensity is monitored during each PCR cycle by the SmartCycler II instrument.

The provided text describes a special 510(k) submission for the Prodesse® ProFAST®+ Assay, primarily focusing on modifications to the internal control and positive control, and an additional reactivity claim for H3N2v. The submission argues for substantial equivalence to a predicate device (K101855, ProFAST 101+ Assay).

Crucially, the document does not present acceptance criteria or detailed results from a study that "proves the device meets the acceptance criteria" in the format of a typical clinical validation study. Instead, it focuses on demonstrating that modifications did not negatively impact performance compared to the previously cleared predicate device.

Here's an attempt to extract the requested information, noting where details are missing based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state quantitative acceptance criteria (e.g., minimum sensitivity, specificity, or agreement percentages) for a clinical performance study of the modified device. Instead, it refers to the previous performance claims of the ProFAST+ Assay (the predicate device for the modifications) and states that the modified assay "continues to meet the performance claims."

The closest to "reported device performance" are the results of the verification/validation studies for the modifications:

Note: The document explicitly states that "the performance characteristics of this device with clinical specimens that are positive for H3N2v influenza virus have not been established." This means for H3N2v, only analytical reactivity was shown, not clinical performance.

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

• Sample Size for Test Set: The document mentions a "retrospective clinical comparison study" for the UIC modification but does not provide the sample size used in this study.
• Data Provenance: The document states "clinical comparison study," implying human patient samples were used. The term "retrospective" indicates that these samples were collected in the past. The country of origin is not specified but is implicitly the US given the FDA submission.

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

Not provided. The nature of the ground truth (e.g., a reference method like viral culture or another FDA-cleared NAT) is not detailed, nor is the number or qualifications of experts, if any, involved in establishing it. It's likely the "ground truth" for the clinical comparison study would have been established by the reference method against which the predicate device's original performance claims were made.

4. Adjudication Method for the Test Set

Not provided. Given that this appears to be a comparison study against a historical reference or predicate, an adjudication method might not have been
explicitly described in this type of submission.

5. If a Multi Reader Multi Case (MRMC) Comparative Effectiveness Study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance

Not applicable. This device is an in vitro diagnostic (IVD) RT-PCR assay for detecting viral nucleic acids, not an AI-assisted diagnostic tool that would be used by "human readers" in the sense of image interpretation. Therefore, an MRMC study with human readers and AI assistance is not relevant to this device. The "reader" here is the instrument interpreting PCR amplification curves.

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

Yes, the device is inherently a standalone algorithm/assay without human-in-the-loop performance influencing its primary result. It provides a qualitative (positive/negative) detection and discrimination of influenza A subtypes. The "retrospective clinical comparison study" would represent the standalone performance of the modified assay.

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

Not explicitly stated in the provided text. For RT-PCR assays, the ground truth for clinical studies is typically established by:

• A "gold standard" laboratory method (e.g., viral culture if available and sensitive enough, or a highly sensitive and specific FDA-cleared reference molecular test).
• A composite reference method combining multiple tests or clinical findings.

Given it's a "clinical comparison study," it implies comparison to established clinical diagnoses or reference lab results, but the specifics are absent.

8. The Sample Size for the Training Set

Not applicable/Not provided. This is an RT-PCR assay, not a machine learning or AI algorithm that requires a "training set" in the conventional sense. The "training" for such a device involves assay optimization and analytical validation using characterized samples (e.g., contrived samples with known viral concentrations, characterized clinical samples) to establish parameters like limit of detection, linearity, and specificity. The document refers to "Analytical Sensitivity, IC Interference, Extractor Equivalency, and Sample Stability studies," which utilize such characterized samples, but a specific "training set sample size" as per AI/ML terminology is not relevant here.

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

Not applicable/Not provided in the AI/ML context. For analytical studies, the "ground truth" (e.g., viral presence and concentration) is established by using characterized stocks, reference materials, or quantified clinical samples whose status is independently verified (e.g., by culture, sequencing, or quantitative PCR methods).

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The CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel is intended for use in real-time RT-PCR (rRT-PCR) assays on an Applied Biosystems (ABI) 7500 Fast Dx Real-Time PCR Instrument in conjunction with clinical and epidemiological information:

• For qualitative detection of influenza virus type A or B from viral RNA in upper respiratory tract clinical specimens (including nasopharyngeal swabs [NPS], nasal swabs [NS], throat swabs [TS], nasal aspirates [NA], nasal washes [NW] and dual nasopharyngeal/throat swabs [NPS/TS]), and lower respiratory tract specimens (including bronchoalveolar lavage [BAL], bronchial wash [BW], tracheal aspirate [TA], sputum, and lung tissue) from human patients with signs and symptoms of respiratory infection and/or from viral culture;
• For determination of the subtype of seasonal human influenza A viruses as seasonal A/H1, A/H3, and/or A/H1pdm09 from viral RNA in upper respiratory tract clinical specimens (including NPS, NS, TS, NA, NW and NPS/TS) and lower respiratory tract specimens (including BAL, BW, TA, sputum and lung tissue) from human patients with signs and symptoms of respiratory infection and/or from viral culture;
• For the presumptive identification of virus in patients who may be infected with influenza A subtype A/H5 (Asian lineage) from viral RNA in human respiratory specimens and viral culture in conjunction with clinical and epidemiological risk factors;
• To provide epidemiological information for surveillance of circulating influenza viruses.

The CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel is used in real-time RT-PCR assays (rRT-PCR) on the ABI 7500 Fast Dx Real-Time PCR Instrument. The device consists of oligonucleotide primers and dual-labeled hydrolysis (TaqMan®) probes to be used in rRT-PCR for the in vitro qualitative detection and characterization of human influenza viruses from viral RNA in respiratory specimens from patients presenting with influenza-like illness (ILI).

Here's a breakdown of the acceptance criteria and study information for the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel, based on the provided 510(k) summary:

1. Table of Acceptance Criteria and Reported Device Performance

The 510(k) is for a modification to an existing device (K111507) to add a new enzyme kit (Quanta qScript™). Therefore, the acceptance criteria are implicitly demonstrating substantial equivalence to the predicate device (the panel using Invitrogen SuperScript™). The performance is shown as agreement with the predicate.

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

• Prospective Clinical Study:

  • Initial Sample Size: 1,002 respiratory specimens.
  • Analyzed Sample Size: 931 specimens (after exclusions for inconclusive results, technician/instrument error, or unspecified specimen type).
  • Data Provenance: Prospective, 2011-2012 influenza season. The country of origin is not explicitly stated but is implied to be the United States (references to U.S. DHHS, World Health Organization and National Respiratory and Enteric Virus Surveillance System (NREVSS) collaborating laboratories in the United States).

• Retrospective Clinical Study (A/H1 supplement):

  • Sample Size: 30 positive specimens for seasonal influenza A/H1N1.
  • Data Provenance: Retrospective, from a previous clinical study conducted during the 2006-2007 influenza season. Implicitly United States.

• Retrospective Clinical Study (A/H5 simulated samples):

  • Sample Size: 36 samples (12 high concentration, 12 moderate concentration, 12 low concentration).
  • Data Provenance: Simulated samples prepared from a characterized and titered stock of influenza A/H5N1 virus and human A549 cells (Lednicky et al. method). This is an in vitro study, not from human patients directly.

• Retrospective Clinical Study (Negative specimens):

  • Sample Size: 50 negative specimens.
  • Data Provenance: Retrospective, from a previous clinical study conducted during the 2006-2007 influenza season. Implicitly United States.

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

The ground truth in this submission is established by the results of the predicate device (CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel using Invitrogen SuperScript™). The submission focuses on equivalency. There is no mention of external subject matter experts being used to establish a separate ground truth for the test set independent of the predicate device's results. The predicate device itself acts as the "gold standard" or "ground truth" for comparison.

4. Adjudication Method for the Test Set

Not applicable in the typical sense for this submission. The ground truth for the comparison is the result obtained by the predicate device. The study design is a comparison of the modified device's performance against the established performance of the predicate device. Discrepancies between the investigational device and the predicate device's results would be analyzed, but there's no mention of a separate expert adjudication panel for individual cases.

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

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not done. This device is an in vitro diagnostic (IVD) molecular assay (RT-PCR) and involves laboratory testing, not human reader interpretation of images or other data where AI assistance would be relevant. The "readers" are the laboratory instruments and trained technicians interpreting quantitative PCR curves, not human experts making diagnostic decisions from primary clinical data with or without AI aid.

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

The device is a diagnostic panel that requires human operation (sample preparation, instrument loading, interpretation of results, although interpretation of the raw PCR data is instrument-assisted). There is no "algorithm only" performance reported in the context of being a standalone diagnostic decision-making system. Its performance is always within a human-operated laboratory workflow. The analytical and clinical studies evaluate the performance of the assay itself when run by trained personnel.

7. The Type of Ground Truth Used (Expert Consensus, Pathology, Outcomes Data, etc.)

For the clinical performance evaluation:

• The primary ground truth for comparison is the results obtained using the predicate device (CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel with Invitrogen SuperScript™). This effectively means the ground truth is established by a previously validated RT-PCR assay.
• The initial patient status (influenza-like illness symptoms, viral culture results) provides context, but the direct comparison is test-to-test.

For the analytical sensitivity and inclusivity studies:

• Characterized influenza virus strains (EID50/mL or TCID30/mL values) were used as the ground truth for establishing the Limit of Detection (LOD) and for inclusivity testing.

8. The Sample Size for the Training Set

This submission does not discuss a "training set" in the context of machine learning or AI models. The device is a RT-PCR diagnostic panel. There's no training phase described for an algorithm. The "training" of the assay itself would refer to the historical development and validation of the primers and probes, which isn't detailed here but precedes this 510(k) modification.

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

Not applicable as there is no "training set" for an algorithm. The development of the RT-PCR panel involves establishing the specificity and sensitivity of the primers and probes against known viral sequences and cultured viruses, which constitute the scientific basis for the assay. This information is assumed to be established for the predicate device.

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The CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel is intended for use in real-time RT-PCR (rRT-PCR) assays on an Applied Biosystems (ABI) 7500 Fast Dx Real-Time PCR Instrument in conjunction with clinical and epidemiological information:

• For qualitative detection of influenza virus type A or B from viral RNA in upper respiratory tract clinical specimens (including nasopharyngeal swabs [NPS], nasal swabs [NS], throat swabs [TS], nasal aspirates [NA], nasal washes [NW] and dual nasopharyngeal/throat swabs [NPS/TS], and lower respiratory tract specimens (including bronchoalveolar lavage [BAL], bronchial wash [BW], tracheal aspirate [TA], sputum, and lung tissue) from human patients with signs and symptoms of respiratory infection and/or from viral culture;
• For determination of the subtype of seasonal human influenza A virus as seasonal A/H1, A/H3, and/or A/H1pdm09 from viral RNA in upper respiratory tract clinical specimens (including NPS, NS, TS, NA, NW and NPS/TS) and lower respiratory tract specimens (including BAL, BW, TA, sputum and lung tissue) from human patients with signs and symptoms of respiratory infection and/or from viral culture;
• For the presumptive identification of virus in patients who may be infected with influenza A subtype A/H5 (Asian lineage) from viral RNA in human respiratory specimens and viral culture in conjunction with clinical and epidemiological risk factors;
• To provide epidemiologic information for surveillance of circulating influenza viruses.

The CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel is used in real-time RT-PCR (rRT-PCR) assays on the ABI 7500 Fast Dx Real-Time PCR Instrument. The panel consists of oligonucleotide primers and dual-labeled hydrolysis (TaqMan®) probes to be used in rRT-PCR for the in vitro qualitative detection and characterization of human influenza viruses from viral RNA in respiratory specimens from patients presenting with influenza-like illness (ILI).

Here's a breakdown of the acceptance criteria and study information for the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel, based on the provided text:

Acceptance Criteria and Device Performance

Study Information:

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

• Sample Size for Clinical Specimen Testing: 165 human respiratory specimens.
• Data Provenance: Retrospective. The specimens were "transferred from U.S. public health laboratories to the CDC for confirmatory testing." This indicates they were previously collected and tested. The country of origin is the U.S.

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 for the test set. It mentions that specimens were "transferred from U.S. public health laboratories to the CDC for confirmatory testing" and that "Results were confirmed through genetic sequence analysis." It also states "Performance characteristics for influenza were established during a season when seasonal influenza viruses A/H1 and A/H3 were the predominant influenza A viruses in circulation and during a season when the A/H1pdm09 influenza virus was the predominant influenza A virus in circulation." This implies the original diagnoses were clinical, but the ground truth for this specific study was molecular.

4. Adjudication method for the test set:

• The document implies that genetic sequence analysis served as the definitive "comparator" or gold standard. There is no mention of a traditional expert adjudication method (like 2+1 or 3+1 consensus) for establishing the ground truth of the clinical samples. The device's results were compared directly to the genetic sequencing (molecular) findings.

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. This is a diagnostic panel for molecular detection, not an AI-based imaging or interpretive device that would typically involve human "readers" or a MRMC study. The study focuses on the device's ability to detect specific viral markers.

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

• Yes, implicitly. The "Performance Summary" describes the analytical sensitivity (LOD, inclusivity) and clinical performance (positive percent agreement) of the "CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel" itself. It is evaluating the direct output of the RT-PCR assay against a ground truth, which constitutes a standalone performance evaluation of the diagnostic panel. The "human-in-the-loop" aspect comes in interpreting the results from InfA, pdmInfA, and H3 markers, but the performance metrics are for the assay's ability to produce those results.

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

• Molecular (Genetic Sequence Analysis). For the clinical specimen testing, the "comparator" used to establish the ground truth was "genetic sequence analysis." For analytical sensitivity and inclusivity, it was based on known concentrations of spiked viral isolates.

8. The sample size for the training set:

• Not specified. The document does not describe a training set for the device's development or a separate validation study using a distinct training set. The performance data presented are for validation/testing of the device itself.

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

• N/A (Not Applicable). As no training set is described, there's no information on how its ground truth would have been established. The device is a molecular diagnostic panel based on RT-PCR technology, not a machine learning algorithm that requires a labeled training set in the typical sense. Its development relies on designing primers and probes specific to target genetic sequences.

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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 Joint Biological Agent Identification and Diagnostic System (JBAIDS) Influenza A Subtyping Kit is intended for the in vitro qualitative detection and differentiation of seasonal Influenza A/H1, seasonal Influenza A/H3, and 2009 H1N1Influenza viral nucleic acids isolated and purified from nasopharyngeal swab (NPS) and nasopharyngeal wash (NPW) specimens from human patients with signs and symptoms of respiratory infection, in conjunction with clinical and epidemiological risk factors. The JBAIDS Influenza A Subtyping Kit contains reverse transcriptase real-time polymerase chain reaction (rRT-PCR) assays for use on the JBAIDS instruments. The Flu A H1. Flu A H3. and Flu A H1 2009 assays of the JBAIDS Influenza A Subtyping Kit target a region of the hemagglutinin (HA) gene of the respective Influenza A virus. The Flu A Sw assay of the JBAIDS Influenza A Subtyping Kit targets a region of the nucleocapsid protein (NP) gene of the 2009 H1N1 Influenza virus, as well as some other Influenza A viruses of swine lineage. This kit is not intended to detect Influenza B or Influenza C viruses.

A negative result for all assays in the JBAIDS Influenza A Subtyping Kit is a presumptive negative result for Influenza A. These results should be confirmed using the JBAIDS Influenza A & B Detection Kit.

Test results are to be used in conjunction with other clinical and epidemiological information. Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions.

Due to low seasonal prevalence, performance characteristics for detection of seasonal Influenza A/H1 were established primarily with retrospective and contrived clinical specimens.

All users, analysts, and any person reporting diagnostic results from use of this device should be trained to perform and interpret the results from this procedure by JBAIDS instructors or designees prior to use. Use of this device is limited to designated Department of Defense (DoD) laboratories equipped with the JBAIDS instruments.

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 departments for testing. Viral culture should not be attempted in these cases unless a biosafety laboratory (BSL) 3+ facility is available to receive and culture specimens.

The JBAIDS Influenza A Subtyping Kit is a real time RT-PCR test kit, which, when used with the JBAIDS instrument and software, allows the qualitative in vitro detection and identification of influenza A subtypes H1, H3, and H1 2009 (swine lineage) viral RNA. The assays have been optimized as freeze-dried assays with primer and fluorescent-probe sets for the detection of influenza A/H1, A/H3, and A/2009 H1 viral RNA. In particular, the Flu A H1, Flu A H3, and Flu A H1 2009 assays target distinct regions of the hemagglutinin gene specific to those subtypes, and the Flu A Sw assay targets a region of the nucleocapsid protein gene as a secondary target for the influenza A/2009 H1(swine lineage) virus. The tests are performed using the JBAIDS instrument and software.

Here's an analysis of the acceptance criteria and study details for the JBAIDS Influenza A Subtyping Kit based on the provided text:

Preamble Regarding Acceptance Criteria:
The document provided, a 510(k) Summary, details performance data but does not explicitly state pre-defined "acceptance criteria" in the format of pass/fail thresholds for the clinical study. Instead, it presents the achieved performance metrics (PPA and NPA with 95% Confidence Intervals) from the clinical and analytical studies, implicitly demonstrating that these achieved results were deemed sufficient for substantial equivalence. For the purpose of this response, I will present the reported performance as the "acceptance criteria met," recognizing that the specific numerical targets for acceptance are not explicitly listed in this summary.

1. Table of Acceptance Criteria and Reported Device Performance

As described above, explicit acceptance criteria (i.e., specific numerical targets prior to testing) were not provided in the 510(k) summary. However, the reported performance from the clinical studies served as the basis for the device's clearance. The table below summarizes the reported clinical performance.

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

• Clinical Performance (Prospective Study):

  • Sample Size: 795 valid specimens (312 NPS, 483 NPW) were analyzed. 6% (44/795) required retesting due to invalid/inconclusive/unsubtypeable initial results.
  • Data Provenance: Prospective, collected from 5 geographically separated military clinical sites in the U.S. during the 2010-2011 influenza season (December 2010 to April 2011).

• Testing of Preselected Archived Samples (for Seasonal Influenza A/H1):

  • Sample Size: 51 NPS specimens (30 known positive seasonal Influenza A/H1, 21 influenza-negative).
  • Data Provenance: Retrospective, pre-selected archived clinical NPS specimens obtained and confirmed at two different clinical study sites in the U.S.

• Testing of Surrogate Clinical Specimens (for Seasonal Influenza A/H1):

  • Sample Size: 136 individual influenza-negative clinical specimens (68 NPS, 68 NPW) were spiked with known concentrations of seasonal Influenza A/H1 virus. Valid results obtained for 128 (62 NPW, 66 NPS).
  • Data Provenance: Contrived clinical samples generated from residual influenza-negative NPS and NPW samples. Sent to two different clinical trial sites in the U.S.

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

The document does not explicitly state the number or qualifications of individual experts who established the ground truth. However, the ground truth for the clinical and archived samples was established using a reference method:

• "The reference method was the CDC rRT-PCT Flu Panel influenza A and influenza B assays, followed by subtype-specific PCR and bi-directional sequencing of amplicons."
• "the presence of Influenza A/H1 viral RNA was confirmed using 'validation' PCR assays. The validation PCR assays were identical to the comparator assays that were used for the prospective clinical evaluation study."

This implies that the "experts" were the personnel performing and interpreting the CDC assays and sequencing results, which would typically be highly trained laboratory professionals and molecular biologists proficient in these techniques, likely adhering to CDC protocols. No specific number of such experts is given.

4. Adjudication Method for the Test Set

The document states: "JBAIDS Software analyzes the fluorescence amplification curves and reports results as positive, negative, or uncertain. A failure of the Positive or Negative Control will result in the entire run being called invalid. Retesting is required to resolve uncertain or invalid results."

For the clinical study, 6% of specimens (44/795) required retesting. Specifically, "Invalid" (32/44), "Inconclusive" (1/44), and "Unsubtypeable" (1/44). "Forty (40) out of 44 samples resolved upon a 1st retest and the remaining 4 samples required a re-extraction and retest and resolved." This indicates a retesting and re-extraction protocol for initial indeterminate or invalid results rather than a multi-expert adjudication panel for interpretive discrepancies. The ground truth itself (CDC rRT-PCR + sequencing) would have its own internal validation/adjudication processes, but this is not detailed for external review.

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

No multi-reader, multi-case (MRMC) comparative effectiveness study comparing human readers with and without AI assistance was performed. This device is a diagnostic kit (real-time RT-PCR assay) that provides automated qualitative results, not an imaging AI diagnostic aid for human interpretation. Therefore, the concept of "human readers improve with AI vs without AI assistance" does not directly apply to this type of device.

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

Yes, the studies described (clinical performance, archived samples, surrogate samples) represent standalone performance of the algorithm/device. The "JBAIDS Software analyzes the fluorescence amplification curves and reports results as positive, negative, or uncertain." While human operators perform the physical tests, the interpretation of the raw PCR data into a final qualitative result (positive/negative/uncertain) is automated by the device's software (algorithm). The clinical performance data presented (PPA and NPA) are a direct measure of this standalone performance against a defined ground truth.

7. Type of Ground Truth Used

The ground truth used was:

• Expert Consensus/Reference Method: For prospective clinical specimens and archived samples, the reference method was the CDC rRT-PCR Flu Panel influenza A and influenza B assays, followed by subtype-specific PCR and bi-directional sequencing of amplicons. This is a highly robust and accepted laboratory method for viral identification and subtyping.
• Known Spiked Concentration: For surrogate clinical specimens, the ground truth for positive samples was established by spiking influenza-negative clinical samples with known concentrations of seasonal Influenza A/H1 virus. Un-spiked negative samples also served as ground truth negatives.

8. Sample Size for the Training Set

The document does not provide details on a specific "training set" sample size or data for the development of the device's algorithm, as would typically be described for machine learning or AI models. This device is a PCR-based assay, where the "algorithm" is primarily the pre-defined thresholds and analysis logic within the JBAIDS software to interpret PCR amplification curves. Performance characteristics are primarily established through analytical validation (LoD, inclusivity, exclusivity) and clinical validation with independent test sets, rather than an explicit training-validation split in the context of an adaptive or learning algorithm. The design of the primers and probes, and the associated software interpretation, would have been iteratively developed and optimized, but a formal "training set" as understood in AI/ML is not mentioned.

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

As noted above, a formal "training set" for an AI/ML algorithm is not described for this diagnostic kit. The underlying principles of the PCR assay (primer/probe design, reaction conditions) are established through extensive analytical studies to ensure specificity and sensitivity. The "ground truth" for developing the analytical performance characteristics (like LoD, exclusivity, inclusivity) would involve testing well-characterized viral strains and non-target organisms with known concentrations, typically obtained from reference collections. This process establishes the analytical sensitivity and specificity that the device is designed to achieve when interpreting a sample.

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The CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel is intended for use in real-time RT-PCR (rRT-PCR) assays on an Applied Biosystems (ABI) 7500 Fast Dx Real-Time PCR Instrument in conjunction with clinical and epidemiological information:

• For qualitative detection of influenza virus type A or B viral RNA in upper respiratory tract clinical specimens (including nasopharyngeal swabs [NPS], nasal swabs [NS], throat swabs [TS], nasal aspirates [NA], nasal washes [NW] and dual nasopharyngeal/throat swabs [NPS/TS]) and lower respiratory tract specimens (including bronchoalveolar lavage [BAL], bronchial wash [BW], tracheal aspirate [TA], sputum, and lung tissue) from human patients with signs and symptoms of respiratory infection and/or from viral culture.
• For determination of the subtype of seasonal human influenza A virus as seasonal A/H1, A/H3, and/or A/H1pdm09 from viral RNA in upper respiratory tract clinical specimens (including NPS, NS, TS, NA, NW and NPS/TS) and lower respiratory tract specimens (including BAL, BW, TA, sputum, and lung tissue) from human patients with signs and symptoms of respiratory infection and/or from viral culture.
• For the presumptive identification of virus in patients who may be infected with influenza A subtype A/H5 (Asian Lineage) from viral RNA in human respiratory specimens and viral culture in conjunction with clinical and epidemiological risk factors.
• To provide epidemiologic information for surveillance of circulating influenza viruses.

The CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel is used in real-time RT-PCR assays (rRT-PCR) on the ABI 7500 Fast Dx Real-Time PCR Instrument. The CDC Human Influenza Virus Real-time RT-PCR Diagnostic Panel is a panel of oligonucleotide primers and dual-labeled hydrolysis (TaqMan®) probes to be used in rRT-PCR for the in vitro qualitative detection and characterization of human influenza viruses from viral RNA in respiratory specimens from patients presenting with influenza-like illness (ILI). Detection of viral RNA not only aids in the diagnosis of illness caused by seasonal and novel influenza viruses in patients with ILI, but also provides epidemiological information on influenza and aids in the presumptive laboratory identification of specific novel influenza A viruses. The CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel is based on technology which is used in many molecular diagnostic assays. rRT-PCR assays are one-tube assays that first reverse-transcribe specific regions of RNA into cDNA copies. The cDNA then serves as a template for a polymerase chain reaction that utilizes a thermocyclic heating and cooling of the reaction to logarithmically amplify a specific region of DNA. The probe anneals to a specific internal target sequence located between the target loci of the forward and reverse primers. During the extension phase of the PCR cycle, the 5' nuclease activity of Tag polymerase degrades any probe molecules hybridized to amplified target sequence, causing the reporter dye to separate from the quencher dye, and generating a fluorescent signal. With each cycle, additional reporter dye molecules are cleaved from their respective probes, increasing the fluorescence intensity. Fluorescence intensity is monitored at each PCR cycle. Amplification of targets is reflected by logarithmic increase in fluorescence over time in comparison to background signal.

The provided text does not contain specific acceptance criteria with numerical thresholds that the device must meet, nor does it detail a dedicated pivotal study with a defined sample size and ground truth establishment methodology for this specific device (K111507) to demonstrate its performance against those criteria.

Instead, the submission for K111507 relies on the "substantial equivalence comparison" to two previously FDA-cleared predicate devices (K080570 and K101564). The performance data cited for K111507 directs the reader to the performance characteristics established for these predicate devices. The document also includes additional performance data collected during the 2010-2011 influenza season to show that recent circulating strains can be detected.

Therefore, the following information is extracted and presented based on the available text:

1. Table of Acceptance Criteria and Reported Device Performance

As specific, quantified acceptance criteria for K111507 are not explicitly stated in the provided text, a direct comparison table cannot be created in the traditional sense. The submission for K111507 primarily relies on the equivalency to predicate devices and additional data showing detection of recent circulating strains.

Reported Device Performance (for K111507, based on additional data for recent circulating strains):

2. Sample Size and Data Provenance for Test Set

• Sample Size for Test Set:
  • For detection of recent seasonal influenza A/H3 and B: 49 original specimens.
  • For detection in lower respiratory tract specimens: 18 original specimens.
• Data Provenance: Retrospective, from US public health laboratories (identified as "original specimens received from US public health laboratories").

3. Number of Experts and Qualifications for Ground Truth

The document does not specify the number or qualifications of experts used to establish the ground truth for the test set. Given the nature of a PCR diagnostic panel, the ground truth would typically be established through a reference method, often a combination of viral culture and/or additional molecular testing performed by qualified laboratory personnel, rather than expert interpretation of images or clinical assessments.

4. Adjudication Method for Test Set

The document does not describe any adjudication method (e.g., 2+1, 3+1) for establishing the ground truth of the test set.

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

• No MRMC study was done. This device is a real-time RT-PCR diagnostic panel, not an AI-assisted diagnostic tool for human readers. Therefore, the concept of human readers improving with AI vs. without AI assistance is not applicable.

6. Standalone Performance Study (Algorithm Only)

• Yes, a standalone study was implicitly done. The performance data presented (detection of influenza A/H3 and B in specimens) represents the algorithm's (the PCR panel's) performance independent of human-in-the-loop diagnostic interpretation beyond standard laboratory procedures. The detection is based on the fluorescent signal generated by the rRT-PCR assay.

7. Type of Ground Truth Used

• The ground truth type is implied to be a reference method for influenza detection and characterization, likely viral culture and/or a validated molecular method accepted as the standard for influenza diagnosis. The text states "Performance characteristics for influenza were established...as demonstrated by analytical testing of the 49 original specimens...". For the lower respiratory specimens, it states the panel "detected influenza A/H3, A/H1pdm09, and influenza B," implying comparison to an established truth.

8. Sample Size for Training Set

• The document does not explicitly mention a "training set" in the context of this diagnostic panel. As a PCR diagnostic kit, its development involves designing primers and probes that target specific viral genetic sequences. This process typically involves bioinformatics analysis and wet-lab analytical validation using known positive and negative controls, rather than a machine learning "training set" in the conventional sense. The "performance characteristics" data mentioned (from predicate devices and the additional 2010-2011 season data) serve as validation or evaluation data.

9. How Ground Truth for Training Set Was Established

• Not applicable, as a discrete "training set" with established ground truth in the context of machine learning is not described or relevant for this type of PCR diagnostic device. The design and analytical validation of PCR primers and probes rely on known viral sequences and synthetic or cultured viral samples with confirmed identities, not a "ground truth" derived from human experts interpreting a diagnostic outcome.

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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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The ProFAST™+ Assay is a multiplex Real Time RT-PCR in vitro diagnostic test for the qualitative detection and discrimination of seasonal Influenza A/H1, seasonal Influenza A/H3 and 2009 H1N1 Influenza viral nucleic acids isolated and purified from nasopharyngeal (NP) swab specimens from human patients with signs and symptoms of respiratory infection in conjunction with clinical and epidemiological risk factors. This assay targets conserved regions of the Hemagglutinin (HA) gene for seasonal Influenza A/H1, seasonal Influenza A/H3 and 2009 H1N1 Influenza Virus, respectively. This assay is not intended to detect Influenza B or Influenza C Viruses.

A negative ProFAST+ Assay result is a presumptive negative result for Influenza A. These results should be confirmed by an FDA cleared nucleic acid-based test (NAT) detecting Influenza A.

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

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 ProFAST+ Assay enables detection and discrimination of Influenza A Virus subtypes: seasonal H1, seasonal H3, and 2009 H1N1.

An overview of the procedure is as follows:

1. Collect nasopharyngeal swab specimens from symptomatic patients using a polyester, ravon, or nylon tipped swab and place into viral transport medium (refer to Materials Required but not Provided section of this Instruction for Use).
2. Add an Internal Control (IC) to every sample to monitor for inhibitors present in the specimens.
3. Perform isolation and purification of nucleic acids using a MagNA Pure LC Instrument (Roche) and the MagNA Pure Total Nucleic Acid Isolation Kit (Roche) or a NucliSENS easyMAG System (bioMérieux) and the Automated Magnetic Extraction Reagents (bioMérieux).
4. Add purified nucleic acids to the ProFAST+ Supermix along with enzymes included in the ProFAST+ kit. The ProFAST+ Supermix contains target-specific oligonucleotide primers and probes. The primers are complementary to conserved regions of the Hemagglutinin (HA) gene for seasonal influenza A/H1, seasonal influenza A/H3 and 2009 H1N1 Influenza Virus (swine-origin), respectively. The probes are dual-labeled with a reporter dye and a quencher dye (see table below).
5. Perform reverse transcription of RNA into complementary DNA (cDNA) and subsequent amplification of DNA in a Cepheid SmartCycler II instrument. In this process, the probe anneals specifically to the template followed by primer extension and amplification. The ProFAST+ Assay is based on Taqman chemistry, which utilizes the 5' - 3' exonuclease activity of the Taq polymerase to cleave the probe thus separating the reporter dye from the quencher. This generates an increase in fluorescent signal upon excitation from a light source. With each cycle, additional reporter dye molecules are cleaved from their respective probes, further increasing fluorescent signal. The amount of fluorescence at any given cycle is dependent on the amount of amplification products present at that time. Fluorescent intensity is monitored during each PCR cycle by the real-time instrument.

Here's a summary of the acceptance criteria and study details for the ProFAST+ Assay based on the provided document:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are implied by the "Positive Percent Agreement" and "Negative Percent Agreement" values, along with their 95% Confidence Intervals. For the reproducibility study, the acceptance criterion is an "overall percent agreement with the expected result" of 99.7%.

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

• Prospective Study:
  • Sample Size: 842 eligible prospective NP swab specimens (initially 874, but 32 were excluded).
  • Data Provenance: U.S. clinical laboratories (4 sites), collected between December 2009 and May 2010. Also included prospectively collected archived samples (from January-March, 2008, February-March, 2009, and October-November, 2009) due to absence of seasonal influenza during the 2009-2010 winter season. These represented "excess, remnants of nasopharyngeal (NP) swab specimens that were prospectively collected from symptomatic individuals suspected of respiratory infection, and were submitted for routine care or analysis by each site, and that otherwise would have been discarded."
• Retrospective Study:
  • Sample Size: 160 retrospective nasopharyngeal (NP) swab samples.
  • Data Provenance: Two clinical sites, collected from January-March, 2008, January-November 2009, and March 2010.

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

The document does not specify the number or qualifications of experts involved in establishing the ground truth. The ground truth was established by a "composite comparator/reference method."

4. Adjudication Method for the Test Set

The document does not describe an explicit adjudication method between human experts, as the ground truth was established by a composite of laboratory tests rather than expert consensus on interpretation. It mentions that samples were "considered as any sample that was tested positive for Influenza A by the ProFlu+ Assay, and had bidirectional sequencing data meeting pre-defined quality acceptance criteria."

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 performed. This is a diagnostic test (RT-PCR assay) intended for standalone use, not an imaging device requiring human reader interpretation, so this type of study would not be applicable.

6. If a Standalone Study Was Done

Yes, a standalone study (algorithm/device-only performance without human-in-the-loop performance) was done. The ProFAST+ Assay's performance was compared directly against a composite reference method. The clinical performance data (Positive and Negative Percent Agreements) reflect the standalone performance of the device.

7. The Type of Ground Truth Used

The ground truth used was a composite comparator/reference method consisting of:

• FDA cleared ProFlu+ Assay (another nucleic acid-based test for Influenza A).
• Individual well-characterized Influenza A subtype-specific RT-PCR assays.
• Bi-directional sequencing (targeting different regions of the hemagglutinin gene than the ProFAST+ Assay).

"True" positives required:

• Positive for Influenza A by ProFlu+ Assay
• And bidirectional sequencing data meeting pre-defined quality acceptance criteria, matching seasonal A/H1, A/H3, or A/2009 H1N1 sequences in the NCBI GenBank database with acceptable E-values.

"True" negatives required:

• Negative for Influenza A by ProFlu+ Assay
• OR positive for Influenza A by ProFlu+ Assay but negative by the respective Influenza A subtype-specific RT-PCR assay followed by bi-directional sequencing.

8. The Sample Size for the Training Set

The document does not explicitly state the sample size used for a training set. The clinical performance studies describe the evaluation of the device using prospective and retrospective samples, implying these are test sets rather than training sets. Typically for an in vitro diagnostic (IVD) assay like this, development and optimization (which might use internal "training-like" datasets) are usually completed before the formal clinical validation described in the 510(k) summary.

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

Since a distinct "training set" is not explicitly mentioned as part of this premarket notification validation, the method for establishing its ground truth is not detailed. For typical IVD development, internal positive and negative controls, spiked samples, and characterized clinical samples would be used during development and optimization phases.

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The CDC Influenza 2009 A(H1N1)pdm Real-Time RT-PCR Panel (CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel) is intended for use in real-time RT-PCR assays on an Applied Biosystems (ABI) 7500 Fast Dx Real-Time PCR Instrument in conjunction with clinical and epidemiological information:

• For the qualitative detection of influenza virus type A viral RNA from nasopharyngeal swabs (NPS), nasal swabs (NS), nasal aspirates (NA), nasal washes (NW), dual nasopharyngeal / throat swabs (NPS/TS), broncheoalveolar lavage (BAL), tracheal aspirate (TA), and bronchial wash (BW), collected from the respiratory tract of human patients with signs and symptoms of respiratory infection and/or from viral culture.
• For differentiation of 2009 H1N1 influenza virus RNA from nasopharyngeal swabs (NPS), nasal swabs (NS), nasal aspirates (NA), nasal washes (NW) dual nasopharyngeal / throat swabs (NPS/TS), broncheoalveolar lavage (BAL), tracheal aspirate (TA), and bronchial wash (BW) collected from the respiratory tract of human patients with signs and symptoms of respiratory infection and/or from viral culture.
• To provide epidemiologic information for surveillance of the 2009 H1N1 influenza virus.

The CDC Influenza 2009 A(H1N1)pdm Real-time RT-PCR Panel (CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel) is a panel of oligonucleotide primers and dual-labeled hydrolysis (TaqMan®) probes which may be used in real-time RT-PCR (rRT-PCR) assays for the in vitro qualitative detection and characterization of human influenza viruses (RNA) in respiratory specimens from patients presenting with influenza-like illness (ILI). Detection of viral RNA not only aids in the diagnosis of illness caused by seasonal and novel influenza viruses in patients with ILI, but also provides epidemiologic information on influenza and aids in the presumptive laboratory identification of specific novel influenza A viruses. The influenza A primer and probe sets are designed for universal detection of type A influenza viruses, Influenza A subtyping primer and probe sets are designed to specifically detect 2009 A (H1N1) influenza virus. The CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel is an updated version of the assay distributed by CDC. during the 2009 A(H1N1) pandemic strain to qualified laboratories (CDC rRT-PCR Swine Flu Panel G090072) under Emergency Use Authorization (EUA) from the FDA in that it has different sequences of primers and probes that are more specific to the currently circulating 2009 A(H1N1) influenza virus. The CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel includes the following primer and probe sets and positive control: InfA detects all influenza A strains, but does not detect influenza B strains. pdm InfA is specific for 2009 influenza A. pdm H1 is specific for 2009 influenza A, subtype H1. RNase P (RP) detects human RNase P and is used with human clinical specimens to indicate that adequate isolation of nucleic acid resulted from the extraction of the clinical specimen. Inactivated Influenza Typing Panel Real-Time RT-PCR Positive Control is a positive control designed to react with all the primer and probe sets including RNase P.

Device Acceptance Criteria and Study Details: CDC Influenza 2009 A(H1N1)pdm Real-Time RT-PCR Panel

1. Table of Acceptance Criteria and Reported Device Performance

• A/New York/18/2009: InfA (10^1.3), pdm InfA (10^0.6), pdm H1 (10^1.3). Final LoD = 10^1.3 (EID50/mL). |
  | Inclusivity | Concordance with diverse 2009 A(H1N1) influenza viruses | Not explicitly stated as a numerical threshold, implied to be high for specific detection. | 100% concordance with all primer and probe sets when testing ten diverse 2009 A(H1N1) influenza viruses at low concentrations. |
  | Exclusivity (Seasonal) | Non-detection of seasonal influenza viruses by pdm InfA and pdm H1 | Complete non-reactivity (negative results) of pdm InfA and pdm H1 with seasonal Inf A and Inf B strains, while Inf A should detect seasonal A. | - Seasonal H1N1: InfA detected all 16 tested strains; pdm InfA and pdm H1 were negative for all.
• Seasonal H3N2: InfA detected all 7 tested strains; pdm InfA and pdm H1 were negative for all.
• Influenza B: InfA, pdm InfA, and pdm H1 were negative for all 12 tested strains. |
  | Exclusivity (H5N1) | Non-detection of H5N1 by pdm InfA and pdm H1, while InfA should detect H5N1 | Complete non-reactivity (negative results) of pdm InfA and pdm H1 with H5N1 strains, while Inf A should detect H5N1. | InfA detected all 10 tested H5N1 strains; pdm InfA and pdm H1 were negative for all, except for one H5N1 strain (A/Chicken/Vietnam/NCVD-016/2008) where pdm H1 result was not provided. |
  | Non-Influenza Pathogens | Non-cross reactivity with common respiratory pathogens and flora | 100% agreement with expected negative results. | 100% concordance with expected results for all primer and probe sets (InfA, pdm InfA, pdm H1) when tested against 34 non-influenza organisms (commensal bacteria, yeast, and non-influenza respiratory viruses). This means all markers were negative. |
  | Reproducibility | Agreement with expected results and %CV for Ct values across sites and operators | Demonstrated reproducibility across six sites, two operators, and five days. Percentage of agreement for moderate, low, "high negative", and negative samples. Low %CV for average Ct values, particularly for moderate and low viral RNA concentrations. | - No Template Control, RP, HSC, Positive Control: 100% agreement (60/60) across all sites.
• Sample 1 (moderate viral RNA): 100% agreement (60/60) for InfA, pdm InfA, pdm H1, and RP. Low %CV for all markers (e.g., InfA 2.59-4.23%, pdm InfA 2.10-3.10%, pdm H1 1.48-3.17%, RP 1.33-2.65%).
• Sample 2 (low viral RNA): High agreement (54/60 to 60/60 total). Some sites showed lower agreement (e.g., 7/10 for pdm H1 at Roche MagNA Pure LC and Compact NA). %CV generally higher than moderate samples (e.g., InfA 1.04-35.61%, pdm InfA 2.48-35.27%, pdm H1 1.27-52.94%, RP 1.34-5.11%).
• Sample 3 ("high negative"): Agreement ranged from 54/60 to 58/60, with some individual site variations. All samples were expected to be negative for InfA, pdm InfA, and pdm H1. |
  | Clinical Performance | | | |
  | Clinical Sensitivity | Greater than a specified percentage | >96% for all markers (upper respiratory specimens).

83% for all markers (lower respiratory specimens). | - Upper Respiratory (NPS/NS): InfA (96.8%), 2009 H1N1 (100%).

• Upper Respiratory (NA/NW): InfA (100%), 2009 H1N1 (100%).
• Upper Respiratory (NPS/TS): InfA (100%), 2009 H1N1 (100%).
• Lower Respiratory (BAL, TA, BW): InfA (83.3%), 2009 H1N1 (100%). Results meet or exceed criteria. |
  | Clinical Specificity | Greater than a specified percentage | >96% for all markers (upper respiratory specimens).

83% for all markers (lower respiratory specimens). | - Upper Respiratory (NPS/NS): InfA (96.2%), 2009 H1N1 (96.7%).

• Upper Respiratory (NA/NW): InfA (99.3%), 2009 H1N1 (98.6%).
• Upper Respiratory (NPS/TS): InfA (100%), 2009 H1N1 (98.3%).
• Lower Respiratory (BAL, TA, BW): InfA (83.3%), 2009 H1N1 (84.0%). Results meet or exceed criteria. |
  | Positive Percent Agreement | Greater than a specified percentage | >96% for all markers (upper respiratory specimens).
  100% for all markers (lower respiratory specimens). | - Upper Respiratory (NPS/NS): InfA (99.4%), 2009 H1N1 (99.7%).
• Upper Respiratory (NA/NW): InfA (97.7%), 2009 H1N1 (99.0%).
• Upper Respiratory (NPS/TS): InfA (100%), 2009 H1N1 (100%).
• Lower Respiratory (BAL, TA, BW): InfA (100%), 2009 H1N1 (100%). Results meet or exceed criteria. |
  | Overall Performance Call Rate | (Clinical Evaluation) | >99% | >99% |

2. Sample Size and Data Provenance (Clinical Performance Test Set)

• Sample Size for Test Set: 1901 total patient specimens.
  • 1191 nasopharyngeal swabs (NPS) and nasal swabs (NS)
  • 50 throat swabs (TS)
  • 519 nasal washes (NW) and nasal aspirates (NA)
  • 99 dual nasopharyngeal / throat swabs (NPS/TS)
  • 42 lower respiratory specimens (broncheoalveolar lavage (BAL), tracheal aspirate (TA), and bronchial wash (BW))
• Data Provenance: Prospective study, supplemented with retrospective specimens. Data collected during the 2009-2010 respiratory virus season (February-April) from 8 U.S. public health laboratories and a Department of Defense (DoD) laboratory. Samples were from individuals symptomatic with influenza-like illness (ILI). Retrospective specimens were used to supplement due to low prevalence of influenza.

3. Number of Experts and Qualifications for Test Set Ground Truth

The document does not explicitly state the "number of experts" used to establish the ground truth in the same way one would delineate expert radiologists for image annotation.

Instead, the ground truth for the clinical performance study was established by standard laboratory methods:

• For InfA (Influenza A detection): Virus culture with Immunofluorescent Antibody (IFA) or Direct Fluorescent Antibody (DFA) was used for screening and identification of influenza type.
• For 2009 H1N1 (subtype detection): Bi-directional sequencing was used for confirmation of 2009 influenza A (H1N1) subtype. (Sequencing was performed only on specimens already identified as positive for influenza A by virus culture).

These methods represent established diagnostic procedures typically performed by trained laboratory personnel, rather than interpretation by "experts" in the context of a consensus reading.

4. Adjudication Method for the Test Set

The document does not describe an explicit "adjudication method" in the context of multiple human readers reconciling differences. The ground truth was established by laboratory methods as described above. If there were discrepancies between initial screening (IFA/DFA) and confirmatory sequencing, the sequencing result would have been the definitive ground truth for subtyping.

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

No, a multi-reader multi-case (MRMC) comparative effectiveness study was not performed. The study evaluates the standalone performance of the RT-PCR panel against laboratory reference methods, not improvement in human reader performance with AI assistance.

6. Standalone (Algorithm Only) Performance Study

Yes, a standalone performance study was done. The entire document focuses on evaluating the analytical and clinical performance of the CDC Influenza 2009 A(H1N1)pdm Real-Time RT-PCR Panel (an algorithm/assay) independent of human interpretation or assistance, against established laboratory reference methods. The results presented for sensitivity, specificity, and agreement are for the device's performance directly.

7. Type of Ground Truth Used

• Clinical Performance Ground Truth:
  • Virus Culture (with IFA/DFA): For the detection of influenza type A (against which the InfA analyte of the device was compared).
  • Bi-directional Sequencing: For the confirmation of 2009 influenza A (H1N1) subtype (against which the pdm InfA and pdm H1 analytes of the device were compared).
• Analytical Performance Ground Truth:
  • Known viral concentrations (EID50/mL, TCID50/mL): For Limit of Detection, Inclusivity, and Exclusivity studies.
  • Known identity of specific influenza and non-influenza organisms: For Inclusivity, Exclusivity, and Reactivity of Non-Influenza Respiratory Viral and Bacterial Pathogens studies.

8. Sample Size for the Training Set

The document does not provide information about a "training set" or its sample size. This type of device (RT-PCR panel) is a diagnostic kit where the primers and probes are designed based on known viral sequences, rather than an AI/ML algorithm that is "trained" on a dataset in the conventional sense. The "development" and "optimization" of such a panel would involve bioinformatic design and iterative testing, not "training" on a sample set like an AI model.

9. How the Ground Truth for the Training Set (if applicable) was Established

As mentioned above, the concept of a "training set" and "ground truth establishment for a training set" as it applies to AI/ML devices is not applicable here. The CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel is a molecular diagnostic assay where the "design" is based on established scientific principles of molecular biology and virology, including viral genome sequencing and primer/probe design. The specificity and sensitivity are intrinsically built into the primer and probe sequences themselves, which are designed to target specific genetic regions of the influenza virus.

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The Focus Diagnostics Simplexa™ Influenza A H1N1 (2009) assay is intended for use on the 3M Integrated Cycler as part of the Microfluidic Molecular System for the in vitro qualitative detection and differentiation of influenza A and 2009 H1N1 influenza viral RNA in nasopharyngeal swabs (NPS), nasal swabs (NS), and nasopharyngeal aspirates (NPA) from human patients with signs and symptoms of respiratory infection in conjunction with clinical and epidemiological risk factors.

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.

Simplexa™ Influenza A H1N1 (2009) test kit is a nucleic acid amplification test that uses real-time reverse transcriptase polymerase chain reaction (RT-PCR) amplification to enable simultaneous and distinct detection of influenza A and 2009 H1N1 influenza in a single reaction from nasophayngeal swabs (NPS), nasal swabs (NS), and nasopharyngeal aspirates (NPA). The assay combines real-time PCR amplification with fluorescent signal detection technology. A bi-functional fluorescent probe-primer is used together with a reverse primer to amplify a specific target (for each analyte and internal control). A fluorescent signal is generated after the separation of the fluorophore from the quencher as a result of the binding of a probe element to the extended RNA fragment synthesized during amplification.

The 3M Integrated Cycler is a rapid real-time Polymerase Chain Reaction thermocycler used for the identification of nucleic acid from prepared biological samples. The instrument utilizes disk media to contain and to process samples. The instrument uses real time flourometric detection to identify targets within the sample wells. The instrument is controlled by an external computer running the Integrated Cycler Studio Software. Together, the instrument, software and test kit are referred to as the "Microfluidic Molecular System."

Here's a breakdown of the acceptance criteria and study details for the Simplexa™ Influenza A H1N1 (2009) assay based on the provided document:

1. Table of Acceptance Criteria and Reported Device Performance

The document doesn't explicitly state "acceptance criteria" in a separate table with target values for clinical performance as commonly seen for medical devices. However, the reported performance metrics (Positive Percent Agreement - PPA, Negative Percent Agreement - NPA) from the clinical agreement studies serve as the de-facto acceptance criteria demonstrated by the device.

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

• Prospective Data:

  • Nasal/Nasopharyngeal Swabs: 299 specimens initially collected. After exclusions due to lack of consensus among reference assays (10 for Influenza A, 9 additional for H1N1 subtype sequencing), the sample sizes used for agreement calculations are:
    • H1N1 Detected: 101
    • H1N1 Not Detected: 179
    • Influenza A Detected: 116
    • Influenza A Not Detected: 173
  • Nasopharyngeal Aspirates: 112 specimens initially collected. After exclusions due to lack of consensus among reference assays (5 for Influenza A, 3 additional for H1N1 subtype sequencing), the sample sizes used for agreement calculations are:
    • H1N1 Detected: 24
    • H1N1 Not Detected: 80
    • Influenza A Detected: 31
    • Influenza A Not Detected: 76
  • Provenance: Collected from patients with signs and symptoms of influenza-like illness at three sites: Austin, TX (September 2009) and the New South Wales region of Australia (July - September 2009). The collected specimens were blinded and randomly distributed to 3 U.S. clinical laboratories for testing.

• Retrospective Data:

  • Nasal/Nasopharyngeal Swabs: 214 specimens initially collected from the Focus Sample Bank. After exclusions due to lack of consensus among reference assays (3 for Influenza A, 1 additional for H1N1 subtype sequencing), the sample sizes used for agreement calculations are:
    • H1N1 Detected: 57
    • H1N1 Not Detected: 153 (including 1 indeterminate)
    • Influenza A Detected: 132 (including 1 indeterminate)
    • Influenza A Not Detected: 79
  • Nasal Washes: 2 specimens (both positive for 2009 H1N1 influenza and Influenza A by both methods).
  • Provenance: Focus Sample Bank (implying archival samples). No specific country of origin is mentioned beyond "Focus Sample Bank."

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

The document does not specify the number of experts or their qualifications for establishing the ground truth. It refers to a "composite reference method" which includes multiple validated assays, but not human expert review for establishing ground truth.

4. Adjudication Method for the Test Set

The ground truth was established using a composite reference method which included:

• Luminex xTAG RVP Flu A target
• A validated PCR assay using primer and probe sequences published by the CDC
• A well-characterized PCR followed by sequencing.

Specimens were excluded from analysis if there was "no consensus among the reference assays" for the influenza A result or if sequencing data was not available for subtyping. This implies a form of consensus/adjudication among the reference methods, but not by human experts in the traditional sense as a separate step.

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 performed. This study evaluates the Simplexa™ Influenza A H1N1 (2009) assay in a standalone capacity against a composite reference (ground truth), not in comparison to or in assistance with human readers. Therefore, there is no effect size reported for human readers improving with AI vs without AI assistance.

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

Yes, this was a standalone performance study. The Simplexa™ Influenza A H1N1 (2009) assay's performance was evaluated by directly comparing its results to the established composite reference ground truth. There is no mention of human-in-the-loop involvement in the performance evaluation.

7. The Type of Ground Truth Used

The ground truth used was a composite reference method comprised of:

• Luminex xTAG RVP Flu A target
• A validated PCR assay using primer and probe sequences published by the CDC
• A well-characterized PCR followed by sequencing.

8. The Sample Size for the Training Set

The document does not explicitly mention a separate "training set" or its sample size for the development of the Simplexa™ Influenza A H1N1 (2009) assay. The provided studies focus solely on the performance of the developed assay, typically referring to validation or test sets.

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

Since a "training set" is not explicitly discussed, the method for establishing its ground truth is also not specified. It can be inferred that similar validated molecular methods would have been used during the assay's development phase.

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