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The Quidel Molecular RSV + hMPV Assay is a multiplex Real-Time PCR (RT-PCR) assay for the qualitative detection and identification of respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) ribonucleic acid (RNA) extracted from nasal and nasopharyngeal swab specimens from patients with signs and symptoms of respiratory infection. This in vitro diagnostic test is intended to aid in the differential diagnosis of RSV and hMPV infections in humans in conjunction with clinical and epidemiological risk factors. This test is not intended to differentiate the two subtypes of RSV or the four genetic sub-lineages of hMPV.

Negative results do not preclude RSV infection and/or hMPV infection and should not be used as the sole basis for diagnosis, treatment or other patient management decisions.

Conversely, positive results do not rule-out bacterial infection or co-infection with other viruses. The agent detected may not be the definite cause of disease. The use of additional laboratory testing and clinical presentation must be considered in order to obtain the final diagnosis of respiratory viral infection.

The Quidel Molecular RSV + hMPV Assay can be performed using either the Life Technologies QuantStudio™ Dx RT-PCR Instrument, the Applied Biosystems® 7500 Fast Dx RT-PCR Instrument, or the Cepheid SmartCycler® II System.

The Quidel Molecular RSV + hMPV Assay detects viral nucleic acids that have been extracted from a patient sample using the bioMérieux NucliSENS easyMAG automated extraction platform. A multiplex RT-PCR reaction is carried out under optimized conditions in a single tube generating amplicons for each of the target viruses present in the sample. This reaction is performed utilizing the Cepheid SmartCycler II, the Applied Biosystems 7500 Fast Dx. or the Life Technologies QuantStudio Dx. Identification of RSV, hMPV, and the process control (PRC) occurs by the use of target-specific primers and fluorescent-labeled probes that hybridize to conserved regions in the genomes of RSV and hMPV and the PRC.

Acceptance Criteria and Study for Quidel Molecular RSV + hMPV Assay

1. Table of Acceptance Criteria and Reported Device Performance

The provided document does not explicitly state pre-defined acceptance criteria (e.g., minimum PPA/NPA percentages or specific LoD values that must be met for approval). However, it implicitly demonstrates acceptable performance by comparing the device to FDA-cleared predicate devices and presenting the results of analytical and clinical studies. We can infer the "reported device performance" from the "Combined Clinical Site Data" table.

Detailed Reproducibility Results (RSV):

Detailed Reproducibility Results (hMPV):

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

• Test Set Sample Size:
  • RSV: 700 nasal or nasopharyngeal swab specimens (after removing 13 invalid specimens from an initial 713).
  • hMPV: 707 nasal or nasopharyngeal swab specimens (after removing 6 invalid specimens from an initial 713).
• Data Provenance: Prospective study conducted during the 2013 respiratory virus season (January to March 2013) at three sites across the United States.

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

The ground truth for the clinical performance study was established by two FDA-cleared RT-PCR assays (Prodesse ProFlu+ and Pro hMPV+), rather than human experts. Thus, information about the number and qualifications of experts is not applicable to this study design.

4. Adjudication Method for the Test Set

The ground truth was established by two FDA-cleared predicate RT-PCR assays. The document does not describe an explicit adjudication method between these predicate devices or between the predicate devices and an independent reference standard. For each virus (RSV and hMPV) separately, the results of the Quidel Molecular assay were compared directly against the respective predicate device's results.

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

No, an MRMC comparative effectiveness study was not done. This study compares the performance of a molecular diagnostic assay against other molecular diagnostic assays, not the performance of human readers with and without AI assistance.

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

Yes, the study describes the "standalone" performance of the Quidel Molecular RSV + hMPV Assay. It is a molecular diagnostic test that produces a qualitative result (positive/negative) based on the detection of viral nucleic acids through RT-PCR, without human interpretation of complex images or data that would typically involve a "human-in-the-loop" decision process. The output is directly generated by the instrument based on the fluorescent signal.

7. The Type of Ground Truth Used

The ground truth for the clinical performance study was established using comparison to FDA-cleared RT-PCR predicate devices. Specifically:

• For RSV, the predicate device was Prodesse ProFlu+.
• For hMPV, the predicate device was Prodesse Pro hMPV+.

8. The Sample Size for the Training Set

The document does not explicitly mention a "training set" for the purpose of algorithm development or machine learning in the conventional sense. This is a molecular diagnostic assay where primers and probes are designed to target specific viral genes. The "development" of the assay involves optimizing reaction conditions, not training a machine learning model. Therefore, providing a sample size for a training set in this context is not applicable.

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

As noted above, the concept of a "training set" for this type of molecular diagnostic assay is not directly applicable. The "ground truth" for developing the analytical performance characteristics (like LoD, inclusivity, specificity) would have been established through controlled laboratory experiments using known quantities and strains of viruses, and known negative samples. These are standard methods in the development of PCR-based diagnostics.

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The Quidel Molecular Influenza A+B Assay is a multiplex Real Time RT-PCR assay for the in vitro qualitative detection and differentiation of influenza A and influenza B viral RNA in nasal and nasopharyngeal swabs from patients with signs and symptoms of respiratory infection. This test is intended for use as an aid in the differential diagnosis of influenza A and influenza B viral infections in humans in conjunction with clinical and epidemiological risk factors. The assay does not detect the presence of influenza C virus.

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

Performance characteristics for influenza A were established during the 2011 and 2013 influenza seasons when influenza A/H3 and 2009 H1N1 influenza were the predominant influenza A viruses 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 assay can be performed using either the Life Technologies QuantStudio™ Dx, the Applied Biosystems® 7500 Fast Dx, or the Cepheid SmartCycler® II.

The Quidel Molecular Influenza A+B Assay detects viral nucleic acids that have been extracted from a patient sample using the NucliSENS® easyMAG® automated extraction platform. A multiplex real-time RT-PCR reaction is carried out under optimized conditions in a single tube generating amplicons for each of the target viruses present in the sample. This reaction is performed utilizing the Life Technologies QuantStudio™ Dx, the Applied Biosystems® 7500 Fast Dx, or the Cepheid SmartCycler® II platform. Identification of influenza A occurs by the use of target specific primers and a fluorescentlabeled probe that hybridizes to a conserved influenza A sequence within the matrix protein gene. Identification of influenza B occurs by the use of target specific primers and fluorescent-fabeled probes that will hybridize to a conserved influenza B sequence within the neuraminidase gene.

The Quidel Molecular Influenza A + B Assay is a multiplex Real Time RT-PCR assay for the in vitro qualitative detection and differentiation of influenza A and influenza B viral RNA in nasal and nasopharyngeal swabs from patients with signs and symptoms of respiratory infection. This device is intended to be used as an aid in the differential diagnosis of influenza A and influenza B viral infections in humans in conjunction with clinical and epidemiological risk factors.

Here's an analysis of the acceptance criteria and study as presented in the document:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are implied by the clinical performance study aiming to demonstrate substantial equivalence to an FDA-cleared RT-PCR influenza detection device. The agreement metrics (Positive Percent Agreement, Negative Percent Agreement) are the key performance indicators. The document does not explicitly state pre-defined acceptance thresholds (e.g., "PPA must be > 90%"). However, the reported performance is presented in comparison to a predicate device.

2. Sample Size and Data Provenance for the Test Set

• Sample Size:
  • Initial specimens: 631 fresh swab specimens
  • Specimens remaining after exclusions for invalid results: 619
• Data Provenance: Prospective study conducted during the 2013 respiratory virus season (January to March 2013) at three sites across the United States.

3. Number of Experts and Qualifications for Ground Truth

The document does not explicitly state that experts were used to establish the ground truth for the clinical test set. Instead, a "high performance FDA-cleared Influenza A and B molecular test" was used as the comparator (reference standard) to determine agreement.

4. Adjudication Method for the Test Set

The document does not describe an adjudication method for reconciling discordant results between the subject device and the comparator device. It simply reports the raw agreement percentages and notes the number of discordant dual infections.

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

No MRMC comparative effectiveness study was done. This study focuses on a standalone diagnostic device performance.

6. Standalone Performance

Yes, a standalone performance study was conducted. The "Clinical Performance" section evaluates the Quidel Molecular Influenza A + B Assay's performance (algorithm only, as it's an in vitro diagnostic test) against an FDA-cleared comparator device without human intervention in the result interpretation from the device itself.

7. Type of Ground Truth Used

The ground truth for the clinical test set was established by a "high performance FDA-cleared Influenza A and B molecular test" (a comparator device). This is a type of reference standard comparison, where the performance of the new device is measured against an established, cleared diagnostic method.

8. Sample Size for the Training Set

The document does not specify a separate training set or its sample size for the clinical performance evaluation. The clinical study described appears to be a validation/test set. For analytical performance (e.g., Limit of Detection, Analytical reactivity), quantified cultures of various influenza strains were used, but these are for analytical validation, not for training a machine learning model. This is a molecular diagnostic assay, not an AI/ML-based device in the sense of requiring a "training set" of patient data for model development.

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

As noted above, the concept of a "training set" for an AI/ML model with associated ground truth from patient data is not applicable here because this is a molecular diagnostic assay. For the analytical studies (e.g., Limit of Detection, Inclusivity), the "ground truth" was established by using quantified cultures of known influenza A and B strains at specified concentrations (TCID50/mL). These cultures serve as the known positive and negative controls at defined viral loads.

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The Sofia RSV FIA employs immunofluorescence for detection of respiratory syncytial virus (RSV) nucleoprotein antigen in nasopharyngeal swab and nasopharyngeal aspirate/wash specimens taken directly from symptomatic patients. This qualitative test is intended for use as an aid in the rapid diagnosis of acute RSV infections in pediatric patients less than 19 years of age. Negative results do not preclude RSV infection and should not be used as the sole basis for treatment or for other management decisions. A negative result is presumptive, and it is recommended these results be confirmed by virus culture or an FDA-cleared RSV molecular assay.

The Sofia RSV FIA test employs immunofluorescence technology that is used with the Sofia Analyzer for the rapid detection of RSV antigens. The Sofia RSV FIA test involves the disruption of RSV viral antigens. The patient specimen is placed in the Reagent Tube, during which time the virus particles in the specimen are disrupted, exposing internal viral nucleoproteins. After disruption, the specimen is dispensed into the Cassette sample well. From the sample well. the specimen migrates through a test strip containing various unique chemical environments. If RSV viral antigens are present, they will be trapped in a specific location.

Note: Depending upon the user's choice, the cassette is either placed inside of the Sofia Analyzer for automatically timed development (Walk Away Mode) or placed on the counter or bench top for a manually timed development and then placed into the Sofia Analyzer to be scanned (Read Now Mode).

The Sofia Analyzer will scan the test strip and measure the fluorescent signal by processing the results using method-specific algorithms. The Sofia Analyzer will display the test results (Positive, Negative, or Invalid) on the screen. The results can also be automatically printed on an integrated printer if this option is selected.

The provided text describes the 510(k) summary for the Sofia® RSV FIA device, but it does not contain the specific acceptance criteria or the detailed results of the multi-center field clinical study that would allow for a complete table of acceptance criteria and reported device performance. The summary mentions that "Sensitivity and specificity were calculated" but does not provide the actual values or the criteria they were measured against.

Therefore, I cannot fulfill Request 1 directly from the provided text. I will answer the other requests based on the available information.

Here's a breakdown of the information that can be extracted and a note on what is missing:

1. A table of acceptance criteria and the reported device performance

MISSING: The document states that "Sensitivity and specificity were calculated" in the multi-center field clinical study, but it does not report the actual sensitivity and specificity values, nor does it specify any pre-defined acceptance criteria that these values were compared against. Without these numbers, a table cannot be created.

2. Sample size used for the test set and the data provenance (e.g. country of origin of the data, retrospective or prospective)

• Sample Size for Test Set: Not explicitly stated in terms of a specific number of patients or specimens for the multi-center field clinical study. It only mentions that "nasopharyngeal swab and nasopharyngeal aspirate/wash specimens, both fresh and after storage in transport media" were used.
• Data Provenance:
  • Country of Origin: Not specified.
  • Retrospective or Prospective: The study is described as a "multi-center field clinical study," which generally implies a prospective collection of data in a real-world clinical setting.

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts (e.g. radiologist with 10 years of experience)

MISSING: The document does not specify the number of experts used or their qualifications for establishing the ground truth for the clinical study.

4. Adjudication method (e.g. 2+1, 3+1, none) for the test set

MISSING: The document does not describe any adjudication method used for the test set.

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/NOT PERFORMED: The Sofia RSV FIA is an instrumented immunofluorescence assay, not an AI-based diagnostic image analysis device or a system requiring human interpretation with or without AI assistance in the way an MRMC study would typically evaluate. The Sofia Analyzer processes results using "method-specific algorithms" and displays "Positive, Negative, or Invalid" results. The study described focuses on the device's analytical and clinical performance against a predicate device, not on human reader performance.

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

YES (Implicitly): The device functions as a standalone diagnostic. The Sofia Analyzer "scans the test strip and measures the fluorescent signal by processing the results using method-specific algorithms" to display "Positive, Negative, or Invalid" results. This is an algorithmic determination without direct human interpretation of the underlying signal, fitting the definition of standalone performance. The clinical study evaluated the performance of this system (device + analyzer algorithm) against a reference standard.

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

The intended use statement specifies that "Negative results do not preclude RSV infection and should not be used as the sole basis for treatment or for other management decisions. A negative result is presumptive, and it is recommended these results be confirmed by virus culture or an FDA-cleared RSV molecular assay."

Based on this, it is highly probable that the ground truth for the clinical study was established by either virus culture or an FDA-cleared RSV molecular assay as a reference method. The document implicitly supports this by recommending these methods for confirmation of negative results.

8. The sample size for the training set

NOT APPLICABLE/NOT PROVIDED: The document does not mention a "training set" in the context of machine learning. The device is an immunofluorescence assay with an analyzer that processes results using "method-specific algorithms." These algorithms are likely pre-programmed and validated, rather than being "trained" on a large dataset in the sense of deep learning models. The studies described are for analytical validation and clinical performance evaluation.

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

NOT APPLICABLE/NOT PROVIDED: As no "training set" for a machine learning algorithm is discussed, this information is not applicable.

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The Sofia hCG FIA is an immunofluorescence-based lateral flow assay intended for the qualitative detection of human Chorionic Gonadotropin (hCG) in urine specimens and is designed to aid early detection of pregnancy. The test is intended for prescription use only, including use at point-of-care sites.

The test kit consists of individually packaged test Cassettes-each containing monoclonal murine antibodies for the capture and detection of hCG; disposable specimen transfer pipettes; and a package insert. The test is a qualitative immunofluorescence-based assay used to detect concentrations of 20 mIU/mL hCG or more in urine.

The Sofia hCG FIA is an immunofluorescence-based lateral flow assay intended for the qualitative detection of human Chorionic Gonadotropin (hCG) in urine specimens to aid early detection of pregnancy. The device is for prescription use only, including at point-of-care sites.

1. Table of Acceptance Criteria and Reported Device Performance:

The document primarily focuses on demonstrating substantial equivalence to a predicate device rather than explicitly stating acceptance criteria for each performance metric as distinct pass/fail thresholds. However, the performance data presented can be interpreted as demonstrating the device meets the implied requirements for accuracy and reliability for its intended use.

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

• Sample Size for Clinical Performance (Test Set): 974 fresh urine specimens.
• Data Provenance: The clinical study was multi-center, conducted by health care personnel at five (5) distinct sites in various geographical regions within the United States. The data is prospective, collected from patients presenting for pregnancy testing.

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

The document does not specify the number or qualifications of experts used to establish the ground truth for the clinical test set. The clinical performance study compares the Sofia hCG FIA to "a commercially available qualitative test," implying that the result from this comparator test served as the reference or ground truth.

4. Adjudication Method for the Test Set:

The document does not describe an adjudication method for the clinical test set. The comparison is directly between the Sofia hCG FIA and a single "commercially available qualitative test."

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

A MRMC comparative effectiveness study was not explicitly performed or described in the provided text. The clinical study compares the device's performance to another commercial test, not to human readers with or without AI assistance. The Sofia hCG FIA is a rapid immunoassay read by an instrument (Sofia Analyzer), not an imaging device typically associated with MRMC studies involving human readers.

6. Standalone Performance:

Yes, standalone performance was assessed. The entire document describes the performance of the Sofia hCG FIA algorithm (via the Sofia Analyzer) in various analytical and clinical studies, independent of human interpretation or intervention in the diagnostic process beyond specimen collection and loading the cassette. The Sofia Analyzer dictates the development time, scans the strip, and analyzes the fluorescent signal using method-specific algorithms to display the result (Positive, Negative, or Invalid).

7. Type of Ground Truth Used:

• Analytical Performance Studies (Reproducibility, Detection Limit, High-Dose Hook Effect, Analytical Specificity): The ground truth was established using known, contrived samples prepared by spiking negative urine with specific, traceable concentrations of hCG (WHO International 4th Standard) or other substances.
• Clinical Performance Study: The ground truth was based on the results obtained from another commercially available qualitative test when evaluating clinical patient samples.

8. Sample Size for the Training Set:

The document does not explicitly state a separate "training set" for the Sofia hCG FIA. As a lateral flow immunoassay read by an analyzer with method-specific algorithms, the "training" would likely refer to the data used during the development and optimization of the instrument's algorithm and the assay reagents themselves. This information is not provided in detail, as the focus is on the validation performance of the finished product.

9. How Ground Truth for Training Set was Established:

As mentioned above, a "training set" is not explicitly described. However, during the development of such a device, a vast number of samples with known hCG concentrations (established through analytical methods, often traceable to international standards) would be used to develop and refine the detection algorithms and thresholds for the Sofia Analyzer. This would involve rigorous calibration and optimization to correctly identify positive and negative results across the intended range of hCG concentrations and ensure specificity. The performance studies detailed in the submission serve as the validation of this developed system.

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The Sofia Influenza A+B FIA employs immunofluorescence to detect influenza A and influenza B viral nucleoprotein antigens in nasal swab, nasopharyngeal swab, and nasopharyngeal aspirate/wash specimens taken directly from symptomatic patients. This qualitative test is intended for use as an aid in the rapid differential diagnosis of acute influenza A and influenza B viral infections. The test is not intended to detect influenza C antigens. A negative test is presumptive and it is recommended these results be confirmed by virus culture or FDA-cleared influenza A and B molecular assay. Negative results do not preclude influenza virus infections and should not be used as the sole basis for treatment or other management decisions. The test is intended for professional and laboratory use.

Performance characteristics for influenza A and B were established during February through March 2011 when influenza viruses A/California/7/2009 (2009 H1N1), A/Perth/16/2009 (H3N2), and B/Brisbane/60/2008 (Victoria-Like) were the predominant influenza viruses in circulation according to the Morbidity and Mortality Weekly Report from the CDC entitled "Update: Influenza Activity--United States, 2010-2011 Season, and Composition of the 2011-2012 Influenza Vaccine". Performance characteristics may vary against other emerging influenza viruses.

If infection with a novel influenza 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. Virus culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.

The Sofia Influenza A+B FIA employs immunofluorescence technology that is used with the Sofia Analyzer to detect influenza virus nucleoproteins.

The Sofia Influenza A+B FIA is a lateral-flow immunoassay that uses monoclonal antibodies that are specific for influenza antigens and have no known cross-reactivity to normal flora or other known respiratory pathogens.

Nasal swab, nasopharyngeal swab, and nasopharyngeal aspirate/wash specimens are used for this test. The patient specimen is placed in the Reagent Tube, during which time the virus particles in the specimen are disrupted, exposing internal viral nucleoproteins. After disruption, the specimen is dispensed into the cassette sample well. From the sample well, the specimen migrates through a test strip containing various unique chemical environments. If influenza viral antigen is present, they will be trapped in a specific location.

• Note: Depending upon the user's choice, the cassette is either placed inside of the Sofia Analyzer for automatically timed development (Walk Away Mode) or placed on the counter or bench top for a manually timed development and then placed into the Sofia Analyzer to be scanned (Read Now Mode).
  The Sofia Analyzer will scan the test strip and measure the fluorescent signal by processing the results using method-specific algorithms. The Sofia Analyzer will display the test results (Positive, Negative, or Invalid) on the screen. The results can also be automatically printed on an integrated printer if this option is selected.

Here's an analysis of the provided text regarding the Sofia® Influenza A+B FIA device:

1. Table of Acceptance Criteria and Reported Device Performance

2. Sample Size for Test Set and Data Provenance

The provided text describes one analytical study (for H7N9 detection) and refers to previously established performance characteristics for common influenza strains.

• H7N9 Study (Analytical): The sample size for this specific study is not explicitly stated. The study focuses on establishing the Limit of Detection (LoD).
• Previous Performance Characteristics (Clinical): The text mentions that performance characteristics for influenza A and B were established during February through March 2011 when specific influenza viruses were prevalent.
  • Data Provenance: The general context indicates this data would be from the United States, as it references the CDC's Morbidity and Mortality Weekly Report regarding influenza activity in the US.
  • Retrospective or Prospective: The phrasing "performance characteristics were established during February through March 2011" suggests prospective data collection during a specific influenza season. However, no details on how many patient samples were tested are provided in this summary.

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

The provided summary does not explicitly state the number of experts used or their qualifications for establishing ground truth for the test set that determined the original performance characteristics.

For the H7N9 analytical study, the nature of establishing "minimum detectable level" implies laboratory testing rather than expert-based ground truth on clinical samples.

4. Adjudication Method for the Test Set

The document does not describe any adjudication method for establishing the ground truth for the clinical test sets (both for the previously established performance and the H7N9 study).

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

No mention of a Multi-Reader Multi-Case (MRMC) comparative effectiveness study or human reader improvement with/without AI assistance. This device is an immunoassay and an analyzer, not an AI-powered diagnostic imaging tool. Therefore, an MRMC study as typically understood in AI/imaging would not be applicable.

6. Standalone (Algorithm Only) Performance

Yes, the device's performance, as described by the "minimum detectable level" for H7N9 and the "performance characteristics" established for other influenza strains, represents its standalone performance. The Sofia Analyzer processes the results using "method-specific algorithms" and displays "Positive, Negative, or Invalid" results. There is no human-in-the-loop component for interpreting the test outcome itself; the human operates the device and reads its output.

7. Type of Ground Truth Used

• H7N9 Analytical Study: The ground truth for the H7N9 study is based on a known concentration of H7N9 virus (Egg Infective Dose (EID)30/mL), which is a common method for establishing the Limit of Detection for in vitro diagnostic tests.
• Previous Performance Characteristics (Clinical): The intended use statement mentions that for negative results, it is "recommended these results be confirmed by virus culture or FDA-cleared influenza A and B molecular assay." This implies that the ground truth for the clinical performance characteristics (established in 2011) was likely based on confirmation by these more definitive laboratory methods.

8. Sample Size for the Training Set

The document does not provide any information about a "training set" or its sample size. This is an in vitro diagnostic device based on immunofluorescence with specific reagent antibodies and an analyzer using "method-specific algorithms." The development process for such a device would involve extensive analytical validation (e.g., sensitivity, specificity, cross-reactivity) rather than a "training set" in the context of machine learning.

9. How Ground Truth for Training Set Was Established

As no training set is mentioned in the context of machine learning, this question is not applicable based on the provided text. The "method-specific algorithms" in the Sofia Analyzer would have been developed and validated through internal R&D and analytical studies, not typically through a machine learning training process with a dedicated training set and labeled ground truth in the same way an AI imaging algorithm would be.

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The QuickVue Influenza A+B test allows for the rapid, qualitative detection of influenza type A and type B antigens directly from nasal swab, nasopharyngeal swab, nasal aspirate, and nasal wash specimens. The test is intended for use as an aid in the rapid differential diagnosis of acute influenza type A and type B viral infections. The test is not intended to detect influenza C antigens. Negative results should be confirmed by cell culture; they do not preclude influenza virus infection and should not be used as the sole basis for treatment or other management decisions. The test is intended for professional and laboratory use.

The QuickVue Influenza A+B test involves the extraction of influenza A and B viral antigens. The patient specimen is placed in the Extraction Reagent Tube, during which time the virus particles in the specimen are disrupted, exposing internal viral nucleoproteins. After extraction, the Test Strip is placed in the Extraction Reagent Tube where nucleoproteins in the specimen will react with the reagents in the Test Strip. If the extracted specimen contains influenza A or B antigens, a pink-to-red Test Line along with a blue procedural Control Line will appear on the Test Strip indicating a positive result. The Test Line for influenza A or B will develop at separate specified locations on the same Test Strip. If influenza A or B antigens are not present, or are present at very low levels, only the blue procedural Control Line will appear.

Here's a breakdown of the acceptance criteria and the study information derived from the provided text for the QuickVue® Influenza A+B test:

1. Table of Acceptance Criteria and Reported Device Performance

Note: The document primarily focuses on demonstrating substantial equivalence to a predicate device and includes only one specific analytical study result for a new influenza strain (H7N9). There are no explicitly stated acceptance criteria with numerical targets for clinical performance metrics (e.g., sensitivity, specificity) within this summary. Instead, the "Conclusion" states "The QuickVue Influenza A+B test is substantially equivalent with the current QuickVue Influenza A+B test," implying that its performance should be comparable to the predicate device.

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

The provided text only details one analytical study for H7N9 detection. It does not specify a separate "test set" in the context of clinical samples, nor does it provide details on the sample size for this analytical study beyond the reported Limit of Detection. The data provenance (country of origin, retrospective/prospective) is also not mentioned for this analytical study.

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

Not applicable. The provided text describes an analytical study for Limit of Detection (LoD), which typically involves laboratory measurements and does not require expert ground truth establishment in the same way clinical studies do.

4. Adjudication Method for the Test Set

Not applicable as there is no described clinical "test set" and no method for adjudicating results.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was done, Effect Size of Human Improvement with AI vs. Without AI Assistance

Not applicable. The QuickVue® Influenza A+B test is a rapid, qualitative immunological test, not an AI-assisted diagnostic device that would involve human readers and requiring an MRMC study to assess AI assistance.

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

This refers to the performance of the device itself (the test strip and reagents) without human interpretation beyond reading the result. The QuickVue® Influenza A+B test is inherently a standalone device in this sense, as it produces visual results that are interpreted directly. The "Summary of Performance Data" details an analytical study of the device's ability to detect the H7N9 virus independently.

7. The Type of Ground Truth Used

For the analytical study concerning H7N9, the "ground truth" would be established by the known concentration of the H7N9 virus (7.90 x 10^6 EID50/mL) in the prepared samples, which is a laboratory standard rather than expert consensus, pathology, or outcomes data.

8. The Sample Size for the Training Set

Not applicable. The QuickVue® Influenza A+B test is a lateral flow immunoassay, not a machine learning or AI-based device that would require a "training set" in the computational sense.

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

Not applicable for the same reasons as point 8.

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The Quidel Molecular Direct C. difficile Assay is a qualitative, multiplexed in vitro diagnostic test for the direct detection of toxin A gene (tcdA) or toxin B gene (tcdB) sequences of toxigenic strains of Clostridium difficile from unformed (liquid or soft) stool specimens collected from patients suspected of having Clostridium difficile-Associated Disease (CDAD).

The Quidel Molecular Direct C. difficile Assay is a real-time PCR test and utilizes proprietary sample preparation with fluorescently labeled primers and probes. The assay can be performed using either the Life Technologies QuantStudio® Dx; the Applied Biosystems 7500 Fast Dx, or the Cepheid SmartCycler II, to detect the toxin gene sequences associated with toxin-producing C. difficile strains.

The assay is intended to be performed directly on CDAD-suspected stool specimens, and is indicated for use as an aid in the diagnosis of CDAD.

The Quidel Molecular Direct C. difficile Assay detects nucleic acids that have been prepared from a patient sample using proprietary sample preparation. A multiplex real-time PCR reaction is performed under optimized conditions in a single well generating amplicons for each of the targets present in the sample. Identification occurs by the use of oligonucleotide primers and probes that are complementary to conserved regions in the tcdA and tcdB genes of the pathogenicity locus (PaLoc).

The Quidel Molecular Direct C. difficile Assay contains sufficient reagents to process 96 specimens or quality control samples.

Here's a summary of the acceptance criteria and study details for the Quidel Molecular Direct C. difficile Assay, based on the provided document:

1. Table of Acceptance Criteria and Reported Device Performance

The document primarily focuses on demonstrating the device's performance against de facto clinical comparison methods rather than explicitly stating pre-defined acceptance criteria with specific numerical thresholds for sensitivity and specificity before the study. However, the reported performance metrics can be considered the demonstrated "acceptance" level based on the study outcomes.

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

• Clinical Performance Studies (for ABI 7500 Fast Dx & Cepheid SmartCycler II): 665 specimens initially, reduced to 653 (vs Direct Culture) or 656 (vs Enriched Culture) due to invalid/indeterminate results.
• Clinical Performance Study (for Life Technologies QuantStudio Dx): 792 specimens initially, reduced to 788 (vs Direct Culture) or 791 (vs Enriched Culture) due to invalid/indeterminate results.
• Provenance: All clinical specimens were collected prospectively between August 2012 and November 2012 from patients suspected of having Clostridium difficile-Associated Disease (CDAD) at four geographically diverse locations within the United States.

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

The document does not explicitly mention the use of human experts to establish the ground truth for the clinical test set. Instead, the ground truth was established by laboratory-based reference assays:

• Direct Culture Cytotoxicity Assay: This is a biological assay, not directly performed by "experts" in the sense of clinical reviewers.
• Enriched Toxigenic C. difficile Culture: This is also a laboratory culture method, not involving expert review of images or clinical data.
• FDA-cleared molecular device: Used for discordant analysis, this is another laboratory test.

Therefore, the concept of "experts" and their qualifications as typically applied to image-based diagnostic devices (e.g., radiologists) does not apply here.

4. Adjudication Method for the Test Set

The document describes an adjudication method for discordant results.

• For discordant results between the Quidel Molecular Direct C. difficile Assay and the reference methods (Direct Culture Cytotoxicity Assay or Enriched Toxigenic Culture), an FDA-cleared molecular device was used to re-test the discordant specimens. The results from this FDA-cleared molecular device were then used to help explain the discrepancies.
• The overall clinical performance was calculated based on the initial test results prior to the discordant analysis.

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

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not conducted. This type of study typically involves multiple human readers assessing cases with and without AI assistance to measure the effect size of AI on human performance. The Quidel Molecular Direct C. difficile Assay is a qualitative PCR test, not an imaging-based diagnostic system that would involve human interpretation of images, so an MRMC study is not relevant.

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

Yes, the primary clinical performance studies (comparison against Direct Culture Cytotoxicity Assay and Enriched Toxigenic Culture) represent a standalone (algorithm only) performance evaluation of the Quidel Molecular Direct C. difficile Assay. The assay directly reports a qualitative result (positive or negative) without requiring human interpretation or involvement in the diagnostic decision once the sample is processed by the instrument.

7. The Type of Ground Truth Used

The ground truth for the clinical performance studies was established using laboratory reference methods:

• Direct Culture Cytotoxicity Assay: This method identifies the presence of C. difficile toxins.
• Enriched Toxigenic C. difficile Culture: This method specifically identifies toxigenic strains of C. difficile.

For discordant analyses, an FDA-cleared molecular device was used as a third reference.

8. The Sample Size for the Training Set

The document does not specify a training set sample size. This is common for molecular diagnostic assays like this one, where the assay design (e.g., primer and probe sequences) is developed based on known genetic sequences of the target organism, and analytical validation (LoD, inclusivity, specificity) is performed. The clinical studies presented are for validation/testing, not for training a machine learning model.

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

As there is no explicitly mentioned "training set" in the context of machine learning for this device, the concept of ground truth for a training set is not directly applicable. The assay's design and analytical characteristics are based on:

• Knowledge of conserved regions in C. difficile tcdA and tcdB genes.
• Quantified cultures of C. difficile strains (ATCC BAA-1870 and ATCC BAA-1872) for analytical sensitivity (LoD).
• Known toxigenic strains for analytical reactivity (inclusivity).
• A panel of known bacterial, viral, and yeast microorganisms, as well as human DNA, for analytical specificity (cross-reactivity).

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The Quidel Molecular RSV + hMPV assay is a multiplex Real Time RT-PCR assay for the in vitro qualitative detection and identification of respiratory syncytial virus and human metapneumovirus viral RNA extracted from nasal and nasopharyngeal swabs specimens from patients with signs and symptoms of respiratory infection. This in vitro diagnostic test is intended to aid in the differential diagnosis of respiratory syncytial virus and human metapneumovirus infections. This test is not intended to differentiate the four genetic sub-lineages of hMPV.

Negative results do not preclude RSV or hMPV infection and should not be used as the sole basis for diagnosis, treatment or other patient management decisions.

The Quidel Molecular RSV + hMPV Assay detects viral nucleic acids that have been extracted from a patient sample using the NucliSENS® easyMAG® automated extraction platform. A multiplex RT-PCR reaction is carried out under optimized conditions in a single tube generating amplicons for each of the target viruses present in the sample. This reaction is performed utilizing either the Cepheid SmartCycler® II or the Applied Biosystems 7500 Fast DX. Identification of RSV and hMPV and the PRC occurs by the use of target-specific primers and fluorescent-labeled probes that hybridize to conserved regions in the genomes of RSV and hMPV and the PRC.

Here's a summary of the acceptance criteria and study details for the Quidel Molecular RSV + hMPV Assay, based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria provided in the document are primarily for analytical performance (LoD, Reproducibility, Inclusivity, Specificity) and clinical performance (Sensitivity and Specificity/Positive and Negative Percent Agreement). The clinical performance is reported compared to predicate devices or established methods.

Device: Quidel Molecular RSV + hMPV Assay

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

• Clinical Performance Test Set Samples:
  • Total samples collected: 1014 specimens (414 fresh, 600 frozen) for RSV comparison.
  • RSV testing (SmartCycler II): 1009 specimens after excluding contaminated cell cultures.
  • hMPV testing (SmartCycler II): 951 specimens after excluding invalid comparative device results.
  • RSV testing (7500 Fast Dx): 1007 specimens after excluding contaminated cell cultures and invalid subject method results.
  • hMPV testing (7500 Fast Dx): 946 specimens after excluding invalid comparative and subject method results.
• Data Provenance: The samples were collected prospectively during the 2012 respiratory virus season (January to March 2012) from symptomatic patients at four sites across the United States. The study specifically used "fresh (414) and frozen (600) swab 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 or qualifications of experts used. However, the ground truth for RSV was established using "DSFA & Cell Culture w/DFA" (Direct Specimen Fluorescent Antibody and Cell Culture with DFA), which implies interpretation by trained laboratory personnel or specialists, although their specific qualifications or number are not detailed. For hMPV, the ground truth was established by comparing it to the "FDA Cleared hMPV molecular test" (Gen-Probe Prodesse Pro hMPV+, K082688), which is a molecular diagnostic method rather than expert interpretation of raw data.

4. Adjudication Method for the Test Set

• RSV Discrepant Results:
  • For the SmartCycler II, all 21 originally discordant specimens (QM RSV + hMPV positive, reference method negative) were positive for RSV by an FDA-cleared RT-PCR assay and by bi-directional sequence analysis.
  • For the 7500 Fast Dx, 25 of 28 originally discordant specimens were positive by an FDA-cleared RT-PCR assay, and 27 of 28 were positive by bi-directional sequence analysis.
• hMPV Discrepant Results:
  • For both the SmartCycler II and the 7500 Fast Dx, all originally discordant specimens (QM RSV + hMPV positive, reference method negative) were positive for hMPV by bi-directional sequence analysis.

This indicates a form of post-hoc adjudication or discrepancy resolution using additional, more definitive molecular methods (FDA-cleared RT-PCR and bi-directional sequencing) for cases where the subject device and the initial reference method disagreed.

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 was not an MRMC study. The device is an in vitro diagnostic (molecular assay) for direct detection of viral RNA, not an imaging device requiring human reader interpretation or AI assistance in interpretation. Therefore, a multi-reader multi-case comparative effectiveness study on human reader improvement with or without AI assistance is not applicable here.

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

Yes, the clinical performance study evaluates the standalone performance of the Quidel Molecular RSV + hMPV Assay. The results are presented as the device's agreement (sensitivity, specificity, positive/negative percent agreement) compared directly to the reference methods, without human interpretation of the assay results impacting the reported performance metrics. The assay itself provides a qualitative (positive/negative) result based on its internal thresholding (e.g., fluorescence achieved by 50 cycles on SmartCycler II or 35 cycles on ABI 7500 Fast Dx).

7. The Type of Ground Truth Used

• For RSV: The ground truth for clinical performance was established using Direct Specimen Fluorescent Antibody (DSFA) and Cell Culture with DFA.
• For hMPV: The ground truth for clinical performance was established using an FDA Cleared hMPV molecular test (Gen-Probe Prodesse Pro hMPV+).
• For discrepant results: Bi-directional sequence analysis and/or another FDA-cleared RT-PCR assay were used as definitive ground truth.

8. The Sample Size for the Training Set

The document does not explicitly state a sample size for a "training set" in the context of machine learning or algorithm development. For this type of molecular diagnostic assay, analytical studies (LoD, inclusivity, specificity) and clinical validation are performed. The LoD study involved replicates of serially diluted viral cultures, and inclusivity/specificity studies used panels of various strains/organisms. These analytical studies are analogous to "training" or "development" data in that they inform and validate the assay's operational parameters, but they are not framed as a classic machine learning training set.

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

Given that this is a molecular diagnostic assay, the "ground truth" for establishing analytical parameters (like LoD, inclusivity, and specificity) is based on:

• Quantified viral cultures (TCID50/mL): Used for LoD studies, where the exact concentration of virus is known.
• Known viral strains or bacterial/yeast cultures: Used for inclusivity (known to contain the target virus) and specificity (known to contain other organisms to test for cross-reactivity) studies. The identity and concentration of these cultures are established through standard microbiological and virological techniques.

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The Quidel® Molecular Influenza A+B assay is a multiplex Real Time RT-PCR assay for the in vitro qualitative detection and differentiation of influenza A and influenza B viral RNA in nasal and nasopharyngeal swabs from patients with signs and symptoms of respiratory infection. This test is intended for use as an aid in the differential diagnosis of influenza A and influenza B viral infections in humans in conjunction with clinical and epidemiological risk factors. The assay does not detect the presence of influenza C virus.

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

Performance characteristics for influenza A were established during the 2010 to 2011 influenza season when influenza A/H3 and 2009 H1N1 influenza were the predominant influenza A viruses 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 Quidel Molecular Influenza A+B Assay detects viral RNA that have been extracted from a patient sample using the NucliSENS® easyMAG® automated extraction platform. A multiplex RT-PCR reaction is carried out under optimized conditions in a single tube generating amplicons for each of the target viruses present in the sample. This reaction is performed utilizing the Cepheid SmartCycler® II platform. Identification of influenza A occurs by the use of target specific primers and a fluorescent- labeled probe that hybridizes to a conserved influenza A sequence within the matrix protein gene. Identification of influenza B occurs by the use of target specific primers and fluorescentlabeled probes that will hybridize to a conserved influenza B sequence within the neuraminidase gene.

The following is a summary of the procedure:

1. Sample Collection: Obtain nasal swabs and nasopharyngeal swabs specimens using standard techniques from symptomatic patients. These specimens are transported, stored, and processed according to established laboratory procedures.
2. Nucleic Acid Extraction: Extract Nucleic Acids from the specimens with the NucliSENS easyMAG System following the manufacturer's instructions using the appropriate reagents.

Prior to the extraction procedure add 20 µL of the Process Control (PRC) to each 180 uL aliquot of specimen. The PRC serves to monitor inhibitors in the extracted specimen, assures that adequate amplification has taken place and that nucleic acid extraction was sufficient.

3. Rehydration of Master Mix: Rehydrate the lyophilized Master Mix using 135uL of Rehydration Solution. The Master Mix contains oligonucleotide primers. fluorophore and quencher-labeled probes targeting highly conserved regions of the influenza A and influenza B viruses as well as the process control sequence. The primers are complementary to highly specific and conserved regions in the genome of these viruses. The probes are dual labeled with a reporter dye attached to the 5'end and a quencher attached to the 3' end. The rehydrated Master Mix is sufficient for eight reactions.
4. Nucleic Acid Amplification and Detection: Add 15 µL of the rehydrated Master Mix to each reaction tube. SuL of extracted nucleic acids (specimen with PRC) is then added to the tube. Then place the tube into the Cepheid SmartCycler® II.

Once the reaction tubes are added to the instrument, the assay protocol is initiated. This protocol initiates reverse transcription of the RNA targets generating complementary DNA, and the subsequent amplification of the target amplicons occurs. The Quidel Molecular Influenza A+B assay is based on TaqMan® chemistry, and uses an enzyme with reverse transcriptase, DNA polymerase, and 5'-3' exonuclease activities. During DNA amplification, this enzyme cleaves the probe bound to the complementary DNA sequence, separating the quencher dye from the reporter dye. This step generates an increase in fluorescent signal upon excitation by a light source of the appropriate wavelength. With each cycle, additional dye molecules are separated from their quenchers resulting in additional signal. If sufficient fluorescence is achieved by 45 cycles, the sample is reported as positive for the detected nucleic acid.

Here's a breakdown of the acceptance criteria and the study results for the Quidel Molecular Influenza A+B Assay, based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are implicitly defined by the results of the comparative clinical study against an FDA-cleared predicate device. The performance characteristics are presented as Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA).

Note: The document explicitly states "good positive and negative percent agreement when compared to a high performance FDA Cleared Influenza A and B molecular test" in the conclusions, which serves as the general acceptance criterion. The precise numerical thresholds for "good" are not explicitly defined but are demonstrated by the presented results.

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

• Prospective Clinical Study:
  • Sample Size: 779 fresh specimens (427 nasal swabs and 352 nasopharyngeal swabs). After removing invalid specimens, 753 specimens were analyzed.
  • Data Provenance: The study was conducted during the 2010-2011 respiratory virus season (January to March 2011) at thirteen sites across the United States. The specimens were collected for routine influenza testing and tested at one central location within 72 hours of collection. This is prospective data.
• Retrospective Clinical Study:
  • Sample Size: 356 frozen nasopharyngeal swab specimens. After removing invalid specimens, 352 specimens were analyzed.
  • Data Provenance: The study used frozen specimens collected during the 2010-2011 respiratory virus season (January to March of 2011). This is retrospective data. The country of origin is not explicitly stated but implied to be the United States, similar to the prospective study, as it's part of the same submission to the FDA in the US.

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

The ground truth for the clinical studies was established using a "high performance FDA Cleared Influenza A and B molecular test" as the comparator method. Thus, it was not established by human experts in the typical sense (e.g., radiologist consensus), but rather by the performance of an already-cleared diagnostic device.

For the discordant results in the prospective study, sequence analysis was used to resolve discrepancies for influenza A (8 specimens that were negative by comparator but positive by Quidel Molecular) and influenza B (26 specimens negative by comparator but positive by Quidel Molecular, and 2 specimens negative by comparator and negative by sequence analysis). The qualifications of those performing the sequence analysis are not detailed.

4. Adjudication Method for the Test Set

For the clinical studies, results were compared directly against the predicate FDA-cleared RT-PCR device. In cases of discordance between the subject device and the comparator device, sequence analysis was performed for some discrepant specimens (specifically, those where the comparator was negative but the Quidel Molecular assay was positive for Influenza A or B). This indicates a form of adjudication where a third, more definitive method (sequencing) was used to assess the comparator's accuracy in those specific cases, rather than an expert panel reviewing the results.

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, an MRMC comparative effectiveness study involving human readers and AI assistance was not done. This submission is for a molecular diagnostic assay (RT-PCR), not an AI-powered image analysis or diagnostic tool that would typically involve human "readers." The comparison is between two molecular tests.

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

Yes, the device was evaluated in a standalone manner. The clinical performance data (Tables 5.8, 5.9, 5.10, 5.11) represent the Quidel Molecular Influenza A+B Assay's performance (algorithm only) compared to another FDA-cleared molecular test.

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

The primary "ground truth" for the clinical performance evaluation was the results of a "high performance FDA Cleared Influenza A and B molecular test" (the predicate comparator device). For specific discordant results, sequence analysis was used as a more definitive method to further evaluate the initial test results.

8. The Sample Size for the Training Set

The document does not explicitly state a sample size for a "training set" in the context of machine learning or AI. This is a molecular diagnostic assay that functions through specific primer and probe binding, not a learning algorithm that requires a distinct training and test set in the AI sense.

However, the analytical performance studies (Limit of Detection, Analytical Reactivity, Analytical Specificity) involve testing various strains and concentrations, which could be considered akin to "training" or "development" data in a broader sense for optimizing assay parameters. For example:

• LoD study: Replicates of 20 per concentration for 3 influenza A strains and 3 influenza B strains.
• Analytical Reactivity: 38 influenza A strains and 15 influenza B strains, tested in triplicate.
• Analytical Specificity: Panels of 26 viral, 24 bacterial, and 1 yeast strain, tested in triplicate.

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

As this is not an AI/ML device with a conventional "training set," the concept of "ground truth for the training set" as it applies to AI is not directly applicable.

Instead, for the analytical studies:

• Limit of Detection (LoD): Ground truth was established by using quantified (TCID50/mL) cultures of known influenza strains serially diluted in negative matrix. The known concentration was the "truth."
• Analytical Reactivity (Inclusivity): Ground truth was established by using known, well-characterized viral strains (Influenza A subtypes and Influenza B strains) at specified TCID50 levels. The knowledge of the specific viral strain and its presence was the "truth."
• Analytical Specificity (Cross-reactivity): Ground truth was established by using known concentrations of specific viral, bacterial, and yeast strains. The knowledge of which organism was present (or not present, if testing for Influenza A/B) was the "truth."

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The Quidel Molecular Influenza A+B assay is a multiplex Real Time RT-PCR assay for the in vitro qualitative detection and differentiation of influenza A and influenza B viral RNA in nasal and nasopharyngeal swabs from patients with signs and symptoms of respiratory infection. This test is intended for use as an aid in the differential diagnosis of influenza A and influenza B viral infections in humans in conjunction with clinical and epidemiological risk factors. The assay does not detect the presence of influenza C virus.

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

Performance characteristics for influenza A were established during the 2010-2011 influenza season when influenza A/H3 and 2009 H1N1 influenza were the predominant influenza A viruses 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 Quidel Molecular Influenza A+B Assay detects viral nucleic acids that have been extracted from a patient sample using the NucliSENS® easyMAG® automated extraction platform. A multiplex real-time RT-PCR reaction is carried out under optimized conditions in a single tube generating amplicons for each of the target viruses present in the sample. This reaction is performed utilizing the Applied Biosystems® 7500 Fast Dx platform. Identification of influenza A occurs by the use of target specific primers and a fluorescentlabeled probe that hybridizes to a conserved influenza A sequence within the matrix protein gene. Identification of influenza B occurs by the use of target specific primers and fluorescent-labeled probes that will hybridize to a conserved influenza B sequence within the neuraminidase gene.

The following is a summary of the procedure:

1. Sample Collection: Obtain nasal swab and nasopharyngeal swab specimens using standard techniques from symptomatic patients. These specimens are transported, stored, and processed according to established laboratory procedures.
2. Nucleic Acid Extraction: Extract Nucleic Acids from the specimens with the NucliSENS easyMAG System following the manufacturer's instructions using the appropriate reagents. Use of other extraction systems with the Quidel Molecular Influenza A+B kit has not been validated. Validation of these systems is the responsibility of the end-user. Prior to the extraction procedure add 20 uL of the Process Control (PRC) to each 180 uL aliquot of specimen. The PRC serves to monitor inhibitors in the extracted specimen, assures that adequate amplification has taken place and that nucleic acid extraction was sufficient.
3. Rehydration of Master Mix: Rehydrate the lyophilized Master Mix using 135uL of Rehydration Solution. The Master Mix contains oligonucleotide primers, fluorophore and quencher-labeled probes targeting highly conserved regions of the influenza A and influenza B viruses as well as the process control sequence. The primers are complementary to highly specific and conserved regions in the genome of these viruses. The probes are dual labeled with a reporter dye attached to the 5-end and a quencher attached to the 3'-end. The rehydrated Master Mix is sufficient for eight reactions.
4. Nucleic Acid Amplification and Detection: Add 15 uL of the rehydrated Master Mix to each reaction plate well. 5uL of extracted nucleic acids (specimen with PRC) is then added to the plate well. Then place the plate into the ABI 7500 FastDx.

Once the plate is added to the instrument, the assay protocol is initiated. This protocol initiates reverse transcription of the RNA targets generating complementary DNA, and the subsequent amplification of the target amplicons occur. The Quidel Molecular Influenza A+B assay is based on TaqMan® chemistry, and uses an enzyme with reverse transcriptase, DNA polymerase, and 5'-3' exonuclease activities. During DNA amplification, this enzyme cleaves the probe bound to the complementary DNA sequence, separating the quencher dye from the reporter dye. This step generates an increase in fluorescent signal upon excitation by a light source of the appropriate wavelength. With each cycle, additional dye molecules are separated from their quenchers resulting in additional signal. If sufficient fluorescence is achieved by 35 cycles during the data collection stage of amplification, the sample is reported as positive for the detected nucleic acid.

This document describes the Quidel Molecular Influenza A+B Assay, a real-time RT-PCR assay for the qualitative detection and differentiation of influenza A and influenza B viral RNA.

Here's an analysis of the acceptance criteria and the study that proves the device meets them:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are not explicitly stated as numerical targets in a single table, but rather are implied by the results presented in the analytical and clinical performance sections when compared to a legally marketed predicate device (Gen-Probe Prodesse ProFlu+).

Here’s a table summarizing the reported device performance, which implicitly suggests the acceptance criteria were met by demonstrating adequate performance comparable to the predicate regarding various analytical and clinical characteristics.

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

• Test Set Sample Size:
  • Analytical Reproducibility: 90 replicates per level for each virus tested across 3 sites (30 per site).
  • Limit of Detection: Replicates of 20 per concentration of virus for each strain.
  • Analytical Reactivity: Triplicate testing for each of the 38 Influenza A strains and 15 Influenza B strains.
  • Analytical Specificity: Triplicate testing for each of the 26 viral, 24 bacterial, and 1 yeast strain.
  • Clinical Performance (Prospective): 668 fresh clinical specimens (373 nasal swabs, 313 nasopharyngeal swabs) after removing invalid results.
  • Clinical Performance (Retrospective): 372 frozen nasopharyngeal swabs after removing invalid results.
• Data Provenance: The document does not explicitly state the country of origin for the clinical samples. The study involved three laboratory sites for reproducibility, which might imply multi-site data collection, potentially from different locations. The clinical studies (prospective and retrospective) are referenced as "clinical studies," implying patient samples. The analytical studies use cultured viral strains and negative matrix.

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

The concept of "experts" in this context refers to the methods and outcomes used to establish the true presence or absence of influenza in clinical samples.

• Clinical Studies Ground Truth: The ground truth for the clinical test set was established by comparing the Quidel Molecular Influenza A+B Assay to a "Comparator: FDA Cleared RT-PCR device" (specifically identified as the Gen-Probe Prodesse ProFlu+ in the device comparison). For discordant results in the prospective study, sequence analysis was used to resolve discrepancies for Influenza A (7 cases) and Influenza B (12 cases). This suggests that a more definitive molecular testing method was considered the "expert" or "gold standard" for resolving these cases, rather than a human expert diagnosis.
• No explicit mention of human experts' qualifications for establishing ground truth, as the ground truth was primarily based on a predicate molecular test and confirmatory sequencing.

4. Adjudication Method for the Test Set

• For the clinical performance studies, the primary comparison was between the Quidel Molecular Influenza A+B Assay and the FDA Cleared RT-PCR device (predicate).
• Discordant results in the prospective clinical study were subjected to sequence analysis for adjudication:
  • For Influenza A: 7 specimens negative by the predicate but positive by the subject device were confirmed positive by sequence analysis. 1 specimen negative by both was also negative by sequence analysis.
  • For Influenza B: 12 specimens negative by the predicate but positive by the subject device were confirmed positive by sequence analysis.
  • This is a form of resolution by a higher-tier method rather than a traditional expert consensus method (e.g., 2+1 or 3+1 review).

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, an MRMC comparative effectiveness study was NOT done. This device is a molecular diagnostic assay, not an imaging or interpretive AI-based diagnostic tool that would involve human "readers" or "interpreters." The performance is evaluated based on the analytical and clinical accuracy of the assay itself.

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

• Yes, the studies evaluate the standalone performance of the assay. The Quidel Molecular Influenza A+B Assay is a laboratory-based real-time RT-PCR test. Its "performance" refers to the accuracy of the assay in detecting viral RNA in a sample. The results presented (reproducibility, LoD, inclusivity, specificity, clinical agreement) are all measures of the device's inherent performance. Human involvement is in sample collection, processing, and executing the assay protocol, but the "performance" itself is the output of the automated assay.

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

• Clinical Ground Truth: Primarily established by comparison to a legally marketed, FDA-cleared molecular diagnostic device (Gen-Probe Prodesse ProFlu+). For discordant results, molecular sequence analysis was used as the confirmatory "gold standard" to establish true positivity/negativity.
• Analytical Ground Truth: For LoD, inclusivity, and specificity studies, the ground truth was based on quantified viral cultures (TCID50/mL) or bacterial/yeast cultures (CFU/mL), which are established analytical standards for concentration and identity.

8. The Sample Size for the Training Set

• The document does not specify a separate "training set" as would be typical for machine learning algorithms. This is a traditional molecular diagnostic assay where performance is characterized through analytical and clinical validation studies. The "development" of the assay involves optimization of primers, probes, and reaction conditions, but not typically a labeled training data set in the sense of AI.

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

• Since there isn't a "training set" explicitly mentioned or evaluated in the context of this traditional diagnostic device, the question of how its ground truth was established is not applicable. The assay's components and parameters would have been developed and optimized using well-characterized viral strains and clinical samples based on established laboratory methods.

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