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The Influenza B Lineage Genotyping Kit contains reagents and controls of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel and 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 the determination of the genetic lineage of human influenza B viruses as B/Victoria or B/Yamagata lineage from · viral RNA in upper respiratory tract clinical specimens (including nasopharyngeal swabs [NS], throat swabs [TS], nasal aspirates [NA], nasal washes [NW] and dual nasopharyngeal/throat swabs [NPS/TS]) from human patients with signs and symptoms of respiratory infection and/or from viral culture;

· To provide epidemiologic information for surveillance of circulating influenza viruses.

Performance characteristics for influenza B lineage genotyping were established during a season when influenza B/ Victoria and B/Yamagata lineages were in circulation.

Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions. Conversely, positive results do not rule out bacterial infection with other viruses. The agent detected may not be the definite cause of disease.

All users, and any person reporting results from use of this device should be trained to perform and interpret the results from this procedure by a competent instructor prior to use. CDC Influenza Division will limit the distribution of this device to only those users who have successfully completed a training course provided by CDC instructors or designees.

The CDC Human Influenza Real-Time RT-PCR Diagnostic Panel is used in real-time RT-PCR (rRT-PCR) assays on the Applied Biosystems® (ABI) 7500 Fast Dx Real-time PCR system. The panel is configured in four separate kits. Each kit consists of oligonucleotide primers. fluorescently labeled hydrolysis probes, and controls which are used in rRT-PCR assays for the in vitro qualitative detection and characterization of influenza virus RNA in respiratory specimens from patients presenting with influenza-like illness (ILI). Oligonucleotide primers and probes included in the Influenza B Lineage Genotyping Kit for detection of influenza B and the two major genetic lineages of influenza B were selected from highly conserved regions of the non-structural (NS) and HA genes, respectively. Oligonucleotide primers and probes to detect the human RNase P gene (RP) in control samples and clinical specimens are also included in the kit.

Here's a summary of the acceptance criteria and study details for the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel: Influenza B Lineage Genotyping Kit, based on the provided document:

This document describes a 510(k) premarket notification for modifications to an existing influenza B lineage genotyping kit (K172091). The changes primarily address recent evolutionary changes in circulating influenza B viruses that could impact the reactivity of the current kit, and the evaluation of an alternative fluorescent hydrolysis probe quencher chemistry (ZEN™). The study aims to demonstrate that the modified device remains substantially equivalent to the predicate device.

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are generally demonstrated through 100% agreement or high percentage sensitivity/specificity with a clear lower bound for the 95% Confidence Interval (CI).

Influenza B Lineage Genotyping Assay (VER 1.1 and VER 2 VIC & YAM assays): All assays demonstrated similar reactivity with apparent LOD endpoints within one 5-fold dilution of each other for various B/Victoria and B/Yamagata strains across both enzyme systems (Invitrogen Superscript™ and Quanta qScript™). For example, LOD for B/Maryland/15/2016 (VER 1.1 & VER 2) was 10^1.7 EID50/mL for both enzymes. |
| Analytical Sensitivity (LOD Confirmation) | Lowest virus concentration where InfB and VIC or InfB and YAM primer and probe sets demonstrated ≥ 95% detection. | Demonstrated LODs for B/Maryland/15/2016 (Victoria) were 10^1.7 EID50/mL for both VER 1.1 and VER 2 with both enzyme systems. Demonstrated LODs for B/Texas/81/2016 (Yamagata) were 10^2.2 EID50/mL (VER 1.1 & VER 2 with Invitrogen) and 10^1.5 EID50/mL (VER 1.1 & VER 2 with Quanta). |
| Analytical Sensitivity (Inclusivity) | 100% positivity (3 out of 3 replicates) for the tested strains at or near the established LOD. | B/Victoria (VER 1.1 & VER 2): All 10 tested B/Victoria strains showed 3/3 positive replicates (100% inclusivity) at or near their LOD for both VER 1.1 and VER 2, across both enzyme systems.
B/Yamagata (VER 1.1 & VER 2): All 10 tested B/Yamagata strains showed 3/3 positive replicates (100% inclusivity) at or near their LOD for both VER 1.1 and VER 2, across both enzyme systems. |
| Analytical Specificity (Cross-Reactivity) | No cross-reactivity detected with the opposite influenza B lineage. | No cross-reactivity was detected with either VER 1.1 or VER 2 VIC and YAM primer/probe sets when tested against 10 high-titer B/Yamagata lineage viruses (for VIC assays) and 10 high-titer B/Victoria lineage viruses (for YAM assays), across both enzyme systems. (Results consistently showed '-') |
| Analytical Specificity (Exclusivity with Influenza A Viruses) | No cross-reactivity detected with various influenza A viruses. | No cross-reactivity was detected with either VER 1.1 or VER 2 VIC and YAM assays when tested against 8 high-titer Influenza A viruses of various human and animal subtypes, across both enzyme systems. (Results consistently showed '-') |
| Analytical Specificity (Exclusivity with Non-Influenza Respiratory Pathogens) | No cross-reactivity detected with common non-influenza respiratory pathogens or flora. | No cross-reactivity was detected with VER 2 VIC and YAM assays when tested against 36 organisms (16 viruses, 19 bacteria, and 1 yeast) at high titers, across both enzyme systems. (Results consistently showed '-') |
| Clinical Performance (Retrospective InfB Assay) | 100% positive and negative agreement with the cleared oligonucleotide probe (BHQ quencher). | 100.0% Positive Agreement (95% CI: 88.7-100.0) for 30/30 positive samples. 100.0% Negative Agreement (95% CI: 92.9-100.0) for 50/50 negative samples. (Both enzyme systems showed identical results). |
| Clinical Performance (VER 2 B/Victoria Prospective Study) | High sensitivity and specificity, typically aiming for >90% sensitivity and >95% specificity with a tight 95% CI. | Sensitivity: 100.0% (5/5 positives) (95% CI: 56.6 – 100.0).
Specificity: 100.0% (574/574 negatives) (95% CI: 99.3 – 100.0). (Note: Low number of positives impacts CI width for sensitivity). |
| Clinical Performance (VER 2 B/Yamagata Prospective Study) | High sensitivity and specificity, typically aiming for >90% sensitivity and >95% specificity with a tight 95% CI. | Sensitivity: 100.0% (38/38 positives) (95% CI: 90.8 – 100.0).
Specificity: 99.8% (540/541 negatives) (95% CI: 99.0 – 100.0). |
| Clinical Performance (Retrospective VER 1.1 & VER 2 VIC & YAM assays) | 100% positive and negative agreement with the FDA-cleared Influenza B Lineage Genotyping Kit. | VIC VER 1.1: 100.0% Positive Agreement (30/30) (95% CI: 88.7 - 100.0); 100.0% Negative Agreement (157/157) (95% CI: 97.6 - 100.0).
YAM VER 1.1: 100.0% Positive Agreement (96/96) (95% CI: 96.2 - 100.0); 100.0% Negative Agreement (91/91) (95% CI: 96.0 - 100.0).
VIC VER 2: 100.0% Positive Agreement (30/30) (95% CI: 88.7 - 100.0); 100.0% Negative Agreement (157/157) (95% CI: 97.6 - 100.0).
YAM VER 2: 100.0% Positive Agreement (96/96) (95% CI: 96.2 - 100.0); 100.0% Negative Agreement (91/91) (95% CI: 96.0 - 100.0). |

2. Sample Size for the Test Set and Data Provenance

Test Set Sample Sizes:

• Analytical Sensitivity (LOD Equivalency & Inclusivity): Varied. Typically, 3 replicates per dilution/strain. For LOD confirmation, 20 extraction replicates per dilution.
• Analytical Specificity (Cross-Reactivity & Exclusivity): For cross-reactivity, 10 B/Yamagata strains for VIC assays and 10 B/Victoria strains for YAM assays. For exclusivity with Influenza A, 8 Influenza A strains. For exclusivity with non-influenza pathogens, 36 organisms (16 viruses, 19 bacteria, 1 yeast). All tested with both enzyme systems.
• Clinical Performance (InfB Assay Retrospective): 30 positive and 50 negative upper respiratory tract clinical samples.
• Clinical Performance (VER 2 Prospective Study):
  • Total collected: 592 samples.
  • Analyzed: 579 upper respiratory tract clinical specimens.
  • B/Victoria: 5 positive, 574 negative.
  • B/Yamagata: 38 positive, 541 negative.
• Clinical Performance (VER 1.1 & VER 2 Retrospective Study):
  • 126 specimens positive for either influenza B/Victoria or B/Yamagata viruses.
  • 61 specimens negative for influenza B viruses.
  • (An additional 4 lung tissue specimens were tested for informational purposes but not included in performance calculations).

Data Provenance:

• Analytical studies: Laboratory-characterized virus stocks and reference strains (e.g., CDC repositories, ATCC).
• Clinical Performance (InfB Assay Retrospective): Retrospective clinical samples collected during the 2011-2012 influenza season.
• Clinical Performance (VER 2 Prospective Study): Prospective clinical investigation conducted at 3 U.S. public health laboratories using upper respiratory tract specimens collected during the 2016-2017 influenza season.
• Clinical Performance (VER 1.1 & VER 2 Retrospective Study): Retrospective upper respiratory tract clinical samples collected during the 2017 and 2017-2018 influenza seasons.

Country of Origin: Primarily the United States (CDC, U.S. public health laboratories) with some virus strains noted as originating from various international locations (e.g., Hong Kong, Bolivia, Laos, Michigan, etc.) for inclusivity and exclusivity testing, reflecting global diversity.

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

The ground truth for the test sets (both analytical and clinical) appears to be established by molecular diagnostic methods using an FDA-cleared predicate device, the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel: Influenza B Lineage Genotyping Kit (K172091), and/or by using well-characterized virus stocks with known titers (EID50/mL or TCID50/mL).

For clinical samples, they were "previously determined to be positive or negative for influenza B virus" or "determined to be positive for influenza B/Victoria or B/Yamagata viruses using the FDA-cleared Influenza B Lineage Genotyping Kit."

The document does not explicitly mention the use of a specific "number of experts" (e.g., clinicians, virologists, or laboratory professionals) for establishing the individual patient-level clinical ground truth for the test sets in the same way one might describe image interpretation by radiologists. Instead, the ground truth is primarily based on the results of the already FDA-cleared predicate diagnostic test or characterized reference materials. The "competent instructor" trained individuals (users, analysts, and those reporting results) on how to perform and interpret the device results.

4. Adjudication Method for the Test Set

The document does not describe an adjudication method for conflicting results in the test set, such as a 2+1 or 3+1 system. This is typical for molecular diagnostic studies where the comparison is generally made against a well-established reference method (the predicate device or characterized stocks). Any discrepancies would likely be investigated to determine the cause (e.g., retesting, review of sample integrity, or confirmation by alternative gold standard) rather than being resolved by expert consensus in a traditional adjudication panel format. For example, in the LOD studies, "3 out of 3 replicates" or "≥ 95% detection" criteria are used, indicating a reliance on consistent assay performance rather than expert adjudication.

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

No, a multi-reader multi-case (MRMC) comparative effectiveness study was not explicitly described. MRMC studies are typically performed for imaging devices or other diagnostics where human interpretation is a key component and the effect of AI assistance on human performance is being evaluated. This submission is for a molecular diagnostic (RT-PCR kit) where the output is a quantitative measurement (Ct values) and qualitative result (positive/negative, lineage determination) interpreted directly by the instrument software or a trained individual following predefined rules, not a subjective interpretation by multiple human readers.

Therefore, there is no mention of an effect size of how much human readers improve with AI vs. without AI assistance.

6. Standalone (Algorithm Only) Performance

Yes, the studies presented primarily represent standalone performance of the device (or its modified components). The analytical performance evaluations (sensitivity, inclusivity, specificity, exclusivity) directly assess the algorithm's (reagent kit's) ability to detect and differentiate influenza B lineages from viral RNA. The clinical performance studies compare the results of the investigational device directly against the FDA-cleared predicate device on clinical samples. While human technicians perform the laboratory procedures, the "algorithm" here refers to the specific RT-PCR assay chemistry, probes, and primer sets that yield a result, which is then interpreted according to established cut-offs/rules, not an AI algorithm that assists human interpretation.

7. Type of Ground Truth Used

The ground truth used for the studies is a combination of:

• Reference standard/Predicate device: For clinical samples, the ground truth was largely established by the "FDA-cleared CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel: Influenza B Lineage Genotyping Kit."
• Characterized virus stocks: For analytical studies (LOD, inclusivity, cross-reactivity, exclusivity), the ground truth was established using well-characterized influenza virus stocks and other respiratory pathogens with known strain designations, subtypes, and titers (EID50/mL, TCID50/mL, CFU/mL, or ng/µL). This implies a high level of confidence in the identity and concentration of the biological material.

8. Sample Size for the Training Set

The document does not explicitly state a sample size for a "training set" in the context of an algorithm's development. This is because the device described is a molecular diagnostic kit (RT-PCR) with specific primers and probes designed based on biological knowledge of viral sequences, rather than a machine learning or artificial intelligence algorithm that requires a distinct training phase with labeled data.

The "modifications" were "primarily to address recent evolutionary changes in circulating influenza B viruses that may impact the reactivity of the current Influenza B Lineage Genotyping Kit. Two design approaches were evaluated that address specific genetic mutations in the targeted hemagglutinin (HA) gene..." This indicates a design process driven by biological knowledge and engineering based on viral genetics, not data-driven machine learning training.

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

Since there's no explicitly defined "training set" for an AI algorithm in the provided text, the concept of establishing ground truth for it doesn't directly apply. The design of the new VER 1.1 and VER 2 assays was based on scientific understanding of circulating influenza B virus evolutionary changes and specific genetic mutations in the HA gene. This informed the selection of the oligonucleotide primers and probes. The efficacy of these new designs was then evaluated using the analytical and clinical studies described in the rest of the document, where the ground truth was established as detailed in point 7.

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The Influenza A Subtyping Kit contains reagents and controls of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel and 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 determination of the subtype of seasonal human influenza A viruses as seasonal A(H3) and/or A(H1)pdm09 from viral RNA in upper respiratory tract clinical specimens (including nasopharyneed 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;

· To provide epidemiologic information for surveillance of circulating influenza viruses.

Performance characteristics for influenza were established during a seasonal influenza viruses A(HIN1) and A(H3N2) were the predominant influenza A viruses in circulation and during a season when the A(H1N) Jodm09 influenza virus was the predominant influenza A virus in circulation. Performance characteristics may vary with other emerging influenza A viruses.

Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions. Conversely, positive results do not rule out bacterial infection with other viruses. The agent detected may not be the definite cause of disease.

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 unless a BSL 3E facility is available to receive and culture specimens.

All users, and any person reporting results from use of this device should be trained to perform and interpret the results from this procedure by a competent instructor prior to use. CDC Influenza Division will limit the distribution of this device to only those users who have successfully completed a training course provided by CDC instructors or designees.

The CDC Human Influenza Real-Time RT-PCR Diagnostic Panel Influenza A Subtyping Kit is a realtime RT-PCR (rRT-PCR) assay that utilizes the Applied Biosystems® (ABI) 7500 Fast Dx Real-time PCR system. The Influenza A Subtyping Kit consists of oligonucleotide primers, fluorescently labeled hydrolysis probes, and controls which are used in rRT-PCR assays for the in vitro qualitative detection and characterization of influenza virus RNA in respiratory specimens from patients presenting with influenza-like illness (ILI). The oligonucleotide primers and probes for detection of influenza A and 2009 influenza A (swine origin) were selected from highly conserved regions of the matrix (M), and the nucleoprotein (NP), respectively. Oligonucleotide primers and probes for characterization and differentiation of influenza A(H3) and A(H1)pdm09 viruses were selected from highly conserved regions of their HA genes. The Influenza A Subtyping kit also contains primers and probes to detect the human RNase P gene (RP) in control samples and clinical specimens.

This document describes the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel, Influenza A Subtyping Kit (VER 2), which is intended for the qualitative detection and characterization of influenza virus RNA. The document outlines changes from a predicate device (K140851) including modifications to the pdmH1 assay and the removal of the H1 assay.

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

1. Table of Acceptance Criteria and Reported Device Performance

The document primarily focuses on demonstrating the analytical and clinical performance of the modified pdmH1 assay and the H3 assay. While explicit acceptance criteria (e.g., "PPA must be >X%") are not formalized in a table of the document, the studies demonstrate a high level of performance. Based on the results, implicit acceptance criteria would involve:

• Analytical Sensitivity (LOD): Consistent detection at low viral concentrations for A(H1)pdm09 and A(H3) assays using both enzyme kits.
• Inclusivity: Successful detection of diverse A(H1)pdm09 strains.
• Clinical Performance (Positive Agreement): High percentage of agreement with previously positive clinical samples for A(H1)pdm09 and A(H3).
• Clinical Performance (Negative Agreement): High percentage of agreement with previously negative clinical samples for influenza A for both pdm and H3 assays.

• BW: 1/1 (100.0% PPA, 95% CI: 20.7-100.0)
• NPS, NS: 34/35 (97.1% PPA, 95% CI: 85.5-99.5)
• NW: 4/4 (100.0% PPA, 95% CI: 51.0-100.0)
• TS: 2/2 (100.0% PPA, 95% CI: 34.2-100.0)
  Quanta qScript™:
• BW: 1/1 (100.0% PPA, 95% CI: 20.7-100.0)
• NPS, NS: 33/33 (100.0% PPA, 95% CI: 89.6-100.0)
• NW: 4/4 (100.0% PPA, 95% CI: 51.0-100.0)
• TS: 2/2 (100.0% PPA, 95% CI: 34.2-100.0) |
  | Clinical Performance - A(H3) Positive Agreement | High positive agreement with previously positive clinical samples (e.g., >95% PPA with tight CI). | Invitrogen SuperScript™:
• NA: 1/1 (100.0% PPA, 95% CI: 20.7-100.0)
• NPS, NS: 30/30 (100.0% PPA, 95% CI: 88.7-100.0)
• NW: 1/1 (100.0% PPA, 95% CI: 20.7-100.0)
  Quanta qScript™:
• NA: 1/1 (100.0% PPA, 95% CI: 20.7-100.0)
• NPS, NS: 30/30 (100.0% PPA, 95% CI: 88.7-100.0)
• NW: 1/1 (100.0% PPA, 95% CI: 20.7-100.0) |
  | Clinical Performance - A(H1)pdm09 Negative Agreement | High negative agreement with previously negative clinical samples (e.g., >95% NPA with tight CI). | Invitrogen SuperScript™:
• NPS: 53/53 (100.00% NPA, 95% CI: 93.2-100.0)
  Quanta qScript™:
• NPS: 52/52 (100.00% NPA, 95% CI: 93.1-100.0) |
  | Clinical Performance - A(H3) Negative Agreement | High negative agreement with previously negative clinical samples (e.g., >95% NPA with tight CI). | Invitrogen SuperScript™:
• NPS: 29/29 (100.00% NPA, 95% CI: 88.3-100.0)
  Quanta qScript™:
• NPS: 28/28 (100.00% NPA, 95% CI: 87.9-100.0) |

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

• Analytical Sensitivity (LOD):
  • pdmH1 assay: For each of the two A(H1)pdm09 strains tested, 20 replicates of the highest virus dilution were used to confirm LOD.
  • H3 assay: A range finding study used 3 replicates per dilution for the LOD equivalency, but the confirmatory LOD for H3 is not explicitly stated in the same manner as pdmH1. However, the study concludes equivalency based on consistent positive results for 3/3 replicates across a range of dilutions.
• Inclusivity Testing: Ten (10) A(H1)pdm09 strains were tested, with each strain tested in triplicate.
• Cross-Reactivity Testing: Five (5) influenza A(H1) virus strains were tested, each in triplicate.
• Clinical Performance Evaluation:
  • Positive A(H1)pdm09: A total of 42 retrospective clinical samples previously determined positive for A(H1)pdm09 were evaluated. The breakdown by specimen type for reported results is 1 BW, 35 NPS/NS, 4 NW, and 2 TS for Invitrogen; and 1 BW, 33 NPS/NS, 4 NW, and 2 TS for Quanta.
  • Positive A(H3): A total of 32 retrospective clinical samples previously determined positive for A(H3) were evaluated. The breakdown by specimen type is 1 NA, 30 NPS/NS, and 1 NW for both enzyme systems.
  • Negative for Influenza A (pdm assays): A total of 53 retrospective clinical samples (NPS) previously determined negative for influenza A were tested for pdm assays.
  • Negative for Influenza A (H3 assay): A total of 30 retrospective clinical samples (NPS) previously determined negative for influenza A were tested for H3 assay.
• Data Provenance: Retrospective clinical samples collected during the 2011-2012, 2013-2014, and 2015-2016 influenza seasons. The country of origin is not explicitly stated but implied to be the US given the CDC context.

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

The document does not explicitly state the number or qualifications of experts used to establish the ground truth for the clinical test set. The ground truth for clinical samples was based on prior determination using the "CDC Human Influenza rRT-PCR Diagnostic Panel" and, for the 2015-2016 samples, confirmation by genetic sequence analysis. This implies the use of laboratory professionals and possibly virologists or epidemiologists, but specific details on their expertise are not provided.

4. Adjudication Method for the Test Set

The document does not describe an explicit adjudication method for the test set results. For the analytical studies, replicates are used, and the standard reporting of "X/X (+)" indicates all replicates were positive. For clinical samples, results were compared against prior determinations and genetic sequencing, suggesting these served as the reference standards rather than a separate adjudication process.

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 device is a diagnostic assay (RT-PCR kit), not an imaging or interpretation device that would typically involve multiple human readers. The study focuses on the analytical and clinical performance of the assay itself.

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

Yes, the studies conducted are standalone performance evaluations of the diagnostic assay. The device is a "Real-Time RT-PCR Diagnostic Panel," meaning the "algorithm" is the biochemical and molecular process of the assay itself, followed by data interpretation based on established thresholds (e.g., Ct values). The results presented are the output of this assay system.

7. The Type of Ground Truth Used

• Analytical Studies (LOD, Inclusivity, Cross-Reactivity): Ground truth was established using characterized viruses of known titers (ID50/mL or EID50/mL).
• Clinical Performance Studies:
  • For positive samples: Ground truth was based on previous positive determination with the "CDC Human Influenza rRT-PCR Diagnostic Panel" and, for some 2015-2016 samples, confirmed by genetic sequence analysis.
  • For negative samples: Ground truth was based on previous negative determination for influenza A with the "CDC Human Influenza rRT-PCR Diagnostic Panel."

8. The Sample Size for the Training Set

The document does not explicitly describe a "training set" in the context of machine learning, as this is a molecular diagnostic assay. The development of the oligonucleotide primers and probes would involve extensive bioinformatics and laboratory work, but these are not referred to as a "training set" in the presented material. The "characterization" of the device involves testing its performance with known inputs rather than training an algorithm.

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

As no explicit "training set" for a machine learning algorithm is described, this question is not directly applicable. The "ground truth" for the development of the assay components (primers and probes) would have been established through molecular biology techniques, genomic sequencing, and epidemiological data on circulating influenza strains to identify conserved regions and ensure specificity. The document mentions that the primers and probes were "selected from highly conserved regions" of the viral genes, implying this foundational work.

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The Influenza A/H5 Subtyping Kit contains reagents and controls of the CDC Human Influenza Virus RT-PCR Diagnostic Panel and 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 the presumptive identification of virus in patients who may be infected with influenza A subtype A/HS (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.

Performance characteristics for influenza were established during a 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. Performance characteristics may vary with other emerging influenza A viruses.

Testing with the influenza H5a and H5b primer and probe sets should not be performed unless the most current U.S. Department of Health and Human Services (DHHS) clinical and epidemiologic criteria for testing suspect A/ H5 specimens. The definitive identification of influenza A/H5 (Asian lineage) either directly from patient or from virus cultures requires additional laboratory testing, along with clinical and epidemiological assessment in consultation with national influenza surveillance experts.

Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions. Conversely, positive results do not rule out bacterial infection with other viruses. The agent detected may not be the definite cause of disease.

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 a state or local health department for testing. Viral culture should not be attempted unless a BSL 3+ facility is available to receive and culture specimens.

All users, and any person reporting results from use of this device should be trained to perform and interpret the results from this procedure by a competent instructor prior to use. CDC Influenza Division will limit the distribution of this device to only those users who have successfully completed a training course provided by CDC instructors or designees.

The CDC Human Influenza Real-Time RT-PCR Diagnostic Panel Influenza A/H5 Subtyping Kit is a real-time RT-PCR) assay that utilizes the Applied Biosystems® (ABI) 7500 Fast Dx Realtime PCR system. The Influenza A/H5 Subtyping Kit consists of oligonucleotide primers, fluorescently labeled hydrolysis probes, and controls which are used in rRT-PCR assays for the in vitro qualitative detection and characterization of influenza virus RNA in respiratory specimens from patients presenting with influenza-like illness (ILI). The oligonucleotide primers and probes for detection of influenza A viruses (InfA) were selected from highly conserved regions of the matrix (M) protein. Oligonucleotide primers and probes for characterization of avian influenza A/H5 (Asian lineage) viruses (H5a and H5b) were selected from highly conserved regions of their HA genes. The Influenza A/H5 Subtyping kit also contains primers and probes to detect the human RNase P gene (RP) in control samples and clinical specimens.

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

Device Name: CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel, Influenza A/H5 Subtyping Kit (VER 3)
K Number: K153148

1. Table of Acceptance Criteria and Reported Device Performance

The document describes the analytical and clinical performance studies, but directly stated "acceptance criteria" for each metric are not explicitly listed in a formatted table with pass/fail thresholds. Instead, the results are presented as the device's performance, implying these values met internal criteria for submission and approval. Inferring from the nature of diagnostic device validation, these would typically be high percentages for sensitivity and specificity.

Based on the provided data, here's a table summarizing the observed performance:

• Invitrogen SuperScript™: 10^3.8 EID50/mL
• Quanta qScript™: 10^2.4 EID50/mL
• A/duck/Vietnam/NCVD-1544/2012 (A/H5N1):
  • Invitrogen SuperScript™: 10^3.1 EID50/mL
  • Quanta qScript™: 10^3.1 EID50/mL
• A/gyrfalcon/Washington/41088-6/2014 (A/H5N8):
  • Invitrogen SuperScript™: 10^3.35 EID50/mL
  • Quanta qScript™: 10^3.35 EID50/mL
    Reported as the lowest concentration where InfA and both H5a/H5b showed uniform detection. |
    | Analytical Inclusivity | Detection of all listed A/H5 (Asian lineage) isolates representative of different geographic locations and phylogenetic clades, at or near the established LOD. | - Reactive with all 15 tested H5 (Asian lineage) isolates (all 3/3 positive for InfA, H5a, and H5b) with both enzyme systems.
• Predicted to be reactive with A/Bangladesh/3222/2011 (in silico analysis). |
  | Analytical Specificity (Cross-Reactivity) | No detection of common human influenza A (non-H5), human influenza B, swine influenza, avian influenza (non-H5), canine influenza, or equine influenza viruses when tested at high titers. | - InfA assay detected all tested human influenza A and various animal influenza A strains (as expected for an InfA primer/probe set).
• H5a and H5b assays showed no cross-reactivity with human influenza A (non-H5), human influenza B, most animal influenza A (non-H5), and specifically did not detect A/duck/Singapore-Q/F119-3/1997 (H5N3) when tested with Invitrogen SuperScript™. (Note: The H5a/H5b assays did detect A/duck/Singapore-Q/F119-3/1997 H5N3, which is an H5 subtype, albeit not specified as Asian lineage. This is an expected positive result for H5 detection.) |
  | Analytical Specificity (Exclusivity) | No detection (no false positives) of common non-influenza human respiratory viruses, respiratory bacteria, and commensal organisms of the human respiratory tract when tested at high concentrations. | - No detection (all negative) for all 18 bacteria, 1 yeast, and 16 non-influenza human respiratory viruses tested with Invitrogen SuperScript™ for InfA, H5a, and H5b assays. |
  | Clinical Performance (Positive Agreement) | High Positive Agreement (e.g., >90% with a reasonable lower bound for 95% CI). | - Quanta qScript™: 100% (95% CI: 92.9 – 100.00) (50/50)
• Invitrogen SuperScript™: 88.0% (95% CI: 76.2 - 94.4) (44/50) |
  | Clinical Performance (Negative Agreement) | High Negative Agreement (e.g., >90% with a reasonable lower bound for 95% CI). | - Quanta qScript™: 100% (95% CI: 94.4 – 100.00) (65/65)
• Invitrogen SuperScript™: 100% (95% CI: 94.4 - 100.00) (65/65) |

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

• Analytical Sensitivity (LOD):

  • Test Set Sample Size: 20 replicates for each enzyme kit (Invitrogen SuperScript™ and Quanta qScript™) for the highest virus dilution for each of the three A/H5 strains to confirm LOD. Initial testing used 3 replicates.
  • Data Provenance: Laboratory-generated (characterized viruses).

• Analytical Inclusivity:

  • Test Set Sample Size: 3 replicates per virus isolate for 15 A/H5 (Asian lineage) viruses for both enzyme systems. One virus was tested in silico. Total real wet-lab tests: 15 viruses * 3 replicates * 3 assays (InfA, H5a, H5b) * 2 enzyme systems = 270 individual reactions.
  • Data Provenance: Laboratory-generated (characterized viruses representing different geographic locations and phylogenetic clades).

• Analytical Specificity (Cross-Reactivity):

  • Test Set Sample Size: 3 replicates per virus for 14 human and animal influenza A and influenza B viruses tested. Total: 14 viruses * 3 replicates * 3 assays (InfA, H5a, H5b) = 126 individual reactions.
  • Data Provenance: Laboratory-generated (characterized viruses).

• Analytical Specificity (Exclusivity):

  • Test Set Sample Size: A single test for each of 35 non-influenza organisms (16 viruses, 18 bacteria, 1 yeast). Total: 35 organisms * 3 assays (InfA, H5a, H5b) = 105 individual reactions.
  • Data Provenance: Laboratory-generated (propagated, titered, and characterized organisms).

• Clinical Performance Evaluation:

  • Test Set Sample Size:
    • Positive Contrived Samples: 50 samples.
    • Negative Specimens: 65 specimens.
  • Data Provenance:
    • Positive Contrived Samples: Laboratory-generated (grown virus added to A549 cell suspension).
    • Negative Specimens: Obtained from a clinical study conducted during the 2011-2012 influenza season. This suggests the negative specimens were retrospective clinical samples (human respiratory specimens), originating likely from the US where CDC studies are typically conducted.

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 to establish the ground truth for the test set.

• For analytical studies (LOD, Inclusivity, Specificity), the ground truth is established by the known characteristics of the laboratory-controlled and characterized viral and bacterial stocks. This does not typically involve human expert consensus in the same way clinical diagnostics do.
• For the clinical performance evaluation:
  • The "positive contrived samples" have their ground truth by definition (known virus added).
  • The "negative specimens" were previously tested as negative for influenza A by the CDC Human Influenza rRT-PCR Diagnostic Panel. The ground truth for these would have been established by the predicate device's results, which were likely interpreted by trained laboratory personnel, rather than experts in a concensus panel for the purpose of this study.

4. Adjudication Method for the Test Set

The document does not describe an adjudication method for the test set.

• For analytical studies, the results are typically quantitative (e.g., Cq values) or qualitative (positive/negative call) based on predetermined assay thresholds. There's no indication of a need for expert adjudication of these raw results.
• For clinical performance, the comparison is made against the known status of contrived samples or the prior result from a predicate device. An adjudication process (like 2+1, 3+1, etc.) is typically used when the ground truth itself is uncertain or to resolve discrepancies between readers/tests, which is not stated as part of this study design.

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.

• MRMC studies are typically used to compare the performance of human readers (e.g., radiologists, pathologists) with and without AI assistance or against another diagnostic method, often looking at reader agreement and diagnostic accuracy.
• This submission describes a RT-PCR diagnostic panel, which is an in vitro diagnostic (IVD) device. The performance is determined by the assay's ability to detect viral RNA, not by human reader interpretation of complex images or clinical data in the same way an MRMC study would apply. Therefore, the concept of "how much human readers improve with AI vs without AI assistance" is not applicable here.

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

Yes, the studies described are essentially standalone performance evaluations of the device (assay) itself.

• The analytical studies (LOD, Inclusivity, Specificity, Exclusivity) inherently assess the algorithm/reagent's ability to detect targets or avoid off-target detection.
• The 'clinical performance evaluation' also tests the device's ability to correctly identify positive and negative contrived/clinical samples.
• While a human laboratory technologist performs the PCR steps and interprets the output (e.g., reads Cq values and applies the interpretation algorithm to call positive/negative), the study evaluates the device's performance characteristics given those outputs, not the technologist's diagnostic ability with or without the device. The data presented is the direct output of the device's reaction, not a human interpretation layer being separately assessed for its impact.

7. The Type of Ground Truth Used

• Analytical Studies (LOD, Inclusivity, Cross-Reactivity, Exclusivity):
  • Known Characteristics of Propagated and Characterized Viruses/Organisms: For these studies, the ground truth is established by the known identity, titer, and genetic makeup of the laboratory-prepared viral and bacterial stocks. For example, an A/H5N1 virus is known to be A/H5N1 because it was characterized as such.
• Clinical Performance Evaluation:
  • Contrived Samples: The ground truth for positive samples was established by the known addition of virus to a cell suspension.
  • Negative Specimens: The ground truth for negative specimens was established by their prior categorization as "negative for influenza A" using the CDC Human Influenza rRT-PCR Diagnostic Panel (likely the predicate device or a similar established method) in a previous clinical study. This is a form of comparator method ground truth.

8. The Sample Size for the Training Set

The document does not explicitly mention a training set or its sample size.

• For IVD assays like RT-PCR, the development process (which would involve "training" or optimization) typically focuses on primer/probe design, reaction conditions, and calibration using initial lab-generated samples. The 'training' in this context is implicit in the assay development and refinement, rather than a distinct, formal training set for a machine learning algorithm.
• The studies described are for validation (testing), not for development or training.

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

As no explicit training set is mentioned (see point 8), the document does not describe how ground truth for a training set was established.

• If an implicit 'training' phase (assay development) occurred, the ground truth would have been established through well-characterized laboratory reference materials and known viral isolates, similar to the ground truth for the analytical validation studies.

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The B. anthracis Real-Time PCR Assay is an in vitro diagnostic test for the qualitative detection of plasmid and chromosomal DNA sequences from B. anthracis. The assay can be used to test human respiratory samples, whole blood, serum, plasma, swabs from lesions, CSF, pleural fluid, and bacterial culture isolates from individuals suspected of having anthrax.

Results generated from direct specimen testing are presumptive for the identification of B. anthracis. Results generated from culture isolate testing should be used in conjunction with other conventional methods for identification of Bacillus anthracis isolates as part of the LRN Bacillus anthracis Testing Algorithm. The diagnosis of anthrax infection must be made based on history, signs, symptoms, exposure likelihood, and other laboratory evidences in addition to the identification of B. anthracis from cultures or detection directly in clinical specimens.

Use is limited to Laboratory Response Network (LRN) designated laboratories.

The B. anthracis Real-time PCR Assay is also intended for environmental specimen testing for biothreat detection and response. FDA has not evaluated claims related to the use of this assay on environmental specimens.

The B. anthracis Real-time PCR Assay uses a fluorogenic probe, consisting of an oligonucleotide with a reporter dye (FAM) attached to the 5' end and a quencher dye (BHQ1) attached at or near the 3' end. The probe anneals to a specific target sequence located between the forward and reverse primers. During the extension phase of the PCR cycle, the 5′ nuclease activity of Tag polymerase degrades the probe causing the reporter dye to separate from the quencher dye and a fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes and the fluorescence intensity is monitored during the PCR. The Tag polymerase used in this assay is inactive at room temperature. It must be activated by incubation at 95°C, which also minimizes the production of nonspecific amplification products.

Each extracted DNA sample is tested with three B. anthracis primer and probe sets run as individual reactions. The primer and probe sets target genes encoding virulence factors as well as conserved regions of DNA from the B. anthracis chromosome. All three primer and probe sets must be positive for the overall result of the B. anthracis Real-time PCR Assay to be interpreted as positive. Any result that is positive for some, but not all three target regions, is still considered equivocal and follow-up laboratory investigation should be performed per the LRN Bacillus anthracis Testing Algorithm.

Here's an analysis of the acceptance criteria and study information for the B. anthracis Real-time PCR Assay, based on the provided 510(k) summary:

1. Acceptance Criteria and Reported Device Performance

The provided document does not explicitly state quantitative acceptance criteria (e.g., sensitivity, specificity thresholds with confidence intervals) for the B. anthracis Real-time PCR Assay. However, the performance characteristics were established by demonstrating its ability to differentiate B. anthracis from other Bacillus species. The document details the types of studies conducted to establish performance but does not provide specific numerical outcomes.

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

The document does not explicitly state the specific sample sizes used for the test sets in each of the clinical performance evaluations (historical collection of Bacillus sp. isolates, known B. cereus isolates, known bacterial isolates).

• Data Provenance: The studies utilized "a historical collection of Bacillus sp. isolates," "known B. cereus isolates," and "known bacterial isolates." The direct country of origin for these specific collections is not mentioned, but the submitting organization is the Centers for Disease Control and Prevention (CDC) in the USA, suggesting the data is likely from the USA or samples curated by the CDC. The studies appear to be retrospective as they involve testing historical and known isolate collections.

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

The document does not specify the "number of experts" or their "qualifications" involved in establishing the ground truth for the test set.

4. Adjudication Method for the Test Set

The document does not describe an adjudication method (such as 2+1 or 3+1) for the test set.

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

A Multi-Reader Multi-Case (MRMC) comparative effectiveness study comparing human readers with and without AI assistance was not done. This device is an in vitro diagnostic test (a PCR assay), not an AI-assisted diagnostic tool that would typically involve human readers interpreting results.

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

Yes, a standalone performance study was done. The B. anthracis Real-time PCR Assay is an automated PCR-based diagnostic test. Its performance characteristics (Limit of Detection, Analytical Sensitivity, Analytical Specificity, and Clinical Performance) were established by evaluating the assay's ability to detect B. anthracis DNA in various samples. This represents the intrinsic performance of the diagnostic algorithm/assay itself, without direct human interpretation of raw data beyond reading the assay's output.

7. Type of Ground Truth Used

The ground truth for the clinical performance evaluations was established using a "battery of tests performed by the CDC, including the gamma phage lysis, conventional culture, microbiological, and biochemical testing." This indicates the use of expert consensus based on established microbiological and biochemical methods as the reference standard.

8. Sample Size for the Training Set

The document does not specify a "training set" or its sample size. As a PCR assay, its development typically involves primer and probe design and optimization, which isn't usually framed in terms of a "training set" in the same way machine learning models are. The performance characteristics were established using "in-house and multicenter sensitivity studies" and evaluations on historical and known bacterial isolate collections.

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

Since the concept of a "training set" in the context of machine learning isn't directly applicable here, the method of establishing ground truth for development/optimization wasn't explicitly stated. However, similar to the test set, it would logically involve established microbiological and molecular techniques to confirm the identity of B. anthracis and other Bacillus species used during the assay's development and validation.

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