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The JBAIDS Influenza A/H5 (Asian lineage) Detection Kit is intended for use in real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) assays on the Joint Biological Agent Identification and Diagnostic System (JBAIDS) instruments for the in vitro qualitative detection of Influenza A/H5 (Asian lineage) viral RNA in patient nasopharyngeal swab (NPS) or throat swab (TS) specimens for the presumptive laboratory identification of Influenza A/H5 (Asian lineage) virus.

Testing with the JBAIDS Influenza A/H5 (Asian lineage) Detection Kit should be in conjunction with other laboratory testing and clinical observations for the following indications:

1. Providing epidemiological information for the surveillance of human infection with Influenza A/H5 (Asian lineage) virus.
2. Identifying patients who may be infected with Influenza A/H5 (Asian lineage) virus based on clinical and epidemiological risk factors.

Testing with the JBAIDS Influenza A/H5 (Asian lineage) Detection Kit should not be performed unless the patient meets the most current U.S. Department of Health and Human Services (DHHS) clinical and epidemiologic criteria for testing suspected A/H5 specimens.

The definitive identification of influenza A/H5 (Asian lineage) either directly from patient specimens 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.

Use is limited to laboratories with appropriate biosafety equipment and containment procedures. It is intended for use by experienced laboratory personnel who have training in standardized molecular testing procedures and expertise in viral diagnosis, and have received training on the JBAIDS Instrument.

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

The Joint Biological Agent Identification and Diagnostic System (JBAIDS) Influenza A/H5 (Asian lineage) Detection Kit is a real-time reverse transcriptase polymerase chain reaction (rRT-PCR) test kit, which, when used with the JBAIDS instrument and software, allows the qualitative in vitro detection of Influenza A/H5 (Asian lineage) viral RNA. The kit contains two freeze-dried assays with primer and fluorescent-probe sets for the detection of Influenza A/H5 (Asian lineage) viral RNA. In particular, the two assays specifically target distinct regions of the influenza A hemagglutinin gene of the highly pathogenic H5N1 viruses from the Asian lineage, without detection of other influenza A virus subtypes, including the North American lineage influenza A/H5 viruses. The tests are performed using the previously FDA-cleared JBAIDS instrument and software. A human gene target assay will be used as an inhibition and extraction control.

Here's an analysis of the acceptance criteria and study details for the JBAIDS Influenza A/H5 (Asian lineage) Detection Kit, based on the provided 510(k) summary:

1. Table of Acceptance Criteria and Reported Device Performance

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

• Analytical Sensitivity (LoD): 20 individual specimens (from independent donors) per specimen type (NPS, TS) per purification kit (VIBE, Platinum Path) for confirmation (total 80 confirmed LoD tests). The provenance is not explicitly stated as country of origin, but it implies laboratory-prepared specimens. This was a prospective study design for LoD determination.
• Analytical Specificity (Inclusivity & Reactivity): 8 Influenza A/H5 (Asian lineage) strains, each spiked into TS specimens at multiple concentrations. The exact number of replicates per concentration is not detailed, but it was "multiple concentrations."
• Analytical Specificity (Exclusivity): 45 non-target organisms, each spiked into TS specimens at high concentrations. The exact number of replicates is not detailed.
• Clinical Performance (Surrogate Clinical Sensitivity):
  • Influenza A/H5 Strains: 8 strains, each at 4 concentrations (LoD, 5x LoD, 10x LoD, 100x LoD).
  • Seasonal Influenza Strains: 6 strains, each at 2 concentrations (LoD, 100x LoD).
  • Unspiked samples: 6 specimens.
  • All panels were spiked into TS or NPS specimens. Each specimen was purified with two different kits (IT 1-2-3 M VIBE and IT 1-2-3 ™ Platinum Path).
  • This results in a total of:
    • 8 (H5 strains) * 4 (concentrations) * 2 (specimen types) * 2 (purification kits) = 128 H5-containing samples.
    • 6 (seasonal strains) * 2 (concentrations) * 2 (specimen types) * 2 (purification kits) = 48 seasonal influenza samples.
    • 6 (unspiked) * 2 (specimen types) * 2 (purification kits) = 24 unspiked samples.
  • Total test set for surrogate clinical sensitivity: 128 + 48 + 24 = 200 specimens. These were laboratory-prepared "surrogate" clinical samples. This indicates a prospective design for testing these surrogate samples.
• Clinical Specificity:
  • NPS specimens: 314 (VIBE purified) + 299 (Platinum Path purified) = 613 specimens.
  • TS specimens: 298 (VIBE purified) + 283 (Platinum Path purified) = 581 specimens.
  • The total number of unique specimens used for clinical specificity is not explicitly stated (some could be both NPS and TS from the same patient, but the numbers suggest separate sample pools).
  • Total specimens tested for clinical specificity = 613 + 581 = 1194.
  • Data Provenance: Frozen, banked NPS and TS specimens previously tested for respiratory pathogens. They were obtained and tested at 3 different test sites. No specific country of origin is mentioned, but "US Department of Health and Human Services" context suggests US-based data. This is a retrospective study design using banked 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 mention the number or qualifications of experts used to establish ground truth for the test set.

• For Analytical Performance (LoD, Inclusivity, Exclusivity) and Surrogate Clinical Sensitivity, the "ground truth" was established by the precise spiking of known quantities of viral strains into samples by the study designers. This is a laboratory-controlled ground truth, not based on expert interpretation.
• For Clinical Specificity, the ground truth was based on the results of a "CDC rRT Flu Panel" (the predicate device) which had previously tested the banked clinical specimens. The document states "Specimens were removed from the study if they did not have a valid result for the CDC comparator assay," implying the CDC panel's results served as the reference. The expertise involved in the original testing of these banked samples by the CDC panel is not detailed here.

4. Adjudication Method for the Test Set

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

• For analytical studies and surrogate clinical sensitivity, the ground truth was defined by the experimental setup (spiking concentrations, known positive/negative samples).
• For clinical specificity, the predicate device's results served as the comparison, implying a direct comparison rather than an adjudication process of ambiguous results. The JBAIDS software provides automated analysis (positive, negative, uncertain), and invalid/uncertain results required retesting, but this is a retest protocol, not an adjudication process involving experts to resolve discrepancies.

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

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not done.

This device is a molecular diagnostic assay (rRT-PCR kit) that provides automated results (positive, negative, uncertain). The output is interpreted by the JBAIDS software, not by multiple human readers. Therefore, there is no "human-in-the-loop" performance that would be assessed in an MRMC study or an effect size demonstrating human reader improvement with AI assistance.

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

Yes, the studies presented are effectively standalone performance studies of the algorithm/device. The device and its accompanying software provide automated interpretation (positive, negative, uncertain) of the rRT-PCR results. The performance characteristics described (LoD, inclusivity, exclusivity, clinical specificity, surrogate clinical sensitivity) represent the device's ability to achieve these results directly from the sample, without human interpretative input into the primary result. Human intervention is limited to operating the instrument, preparing samples, and initiating retesting for uncertain/invalid results, not interpreting the raw amplification curves as "positive" or "negative."

7. The Type of Ground Truth Used

• Analytical Studies (LoD, Inclusivity, Exclusivity): Laboratory-controlled ground truth (known viral strains spiked at specific concentrations).
• Surrogate Clinical Sensitivity: Laboratory-controlled ground truth (known viral strains spiked into clinical matrices at specific concentrations).
• Clinical Specificity: Comparator Assay ground truth. The "CDC rRT Flu Panel" was used as the reference standard for the banked clinical specimens.

8. The Sample Size for the Training Set

The document does not provide information about a separate "training set" or its sample size. This is common for molecular diagnostic kits like this. The device is likely developed and validated using a structured process that involves internal data for assay optimization and parameter setting (which could broadly be considered "training" in a development sense), but there isn't a formally defined "training set" in the context of machine learning model development as typically described in AI/ML device studies. The performance characteristics are derived from the test sets described above.

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

Since no specific "training set" is identified in the document, there's no information on how its ground truth would have been established. Any internal development and optimization would likely have relied on laboratory-controlled samples with known viral presence/absence and concentration.

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The Human Influenza Virus Real-time RT-PCR Detection and Characterization Panel (rRT-PCR Flu Panel) is intended for use in Real-time RT-PCR assays on an AB1 7500 Fast Dx Real-time PCR instrument in conjunction with clinical and epidemiological information:

• for qualitative detection of influenza virus type A or B in symptomatic patients from viral RNA in nasopharyngeal and/or nasal swab specimens,
• for determination of the subtype of seasonal human influenza A virus, as seasonal A/H1 or A/H3, if present, from viral RNA in nasopharyngeal and/or nasal swab specimens,
• for presumptive identification of virus in patients who may be infected with influenza A subtype A/H5 (Asian lineage) from viral RNA in human respiratory specimens and viral culture in conjunction with clinical and epidemiological risk factors.
• to provide epidemiologic information for surveillance for influenza viruses.

The CDC Human Influenza Virus Real-time RT-PCR Detection and Characterization Panel (rRT-PCR Flu Panel) is a panel of oligonucleotide primers and dual-labeled hydrolysis (TagMan®) probes which may be used in real-time RT-PCR assays using the ABI 7500 Fast Dx Real-Time PCR instrument for the in vitro qualitative detection and characterization of human influenza viruses (RNA) in respiratory specimens from patients presenting with influenza-like illness (ILI). Detection of viral RNA not only aids in the diagnosis of illness caused by seasonal and novel influenza viruses in patients with ILI, but also provides epidemiologic information on influenza and aids in the presumptive laboratory identification of specific novel influenza A viruses.

Acceptance Criteria and Device Performance for CDC Human Influenza Virus Real-time RT-PCR Detection and Characterization Panel

1. Table of Acceptance Criteria and Reported Device Performance

Study Details:

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

• Clinical Performance (Seasonal Influenza - InfA, InfB, H1, H3):
  • Sample Size: 415 total prospective specimens.
  • Data Provenance: Prospective collection from US state public health laboratories during the 2006-2007 respiratory virus season (February-April).
  • Specimen Type: Nasal and nasopharyngeal swabs.
• Clinical Performance (Influenza A/H5N1):
  • Clinical Specimens: 24 retrospective A/H5 influenza samples.
  • Data Provenance: Retrospective from suspect positive cases received at CDC, "diverse geographic locations."
  • Cultured Specimens: Not explicitly stated sample size, but also used for H5 detection with 100% agreement.
  • Prospective Clinical Specimens (for negative agreement): Implied to be part of the 415 prospective seasonal specimens, where no H5 was detected.

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

• No specific number or qualifications of experts are explicitly mentioned for establishing ground truth.
• The reference method for prospective seasonal influenza samples was "rapid culture (shell vial) followed by direct fluorescent antibody screening and identification." This implies laboratory personnel with expertise in virology and cell culture.
• Influenza A/H5 and discrepant prospective seasonal influenza samples were "further analyzed by bidirectional sequencing," suggesting molecular biology experts.

4. Adjudication Method for the Test Set:

• No formal adjudication method (e.g., 2+1, 3+1) is explicitly described.
• The ground truth for prospective seasonal influenza was based on rapid culture (shell vial) followed by direct fluorescent antibody screening and identification.
• Discrepant prospective seasonal influenza samples and influenza A/H5 samples were further analyzed by bidirectional sequencing. This serves as an additional, higher-resolution method for resolving discrepancies or confirming difficult cases.

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

• No, an MRMC comparative effectiveness study was not done. This device is a diagnostic assay (RT-PCR panel) for detecting and characterizing influenza viruses, not an imaging device or a device that requires human interpretation in the same way an AI-powered image analysis tool would. Therefore, the concept of "human readers improve with AI vs. without AI assistance" does not apply here.

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

• Yes, the performance presented for the rRT-PCR Flu Panel is standalone (algorithm only, without human-in-the-loop performance). This device is a molecular diagnostic kit, and its performance metrics (sensitivity, specificity, LoD, etc.) reflect the intrinsic analytical and clinical capabilities of the assay itself when executed according to protocol. Human involvement is in sample preparation, running the assay, and interpreting the raw output (e.g., Ct values), but the "diagnosis" of the presence and type of virus is determined by the output of the PCR reaction.

7. The Type of Ground Truth Used:

• Expert Consensus and "Gold Standard" Laboratory Methods:
  • For prospective seasonal influenza, the ground truth was established by gold standard virus culture testing (rapid culture (shell vial) followed by direct fluorescent antibody screening and identification).
  • For influenza A/H5 and discrepant prospective seasonal influenza samples, the ground truth was further analyzed and confirmed by bidirectional sequencing. This suggests a form of expert-validated, highly accurate laboratory confirmation.

8. The Sample Size for the Training Set:

• Not Applicable / Not Explicitly Stated for a "training set" as understood in machine learning. This device is a molecular diagnostic panel based on designed primers and probes, not a machine learning model that undergoes explicit training. The "development" and "optimization" would have involved testing various primer/probe combinations against known virus strains, but this isn't analogous to a "training set" for an AI algorithm.
• However, for Analytical Specificity (Inclusivity), the document mentions initial testing with "ten (10) influenza virus strains of A/H/N1, A/H3N2, and influenza B" and "24 influenza A/H5N1 clinical samples tested retrospectively" to demonstrate the panel's ability to detect different strains. This could be considered akin to an internal validation set during development.

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

• As above, the concept of a "training set" for this type of medical device (RT-PCR panel) is not directly applicable in the same way it would be for an AI algorithm.
• The "ground truth" for the various influenza virus strains and non-influenza pathogens used in analytical studies would have been primarily established through known viral cultures, characterized bacterial/yeast cultures, and molecular sequencing (e.g., 16S ribosomal RNA bi-directional sequencing for bacteria, bi-directional sequencing for non-influenza respiratory viruses), all validated by standard laboratory practices.

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The Influenza A/H5 (Asian Iineage) Virus Real-time RT-PCR Primer and Probe Set is intended for the in vitro qualitative detection of Influenza A/H5 (Asian lineage) virus RNA either directly in patient respiratory specimens or in viral cultures for the presumptive laboratory identification of Influenza A/H5 (Asian lineage) virus.

Testing with the Influenza A/H5 (Asian lineage) Virus Real-time RT-PCR Primer and Probe Set should be used in conjunction with other laboratory testing and clinical observations for the following indications:

• Providing epidemiological information for the surveillance of human infection with Influenza A/H5 (Asian lineage) virus
• Identifying patients who may be infected with Influenza A/H5 (Asian lineage) virus based on clinical and epidemiological risk factors
  The definitive identification of influenza A/H5 (Asian lineage) either directly from patient specimens or from viral 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.

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

The Influenza A/H5 (Asian lineage) Virus Real-time RT-PCR Primer and Probe Set is composed of 2 primer pairs (4 unlabeled oligonucleotides) and 3 labeled probes, along with an inactivated virus control. These reagents are for use in single-tube realtime RT-PCR testing to directly detect novel Influenza A virus specific RNA in human respiratory specimens, or in viral cultures. The real-time PCR process simultaneously amplifies and detects nucleic acid targets in the same reaction.

The primer and probe sets (FluA2 and FluA3) target two distinct RNA regions that are both present in the influenza A/H5 (Asian lineage) hemagglutinin (HA) gene of highly pathogenic H5N1 viruses from the Asian lineage. These target regions were chosen to allow specific detection of Asian lineage influenza A/H5 viruses without detection of other influenza virus subtypes, including the North American lineage influenza A/H5 viruses (e.g., avian H5N2 strains).

Note: There are two lineages of avian influenza A/H5 viruses: the Eurasian (Asian) and North American (American) lineages. Viruses from these two lineages are genetically different. All known human influenza H5 infections have been caused by highly pathogenic viruses of the Asian lineage.

Primers/Probes: The 2 primer and probe sets (FluA2 and FluA3), each target a different region within the HA gene. The FluA2 target is in the HA2 region of the HA gene and the FluA3 target spans the cleavage site of the HA gene. These sets were selected from multiple candidates based on preliminary assessment of reaction efficiency and primer-dimer effects. FluA2 contains an equal mixture of two probes, to ensure optimum homology with viruses within both clades. The probes are labeled with FAM (6-carboxyfluorescein) and Blackhole Quencher™ 1 (BHQTM1). The BHQ chemistry is designed to minimize non-specific fluorescence. Experimental efficiency values of 100% +/- 5% are considered optimal. Tagman reaction efficiencies of FluA2 and FluA3 sets were demonstrated to be 100.6% (R2=0.996) and 100.3% (R2=0.996).
Influenza A/H5N1 positive control (500 µL of virus suspended in 0.01 M PBS): a reverse-engineered vaccine candidate virus that may safely be handled in BSL-2. Inactivated with beta- propiolactone (0.05%). The reassortant virus is noninfectious in chickens and USDA has removed it from the select agent list, classifying it as a BSL2 infectious agent. Additionally, the inactivated virus preparation is non-infectious in embryonated chicken eggs.

Other reagents or accessories required to perform testing with the device are:

Qiagen QuantiTect™ Probe RT-PCR Kit (Qiagen), a master-mix for reversetranscription and cDNA amplification.
Materials for extraction and purification of specimens (three commercial extraction kits are suggested for use in isolating RNA in the test procedure based on specifications meeting the requirements for quality of RNA to be used in the FluA2 and FluA3 reactions). These extraction procedures were used during development and testing of the Influenza A/H5 (Asian lineage) Virus Real-time RT-PCR Primer and Probe Set.
An RNase P (RP) Real-time PCR Primer and probe set that targets the human ribonuclease P (RP) sequence. Extracted clinical specimens should contain human RNA. The RP assay thus serves as a control to ensure RNA resulted from the extraction of clinical specimens.

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

The document does not explicitly state pre-defined acceptance criteria for device performance. However, based on the reproducibility study results, the implied criteria for positive detection (presumptive positive) appear to relate to the virus concentration and agreement with expected results. For negative samples, the acceptance criterion is a 100% agreement with expected results or near 100% given the one equivocal observation.

• 400 TCID50/mL: All 36 samples detected (100%)
• 40 TCID50/mL: 47/55 (85.5%) positive; 52/55 (94.5%) positive or equivocal
• 4 TCID50/mL: 32/36 (88.9%) positive; 35/36 (97.2%) positive or equivocal
  Specific Instrument Performance (Agreement with Expected Results for Positive Samples):
• ABI 7000: 100% at 400, 40, and 4 TCID50/mL (Swab, low 0.4 TCID50/mL: 10% positive, 30% equivocal, 70% negative)
• ABI 7700: 100% at 400, 40, and 4 TCID50/mL (Swab, low 0.4 TCID50/mL: 0% positive, 28.5% equivocal, 71.5% negative)
• LightCycler®: 100% at 400 TCID50/mL, 78% at 40 TCID50/mL, 89% at 40 TCID50/mL (liquid), 67% at 4 TCID50/mL
• SmartCycler®: 100% at 400 TCID50/mL, 67% at 40 TCID50/mL, 100% at 40 TCID50/mL (liquid), 90% at 4 TCID50/mL |
  | Reproducibility (Negative Samples) | High agreement with expected negative results (e.g., 100% negative). | Overall (across all instruments): 71 negative control samples had 1 equivocal result, yielding 98.6% agreement with expected results.
  Specific Instrument Performance (Agreement with Expected Results for Negative Samples):
• ABI 7000: 100% for both liquid and swab negative samples
• ABI 7700: 100% for both liquid and swab negative samples
• LightCycler®: 100% for liquid negative, 89% for swab negative (due to 1 equivocal)
• SmartCycler®: 100% for both liquid and swab negative samples |
  | Detection Limit | Not explicitly stated as a criterion, but values are given as a performance characteristic. | - ABI 7000 and ABI 7700: 4 TCID50/mL for each target
• LightCycler® and SmartCycler®: 40 TCID50/mL for each target |
  | Analytical Specificity (Cross-reactivity) | No cross-reactivity with non-H5 Asian HA types, non-influenza respiratory viruses, or bacteria. | No cross-reactivity observed with the tested viral strains or bacterial species (17 stock strains of non-H5 influenza, 8 non-influenza respiratory viruses, 11 bacterial species). |
  | Purity of Virus Control | Non-infectious | The inactivated virus control is non-infectious in chickens and embryonated chicken eggs. |
  | Clinical Sensitivity | Not estimable due to rarity of Asian H5 virus infections. | Not estimated. |
  | Clinical Specificity | Not estimable due to rarity of Asian H5 virus infections. | Not estimated. |
  | Agreement with Lab-Confirmed Cases (H5N1) | High agreement with confirmed patient diagnosis. | 10/22 (45%) specimens were presumptive positive, and another 9 were equivocal. Thus, 19/22 (86%, 65.1-97.1% CI) specimens showed A/H5 (Asian lineage) virus detection in one or both probe-set reactions, agreeing with the patient case diagnosis (from 10 patients). All lab-confirmed cases had at least one positive test when multiple specimens were tested. |
  | Agreement with Banked Respiratory Specimens (Negative) | High agreement with expected negative results. | Overall agreement was 98.5% (201/204, 95.8-99.7% CI). One sample was positive (confirmed H5N1), and two were equivocal (contained A/H3). |
  | Agreement with Banked Tissue Culture Samples (Negative) | High agreement with expected negative results. | Agreement was 95.2% (20/21, 76.2-99.9% CI). One sample was equivocal (contained A/H3). |

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

• Reproducibility Study (Simulated Specimens):

  • Test Set Size: 6 simulated respiratory specimens (3 swabs, 3 liquid) at various virus concentrations (including negative), tested in 10 laboratories (ABI 7000: 60 tests), 7 laboratories (ABI 7700: 42 tests), 9 laboratories (LightCycler®: 54 tests), and 10 laboratories (SmartCycler®: 60 tests).
  • Total Samples Tested: 216 tests distributed across the instruments. Each simulated specimen type was likely tested multiple times within each lab or across labs.
  • Data Provenance: The simulated specimens were "spiked with both human cellular material and titered amounts of the virus control material." The control virus itself is a "reverse-engineered vaccine candidate virus." This indicates contrived samples, likely prepared in a laboratory setting (e.g., CDC). This is a prospective study design for performance evaluation.

• Clinical Study (Laboratory-confirmed patient cases):

  • Test Set Size: 27 specimens from 10 laboratory-confirmed H5N1 patients and 2 specimens from 1 contact patient (not confirmed for H5N1).
  • Data Provenance: Retrospective. Specimens were from "laboratory-confirmed patient cases (from Indonesia) of influenza H5N1 virus."

• Clinical Study (Banked human respiratory specimens):

  • Test Set Size: 204 banked human respiratory specimens.
  • Data Provenance: Retrospective. Specimens were received by CDC Influenza Branch Laboratory from Vietnam (168), United States (21), Thailand (11), Kuwait (4).

• Clinical Study (Banked tissue culture samples):

  • Test Set Size: 21 banked tissue culture samples.
  • Data Provenance: Retrospective. Received by CDC Influenza Branch Laboratory from international locations (18) and the United States (3).

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts

• Reproducibility Study: No human experts were involved in establishing ground truth for the simulated test set. The ground truth was based on the known concentration of the spiked virus and the expected negative status of non-spiked samples.

• Clinical Study (Laboratory-confirmed patient cases):

  • Number of Experts: Not explicitly stated, but "Confirmation by independent (WHO or Indonesian) laboratory or sequence analysis" indicates multiple experts and methodologies were involved. "Multiple WHO methods (culture, sequencing, RT-PCR testing for multiple HA targets) were used to characterize H5N1 in these patients."
  • Qualifications: "National influenza surveillance experts" and staff in "WHO-recognized RT-PCR assays" and the "WHO Collaborating Center for Surveillance, Epidemiology and Control of Influenza" are implied. These would be highly qualified virologists, microbiologists, and molecular biologists.

• Clinical Study (Banked human respiratory specimens):

  • Number of Experts: Unspecified, but confirmed by "Influenza Branch4 Lab Real-time RT-PCR Panel Result." This refers to the CDC's Influenza Branch Laboratory, a WHO Collaborating Center, implying a team of experts.
  • Qualifications: Experts at a "WHO Collaborating Center for Surveillance, Epidemiology and Control of Influenza" and a "Bioterrorism Rapid Response and Advanced Technology Laboratory" would be highly qualified specialists in virology, molecular diagnostics, and public health.

• Clinical Study (Banked tissue culture samples):

  • Number of Experts: Unspecified, but "Influenza Branch3 Lab Real-time RT-PCR Panel Result" implies experts from the CDC Influenza Branch Laboratory.
  • Qualifications: Similar to the above, highly qualified specialists in virology and molecular diagnostics.

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

• Reproducibility Study: No adjudication method was necessary for the simulated test set, as the ground truth was based on engineered virus concentrations.

• Clinical Studies (Laboratory-confirmed patient cases, Banked human respiratory specimens, Banked tissue culture samples):

  • The term "adjudication" in the sense of resolving discrepancies between multiple readers is not directly applicable. For patient cases, confirmation was achieved through "independent (WHO or Indonesian) laboratory or sequence analysis" and "Multiple WHO methods (culture, sequencing, RT-PCR testing for multiple HA targets)." This suggests a consensus-based approach or reconciliation of results by reference methods rather than an explicit "X+Y" adjudication.
  • For banked specimens, the "Influenza Branch Lab Real-time RT-PCR Panel Result" serves as the reference standard, implying a thorough process by a specialized laboratory.

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

• There was no MRMC comparative effectiveness study done.
• This device is a diagnostic kit (RT-PCR primer and probe set), not an AI or imaging device that would typically involve human "readers" in the sense of interpreting outputs that AI could aid. The output is a quantitative (CT value) and qualitative (positive/equivocal/negative) result based on amplification.

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

• Yes, the performance studies primarily assess the standalone performance of the diagnostic kit. The "reproducibility study" evaluates the kit's performance across different laboratories and instruments, and the "clinical studies" evaluate its ability to detect the virus in patient and banked samples, independent of human interpretation improvements.
• While laboratory technicians operate the equipment, the interpretation of the RT-PCR results (based on crossing thresholds and specific target detection) follows established protocols without a human "in-the-loop" decision-making process in the same way an AI-assisted diagnostic device might be evaluated. The result is directly derived from the reaction.

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

• Reproducibility Study: Engineered/Known Concentration. The ground truth was based on the known, spiked concentrations of the Influenza A/H5N1 virus into simulated respiratory specimens.

• Clinical Study (Laboratory-confirmed patient cases): Multi-method Laboratory Confirmation / Consensus. The ground truth was established by "independent (WHO or Indonesian) laboratory or sequence analysis" and "Multiple WHO methods (culture, sequencing, RT-PCR testing for multiple HA targets)." This represents a form of expert-driven, multi-modal laboratory confirmation, akin to an "expert consensus" based on advanced diagnostic techniques.

• Clinical Studies (Banked human respiratory specimens and tissue culture samples): Reference Laboratory Panel Result. The ground truth was established by the "Influenza Branch Lab Real-time RT-PCR Panel Result" at CDC (a WHO Collaborating Center). This acts as a highly reliable reference standard, representing the most definitive laboratory diagnosis available.

8. The sample size for the training set

• The document describes a diagnostic kit (reagents), not an AI or machine learning model. Therefore, there is no specific "training set" in the conventional sense of data used to train an algorithm.
• The "development" of the primer and probe sets involved selecting them from "multiple candidates based on preliminary assessment of reaction efficiency and primer-dimer effects." This involved testing, but it's part of the assay design and optimization, not machine learning model training.

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

• As stated above, there is no "training set" for an algorithm in this context. The development process for the primer and probe sets involved analytical testing with known viral strains and concentrations to ensure specificity and efficiency, which inherently relies on established knowledge of viral genetics and laboratory standards (e.g., known positive controls, established culture methods).

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