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Adenovirus R-gene® US Assay is a Real Time PCR in vitro diagnostic test for the rapid and qualitative detection of Adenovirus viral DNA isolated and purified from nasopharyngeal swab or nasopharyngeal wash/aspirate specimens obtained from individuals exhibiting signs and symptoms of acute respiratory infection. The intended use for this test is to aid in the diagnosis of respiratory Adenovirus infection in humans in conjunction with other clinical and laboratory findings. The test detects, but does not differentiate, Adenovirus species (A, B, C, D, E, F and G). Negative results do not preclude Adenovirus infection and should not be used as the sole basis for treatment or other patient management decisions.

The ADENOVIRUS R-gene® US Assay is a Taqman based Real Time PCR Assay that enables detection of human Adenovirus DNA and Internal Control 2. An overview of the procedure is as follows: 1. Collect nasopharyngeal specimens from symptomatic patients. Two transport media may be used UTM or M4RT (not provided with kit). 2. Add an Internal Control 2 (IC2) to every sample and to the W0 reagent, to monitor for inhibitors present in the specimens and check the extraction step. 3. Perform extraction and purification of nucleic acids using a NucliSENS® easyMAG™ System (bioMérieux) and the Automated Magnetic Extraction Reagents (bioMérieux). 4. Add purified nucleic acids to the ready to use R10 amplification premix included in the ADENOVIRUS R-gene® US kit. The R10 amplification premix contains oligonucleotide primers and target-specific oligonucleotide probes. The primers and probes are complementary to a fragment located in the Hexon gene region and to the internal control 2 DNA sequence. The probes are duallabeled with a reporter dye attached to the 5'-end and a quencher dye attached to the 3'-end. 5. Perform amplification of DNA in a Cepheid SmartCycler® 2.0 instrument. In this process, the probe anneals specifically to the DNA template followed by primer extension and amplification. The 5' - 3' exonuclease activity of the Tag polymerase cleaves the probe separating the reporter dye from the quencher. This generates an increase in fluorescent signal upon excitation from a light source. With each cycle, additional reporter dye molecules are cleaved from their respective probes, further increasing fluorescent signal. The amount of fluorescence at any given cycle is dependent on the amount of amplification products present at that time. Fluorescent intensity is monitored during each PCR cycle by the real-time instrument. 6. Interpretation.

Acceptance Criteria and Study for Adenovirus R-gene® US Assay

Here's a breakdown of the acceptance criteria and study details for the Adenovirus R-gene® US Assay based on the provided 510(k) summary:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are not explicitly stated as clear pass/fail thresholds in the provided document, but rather implied through the presentation of performance studies. The reported device performance is presented as sensitivity and specificity values against a reference method.

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

• Test Set Sample Size:

  • Clinical Study: 1576 samples (1183 swab specimens, 393 NP wash/aspirate specimens).
  • LoD Study: 4 to 6 dilutions for each of the 22 Adenovirus types tested, for a total of 60 to 90 amplifications/detections.
  • Analytical Specificity: Panels of 61 potentially cross-reacting microorganisms.
  • Carry-Over/Cross-Contamination: 55 High Positive and 55 Negative samples over 5 extraction runs and 4 amplification runs.
  • Precision (Within-Laboratory): 150 Moderate Positive, 120 Low Positive, 90 High Negative total replicates.
  • Precision (Between-Laboratory): 450 Moderate Positive, 360 Low Positive, 270 High Negative total replicates across 3 sites.
  • Cut-off Determination: 218 negative and high negative samples; 184 clinical samples (preliminary study, France); 1576 clinical samples (US clinical studies).

• Data Provenance:

  • Clinical Study: Multi-center prospective investigational study conducted across 3 geographically diverse institutions in the US.
  • Preliminary Cut-off Study: Performed at Caen Hospital (France).
  • Analytical, Carry-over, Interference, and Within-Laboratory Precision studies: Performed at bioMérieux SA – ARGENE site (France).
  • Between-Laboratory Precision: Performed at 3 external sites in the US.
  • The document implies the data is mostly prospective for the clinical evaluation, as it describes a multi-center investigational study with samples collected from September 2010 - November 2011.

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

The document does not specify the number or qualifications of experts used to establish the ground truth for the test set.

4. Adjudication Method for the Test Set

The primary reference method used for the clinical study was "rapid culture (shell vial) followed by direct fluorescent antibody (DFA) screening and identification" using the D3Ultra™ DFA Respiratory Virus screening & ID kit from Diagnostic Hybrids (DHI).

For discrepant results, a "confirmed as positive for adenovirus using Real Time PCR" method was noted, implying an additional confirmatory PCR outside of the device being evaluated. The details of this confirmatory PCR (e.g., specific assay, blinding, adjudication process) are not provided.

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. The study described compares the performance of the Adenovirus R-gene® US Assay (an automated test) against a reference laboratory method (culture + DFA), not against human readers with and without AI assistance.

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

Yes, the studies described are for the standalone performance of the Adenovirus R-gene® US Assay. This is an in vitro diagnostic (IVD) test, where the "algorithm" (the PCR assay and associated interpretation rules) operates independently to generate a result. There is no human interaction with an AI component for interpretation as would be the case in an imaging diagnostic AI device.

7. The Type of Ground Truth Used

The primary ground truth for the clinical studies was viral culture followed by direct fluorescent antibody (DFA) staining. For discrepant results from this primary method, "Real Time PCR" was used as a confirmatory method, which can be considered a form of expert consensus if adjudicated by laboratory experts, or a higher-sensitivity reference method. For the Limit of Detection (LoD), titered viral strains (TCID50/mL) and quantified plasmids (copies/mL) were used.

8. The Sample Size for the Training Set

The document does not explicitly state a separate "training set" size for the clinical performance evaluation. For IVD devices, analytical studies (LoD, specificity, interference, precision) often contribute to the development and optimization of the assay itself, which can be seen as an iterative "training" process for the assay's design and interpretation rules (e.g., cut-off determination). The document mentions "preliminary study, performed at Caen Hospital (France), on 184 clinical samples, prior to US clinical studies" for cut-off determination, which could be considered part of the development/training phase for establishing the assay's parameters.

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

For the preliminary cut-off study using 184 clinical samples at Caen Hospital (France), the method for establishing ground truth is not explicitly detailed. However, given the context of molecular assays, it would likely have involved an established laboratory reference method for Adenovirus detection, similar to or the same as the one used for the main clinical study (culture + DFA or another validated PCR). For the analytical studies, the ground truth was established by using well-characterized and quantified viral strains or plasmids with known concentrations.

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The Diagnostic Hybrids, Inc. device, D3 FastPoint L-DFA RSV/MPV Identification Kit is intended for the qualitative identification of respiratory syncytial virus and human metapneumovirus in nasal and nasopharyngeal swabs and aspirates/washes specimens from patients with signs and symptoms of respiratory infection by direct detection of immunofluorescence using monoclonal antibodies (MAbs).

It is recommended that specimens found to be negative for respiratory syncytial virus after examination of the direct specimen result be confirmed by cell culture. Specimens found to be negative for human metapneumovirus after examination of the direct specimen results should be confirmed by an FDA-cleared human metapneumovirus molecular assay. Negative results do not preclude respiratory syncytial virus and human metapneumovirus infection and should not be used as the sole basis for diagnosis, treatment or other management decisions.

The D3 FastPoint L-DFA RSV/MPV Identification Kit uses a blend (called a "L-DFA Reagent'") of viral antigen-specific murine monoclonal antibodies that are directly labeled with either R-phycoerythin (PE) (respiratory syncytial virus) or fluorescein isothiocyanate (FITC) (human metapneumovirus) for the rapid identification of respiratory syncytial virus and human metapneumovirus in nasal and nasopharyngeal swabs and aspirates from patients with signs and symptoms of respiratory infection.

The cells to be tested are derived from respiratory specimens from patients with signs and symptoms of respiratory infection. The cells are permeabilized and stained concurrently in a liquid suspension format with the L-DFA Reagent. After incubating at 35℃ to 37℃ for 5-minutes, the stained cell suspensions are rinsed with 1X PBS. The rinsed cells are pelleted by centrifugation and then re-suspended with the Resuspension Buffer and loaded onto a specimen slide well. The cells are examined using a fluorescence microscope. Cells infected with RSV will exhibit golden-yellow fluorescence due to the PE. Cells infected with hMPV will exhibit apple-green fluorescence due to the FITC. Non-infected cells will exhibit red fluorescence due to the Evans Blue counter-stain. Nuclei of intact cells will exhibit orange-red fluorescence due to the propidium iodide.

Acceptance Criteria and Study for D3 FastPoint L-DFA RSV/MPV Identification Kit

1. Table of Acceptance Criteria and Reported Device Performance

The provided document doesn't explicitly state numerical acceptance criteria for all performance metrics. However, for a 510(k) submission, implied acceptance is often "substantially equivalent" to predicate devices, and for clinical performance, high sensitivity and specificity are expected. The reproducibility study explicitly aims for 100% agreement.

Note: The "acceptance criteria" are inferred based on standard expectations for diagnostic device performance and the detailed reporting of study outcomes, particularly the 100% agreement for reproducibility and the very high sensitivity/specificity for RSV. The lower sensitivity for hMPV in NP swab samples might be within acceptable limits given the context of medical device approval for challenging targets.

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

• Reproducibility Test Set:
  • Sample Size: A reproducibility panel consisting of 5 members (low RSV, low hMPV, mixed RSV/hMPV, mixed hMPV/RSV, negative). Each panel member was tested daily in two separate runs for 5 days by 4 different laboratories, resulting in 40 total runs. This yielded 280 total tests (across all panel members and runs) with individual results reported for expected positive and negative wells.
  • Data Provenance: The study was conducted at four different laboratories. The document does not specify the country of origin but implies U.S. clinical laboratories (referencing "U.S. clinical laboratories" for clinical performance). It's a prospective study in the sense that the testing itself was performed to assess reproducibility.
• Limit of Detection (LoD) Test Set:
  • Sample Size: Dilution series of infected model cells were used. For each virus (RSV and hMPV A1), 10 replicate microscope slides were prepared for each dilution level. The specific number of dilutions isn't explicitly stated as a single number but spanned from 1000 infected cells/mL down to 0.8 or 1.5 infected cells/mL, with 10 replicates for each dilution.
  • Data Provenance: Laboratory study, likely internal to the manufacturer or a contracted lab.
• Analytical Reactivity (Inclusivity) Test Set:
  • Sample Size: 3 RSV virus strains and 4 hMPV virus strains were evaluated. For each strain, "low concentration infected cell suspensions (approximately 4% cells infected, 25 to 50 infected cells)" were prepared.
  • Data Provenance: Laboratory study.
• Clinical Performance Test Set:
  • Sample Size: 1519 total respiratory specimens (nasal and nasopharyngeal swabs and aspirates/washes).
  • Data Provenance: Prospective studies at 4 geographically diverse U.S. clinical laboratories during the 2009 respiratory virus seasons (January 2009 - March 2009). The specimens were "excess, remnants of respiratory specimens that were prospectively collected from symptomatic individuals suspected of respiratory infection, and were submitted for routine care or analysis by each site, and that otherwise would have been discarded." Individual specimens were delinked from patient identifiers.

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

The document does not explicitly state the number or qualifications of experts for establishing ground truth as a separate role. Instead, the ground truth for clinical performance was established using a composite comparator method:

• RSV: Direct Specimen Fluorescent Antibody (DSFA) test with an FDA-cleared predicate device, followed by viral culture confirmation of all negatives from the comparator DSFA test.
• hMPV: DSFA with an FDA-cleared predicate device, followed by confirmation of all negative specimens from the comparator DSFA test using a validated hMPV real-time RT-PCR, which was then followed by bi-directional sequencing analysis.

This implies that the "ground truth" was determined by the results of these established and confirmed laboratory methods, rather than by human expert consensus or adjudication of raw images/output from the test device solely.

4. Adjudication Method for the Test Set

For the clinical studies, an explicit "adjudication method" involving human experts reviewing conflicting results is not detailed. Instead, a composite comparator algorithm was used to define "true positive" and "true negative":

• "True positive" for RSV was defined as any sample that tested positive by the comparator DSFA test or viral culture.
• "True positive" for hMPV was defined as any sample that tested positive by the comparator DSFA test OR had bi-directional sequencing data meeting pre-defined quality acceptance criteria that matched hMPV sequences in GenBank.
• "True negative" was defined as any sample that tested negative by both the comparator DSFA test and either viral culture (for RSV) or the hMPV real-time RT-PCR comparator assay (for hMPV).

This approach essentially pre-defines how discordant results between screening and confirmatory tests contribute to the final ground truth, replacing a separate human adjudication step.

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

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not conducted. This study is an evaluation of an in-vitro diagnostic device (IVD), specifically a direct fluorescent antibody (DFA) test, which is read by trained laboratory personnel, but the study focuses on the device's performance against comparator methods, not on comparing reader performance with and without AI assistance (as it is not an AI device).

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

Yes, the studies presented are essentially standalone performance evaluations of the device. The D3 FastPoint L-DFA RSV/MPV Identification Kit is an immunofluorescent assay where a human technician observes fluorescent staining patterns under a microscope. However, the performance metrics (sensitivity, specificity) are for the device's ability to detect the viral antigens in specimens, without involving a study design where human readers using the device are compared to human readers using another method, or AI assistance. The results in the tables reflect the diagnostic performance of the kit itself when used according to its instructions.

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

The ground truth used for the clinical performance studies was based on a composite comparator method combining:

• FDA-cleared predicate DFA devices
• Viral culture (for RSV)
• Validated real-time RT-PCR with bi-directional sequencing analysis (for hMPV)

This is a form of reference standard composite, aiming for a highly accurate and confirmed diagnosis of the presence or absence of the target viruses.

8. The Sample Size for the Training Set

The document does not explicitly describe a separate "training set" or "validation set" in the context of machine learning. This is a traditional in vitro diagnostic device, not an AI/ML-based device.

All the described studies (reproducibility, LoD, analytical reactivity, clinical performance) contribute to the overall evidence for the device. The 1519 clinical specimens (fresh nasal/nasopharyngeal wash/aspirate and swab specimens) can be considered the test set for evaluating clinical performance.

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

As noted above, there is no explicit "training set" mentioned in the context of machine learning. The studies assess the performance of the pre-developed D3 FastPoint L-DFA RSV/MPV Identification Kit using the specified ground truth methods.

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The Diagnostic Hybrids, Inc. device, D3 FastPoint L-DFA Respiratory Virus Identification Kit is intended for the qualitative identification of influenza A virus, influenza B virus, respiratory syncytial virus, human metapneumovirus, adenovirus and to screen for the presence of parainfluenza virus types 1, 2, and 3 in nasal and nasopharyngeal swabs and aspirates/washes specimens from patients with signs and symptoms of respiratory infection by direct detection of immunofluorescence using monoclonal antibodies (MAbs).

It is recommended that specimens found to be negative for influenza A virus, influenza B virus, respiratory syncytial virus, adenovirus or parainfluenza viruses after examination of the direct specimen result be confirmed by cell culture. Specimens found to be negative for human metapneumovirus after examination of the direct specimen results should be confirmed by an FDA cleared human metapneumovirus molecular assay. Negative results do not preclude respiratory virus infection and should not be used as the sole basis for diagnosis, treatment or other management decisions.

Performance characteristics for influenza A virus detection and identification were established when influenza A (H3N2) and influenza A (H1N1) were the predominant influenza A strains circulating in the United States. Since influenza strains display antigenic drift and shift from year to year, performance characteristics may vary. If infection with a novel influenza A virus is suspected, based on clinical and epidemiological screening criteria communicated by public health authorities, collect specimens following appropriate infection control precautions and submit to state or local health departments, for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.

The D3 FastPoint L-DFA Respiratory Virus Identification Kit uses three blends (each called a "L-DFA Reagent") of viral antigen-specific murine monoclonal antibodies that are directly labeled with either R-PE (influenza A virus, respiratory syncytial virus, and parainfluenza virus) or fluorescein (influenza B virus, metapneumovirus, and adenovirus) for the rapid identification of respiratory viruses in nasal and nasopharyngeal swabs and aspirates from patients with signs and symptoms of respiratory infection.

Kit Components:

1. D3 FastPoint L-DFA Influenza A/Influenza B Reagent, 4.0-mL. One dropper bottle containing a mixture of PE-labeled murine monoclonal antibodies directed against influenza A virus antigens and FITC-labeled murine monoclonal antibodies directed against influenza B virus antigens. The buffered, stabilized, aqueous solution contains Evans Blue and propidium iodide as counter-stains and 0.1% sodium azide as preservative.
2. D3 FastPoint L-DFA RSV/MPV Reagent, 4.0-mL. One dropper bottle containing a mixture of PE-labeled murine monoclonal antibodies directed against respiratory syncytial virus antigens and FITC-labeled murine monoclonal antibodies directed against metapneumovirus antigens. The buffered, stabilized, aqueous solution contains Evans Blue and propidium iodide as counter-stains and 0.1% sodium azide as preservative.
3. D3 FastPoint L-DFA PIV/Adenovirus Reagent, 4.0-mL. One dropper bottle containing a mixture of PE-labeled murine monoclonal antibodies directed against parainfluenza virus types 1, 2, or 3 antigens and FITClabeled murine monoclonal antibodies directed against adenovirus antigens. The buffered, stabilized, aqueous solution contains Evans Blue and propidium iodide as counter-stains and 0.1% sodium azide as preservative.
4. 40X PBS Concentrate, 25-mL. One bottle of 40X PBS concentrate containing 4% sodium azide (0.1% sodium azide after dilution to 1X using de-mineralized water).
5. Re-suspension Buffer, 6.0-mL. One bottle of a buffered glycerol solution and 0.1% sodium azide.
6. D3 FastPoint L-DFA Respiratory Virus Antigen Control Slides, 5-slides. Five individually packaged control slides containing 6 wells with cell culture-derived positive and negative control cells. Each positive well is identified as to the virus infected cells present, i.e., influenza A virus, influenza B virus, respiratory syncytial virus, metapneumovirus, parainfluenza virus, and adenovirus. The negative wells contain noninfected cells. Each slide is intended to be stained only one time.

The cells to be tested are derived from respiratory specimens from patients with signs and symptoms of respiratory infection. The cells are permeabilized and stained concurrently in a liquid suspension format in 3 separate vials, each containing one of the 3 above reagents. After incubating at 35℃ to 37℃ for 5 minutes, the stained cell suspensions are rinsed with 1X PBS. The rinsed cells are pelleted by centrifugation and then re-suspended with the resuspension buffer and loaded onto a specimen slide well. The cells are examined using a fluorescence microscope. Cells infected with influenza A virus, respiratory syncytial virus, or parainfluenza virus types 1, 2 and 3 will exhibit goldenyellow fluorescence due to the PE. Cells infected with influenza B virus, metapnemovirus or adenovirus will exhibit apple-green fluorescence due to the FITC. Non-infected cells will exhibit red fluorescence due to the Evans Blue counter-stain. Nuclei of intact cells will exhibit orange-red fluorescence due to the propidium iodide.

Here's a summary of the acceptance criteria and study details for the D3 FastPoint L-DFA Respiratory Virus Identification Kit, based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state pre-defined acceptance criteria (e.g., "Sensitivity must be >X%"). Instead, it presents the results of reproducibility and clinical performance studies, implying that these results met the necessary standards for clearance. The "Reported Device Performance" column reflects the results from the clinical sensitivity and specificity studies presented in Tables 5.10-5.15 (for NP wash/aspirate) and 5.16-5.21 (for NP swab). For analytical performance, the reproducibility and LOD results are provided.

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

• Clinical Test Set:

  • Total Specimens Evaluated: 1519
  • Provenance: Prospectively collected excess remnants of respiratory specimens from symptomatic individuals suspected of respiratory infection.
  • Country of Origin: 4 geographically diverse U.S. clinical laboratories.
  • Retrospective or Prospective: Prospective studies (January 2009 - March 2009).
  • Specific Breakdown for Clinical Performance Tables:
    • NP Wash/Aspirate (Sites 1, 2, and 3 combined): Number of specimens varies per virus (e.g., 637 for Flu A, 694 for hMPV).
    • NP Swab (Sites 3 and 4 combined): Number of specimens varies per virus (e.g., 689 for Flu A, 675 for hMPV).

• Analytical Test Set (Reproducibility):

  • Sample Size: 5 randomized panel members for each of the 3 panels (Influenza A/B, RSV/hMPV, HPIV/Adenovirus). Each panel was tested daily in two separate runs for 5 days by four different laboratories (40 total runs per virus group). This means 40 replicates for each panel member for a given virus.
  • Provenance: Proficiency-level antigen control slides with infected cells.

• Analytical Test Set (Limit of Detection):

  • Sample Size: 10 replicate microscope slides for each dilution level of 8 characterized respiratory virus isolates.
  • Provenance: Dilution series of infected model cells.

• Analytical Test Set (Analytical Reactivity/Inclusivity):

  • Sample Size: Low concentration infected cell suspensions (approximately 4% cells infected, 25-50 infected cells) for various viral strains for each of the 3 reagents.
  • Provenance: Culture isolates of various influenza A (13 strains), influenza B (7 strains), RSV (3 strains), hMPV (4 strains), HPIV (3 strains), and Adenovirus (10 strains).

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

The document does not specify the number or qualifications of experts used to establish the ground truth for the clinical test set. Instead, it defines "True" positive and "True" negative based on a composite comparator method:

• For Influenza A, Influenza B, RSV, Parainfluenza, and Adenovirus: Direct Specimen Fluorescent Antibody (DSFA) test with an FDA cleared device, and viral culture confirmation of all negatives (as determined by the comparator DSFA test).
• For Human Metapneumovirus (hMPV): DSFA with an FDA cleared device, and confirmation of all negative specimens (as determined by the comparator DSFA test) using a validated hMPV real-time RT-PCR followed by bi-directional sequencing analysis.

4. Adjudication Method for the Test Set

The adjudication method used for establishing the ground truth for the clinical test set was a composite comparator method. This means results from multiple established methods (DSFA, viral culture, and for hMPV, RT-PCR with sequencing) were combined to determine the "true" status of a specimen. There is no mention of a specific expert panel adjudication method like "2+1" or "3+1" for interpreting these comparator results in case of discrepancies; rather, the definition of "true" positive/negative indicates the hierarchy of methods (e.g., positive by DSFA or viral culture is "true" positive).

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 MRMC comparative effectiveness study was done. This device is a diagnostic kit read by a human using a fluorescence microscope, but the study focuses on the kit's performance against comparator methods, not on human reader improvement with or without AI assistance.

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

This is not applicable as the D3 FastPoint L-DFA Respiratory Virus Identification Kit is a diagnostic kit that relies on a human reading the results under a fluorescence microscope. It is not an AI algorithm. The performance presented is of the kit as used by a human.

7. The Type of Ground Truth Used

The ground truth used for the clinical test set was a composite comparator method, which included:

• FDA cleared Direct Specimen Fluorescent Antibody (DSFA) tests
• Viral Culture
• Validated hMPV real-time RT-PCR followed by bi-directional sequencing analysis (for hMPV only)
• NCBI GenBank database matching with acceptable E-values for bi-directional sequencing data.

8. The Sample Size for the Training Set

The document does not explicitly mention a "training set" in the context of machine learning or AI development. This device is a diagnostic reagent kit, not an AI algorithm. The studies conducted are for analytical and clinical validation of the kit itself.

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

As there is no "training set" in the context of this diagnostic kit, this question is not applicable. The ground truth for the comparator methods was established using established laboratory techniques and FDA-cleared devices, as described in point 7.

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The Diagnostic Hybrids, Inc. device, D3 Duet DFA RSV/Respiratory Virus Screening Kit, is intended for the qualitative detection and identification of respiratory syncytial virus, while screening for influenza A virus, influenza B virus, adenovirus, and parainfluenza virus types 1, 2 and 3 viral antigens, in nasal and nasopharyngeal swabs and aspirates or in cell culture. The assay detects viral antigens by immunofluorescence using monoclonal antibodies (MAbs), from patients with signs and symptoms of respiratory infection.

It is recommended that specimens found to be negative after examination of the direct specimen result be confirmed by cell culture. Negative results do not preclude influenza virus infection and should not be used as the sole basis for diagnosis, treatment or other management decisions.

Performance characteristics for influenza A virus detection and identification were established when influenza A (H3N2) and influenza A (H1N1) were the predominant influenza A strains circulating in the United States. Performance characteristics for influenza A virus detection and identification were established when influenza A H3N2 and influenza A H1N1 were the predominant influenza A strains circulating in the United States. 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 a 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 Diagnostic Hybrids, Inc. device, D3 Duet DFA RSV/Respiratory Virus Screening Kit, uses a blend of viral antigen-specific murine MAbs. MAbs for RSV are directly labeled with R-phycoerythrin (R-PE) for the rapid detection and identification of RSV. MAbs for influenza A virus, influenza B virus, adenovirus, and parainfluenza virus types 1, 2, and 3 are directly labeled with fluorescein isothiocyanate (FITC), for rapid detection of these agents.

Kit components:

• D3 Duet DFA RSV/Respiratory Virus Screening Reagent
• Normal Mouse Gamma Globulin DFA Reagent
• Respiratory Virus Antigen Control Slides
• Wash Solution Concentrate
• Mounting Fluid

The cells to be tested, derived from a clinical specimen or cell culture, are placed onto a glass slide and allowed to air dry. The cells are fixed in acetone. The D3 Duet DFA RSV/Respiratory Virus Screening Reagent is added to the cells which are then incubated for 15 to 30 minutes at 35° to 37°C in a humidified chamber or humidified incubator. The stained cells are then washed with the diluted wash solution, a drop of the supplied Mounting Fluid is added and a coverslip is placed on the prepared cells. The cells are examined using a fluorescence microscope. The respiratory syncytial virus infected cells will fluoresce golden-yellow, while cells infected with any of the other six viruses will fluoresce apple-green. Uninfected cells will contain no fluorescence but will be stained red by the Evans Blue counter-stain. If only golden-yellow fluorescent cells are present the specimen can be reported as positive for respiratory syncytial virus antigen. If only apple-green fluorescent cells are present, the particular virus is identified using the individual reagents from the D3 Ultra DFA Respiratory Virus Screening & ID Kit (D3 Ultra) on new, separate cell preparations. If both golden-yellow and apple-green are present, the additional virus may be identified using the individual reagents from the D3 Ultra on new, separate cell preparations.

Here's a breakdown of the acceptance criteria and study details for the D' DUET DFA RSV/RESPIRATORY VIRUS SCREENING KIT:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are not explicitly stated as numerical targets for sensitivity and specificity in the provided text. Instead, the study uses the legally marketed predicate device (D3 Ultra DFA Respiratory Virus Screening & ID Kit) as the comparator, and "100% agreement" is repeatedly cited as the performance goal versus this comparator. The device met this implicit acceptance criterion by demonstrating 100% agreement in both positive and negative percentage agreements across various virus types and specimen sources.

*IAFVBAPF123 refers to Influenza A virus, Influenza B virus, Adenovirus, and Parainfluenza virus types 1, 2, and 3.

2. Sample Sizes and Data Provenance

• Direct Fresh Specimens (Test Set):
  • Total Initial Specimens: 1203
  • Excluded Specimens: 17 (due to site deviations, duplicate specimen, insufficient cell numbers, or high background)
  • Analyzed Specimens: 1187
  • Data Provenance: Prospective, collected at three study sites in the United States.
• Cultured Specimens (Test Set):
  • Total Specimens: 298 frozen specimens
  • Data Provenance: Retrospective (frozen specimens), from a fourth study site. All specimens were derived from nasopharyngeal specimens. The age distribution of individuals is provided on page 13.

3. Number of Experts and Qualifications

The document does not explicitly state the number of experts used to establish ground truth for the test set or their specific qualifications (e.g., "Radiologist with 10 years of experience"). However, it mentions that the D3 Duet device was compared against a "cleared DSFA device" (D3 Ultra DFA Respiratory Virus Screening & ID Kit) as the comparator. This implies that the ground truth for the clinical studies was established by laboratory personnel using the predicate device, which is a standard immunofluorescence assay.

4. Adjudication Method

The document does not specify an adjudication method like 2+1 or 3+1. The results are presented as direct comparisons between the D3 Duet and the "D3 Ultra Final Identification," implying that the D3 Ultra's results served as the reference standard without further expert adjudication beyond its own established interpretation protocol.

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

No multi-reader multi-case (MRMC) comparative effectiveness study was done. This device is a diagnostic kit read by a single trained technician, not an AI system designed to assist human readers. Therefore, the effect size of human readers improving with AI vs. without AI assistance is not applicable.

6. Standalone Performance

Yes, a standalone performance study (algorithm only without human-in-the-loop performance) was done for the D3 Duet. The entire clinical performance section (pages 11-14) reports the performance of the D3 Duet device independently against the predicate device (D3 Ultra) as the gold standard.

7. Type of Ground Truth Used

The ground truth for the clinical studies was established using a cleared comparator device, specifically the D3 Ultra DFA Respiratory Virus Screening & ID Kit. This is a form of reference standard testing using an established laboratory method (immunofluorescence assay) for viral antigen detection. For the cultured specimens, cell culture methods were also employed.

8. Sample Size for the Training Set

The document does not explicitly mention a "training set" in the context of an AI/algorithm. Instead, the device is a diagnostic kit based on monoclonal antibodies. Therefore, there is no sample size for an algorithm training set. The various analytical and non-clinical performance studies (precision, detection limit, analytical reactivity, analytical specificity) act as development and validation steps for the kit itself, rather than training for an AI.

9. How Ground Truth for the Training Set Was Established

As there is no "training set" for an AI/algorithm, this point is not applicable. The development and validation of the D3 Duet kit relied on established laboratory techniques for virus isolation, identification using cell cultures, reference viral strains, and bacterial/fungal cultures. These methods served as the "ground truth" during the development and analytical validation of the antibody blend and assay performance.

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