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The ProParaflu+ Assay is a multiplex Real Time RT-PCR in vitro diagnostic test for the qualitative detection and discrimination of Parainfluenza 1 Virus, Parainfluenza 2 Virus and Parainfluenza 3 Virus (HPIV-1, HPIV-2 and HPIV-3) nucleic acids isolated and purified from nasopharyngeal (NP) swab specimens obtained from individuals exhibiting signs and symptoms of respiratory tract infections. This assay targets the conserved regions of the Hemagglutinin-Neuraminidase (HN) gene of HPIV-1, HPIV-2 and HPIV-3. respectively. The detection and discrimination of HPIV-1, HPIV-2 and HPIV-3 nucleic acids from symptomatic patients aid in the diagnosis of human respiratory tract parainfluenza infections if used in conjunction with other clinical and laboratory findings. This test is not intended to detect Parainfluenza 4a or Parainfluenza 4b Viruses.

Negative test results are presumptive and should be confirmed by cell culture. Negative results do not preclude Parainfluenza 1, 2 or 3 virus infections and should not be used as the sole basis for treatment or other management decisions.

The ProParaflu+ Assay enables the detection and differentiation of Parainfluenza 1 Virus, Parainfluenza 2 Virus, Parainfluenza 3 Virus and an Internal Control (IC) nucleic acid. Nasopharyngeal swab specimens from symptomatic patients using a polyester, rayon or nylon tipped swab and place into viral transport medium. The IC is added to every sample prior to nucleic acid extraction to monitor for inhibitors present in the specimens.

Isolation and purification of nucleic acids is performed using the bioMérieux NucliSENS easyMAG automated extractor and the Automated Magnetic Extraction Reagents or the Roche MagNA Pure LC Instrument and the MagNA Pure Total Nucleic Acid Isolation Kit.

The purified nucleic acids are added to the ProParaflu+ Supermix along with enzymes included in the ProParaflu+ Detection Kit. The ProParaflu+ Supermix contains oligonucleotide primers that are complementary to highly conserved regions of hemagglutinin neuraminidase gene for each human Parainfluenza type (1, 2 and 3). The probes are dual-labeled with a reporter dye attached to the 5'-end and a quencher dye attached to the 3'-end.

RT-PCR amplification is performed in a Cepheid SmartCycler® II instrument. During this process, the primers and probes anneal specifically to the template (if present) followed by primer extension and amplification. The ProParaflu+ Assay is based on Taqman chemistry, which utilizes the 5' – 3' exonuclease activity of the Taq polymerase to cleave the probe thus separating the reporter dye from the quencher. This generates an increase in fluorescent signal upon excitation from a light source. With each cycle, additional reporter dye molecules are cleaved from their respective probes, further increasing the fluorescent signal. The amount of fluorescence at any given cycle is dependent on the amount of amplification product present at that time. Fluorescent intensity is monitored during each PCR cycle by the real-time instrument. Results are analyzed and interpreted as presented by the software.

Here's an analysis of the ProParaflu+ Assay's acceptance criteria and the studies performed, based on the provided document:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state pre-defined acceptance criteria in terms of specific sensitivity and specificity thresholds. Instead, it presents the calculated sensitivity and specificity with 95% confidence intervals from the clinical studies. For the purpose of this summary, the reported performance metrics can be considered the demonstrated performance against which regulatory acceptance was evaluated.

Reproducibility:

• Overall percent agreement with expected result: 97.8% for the initial reproducibility study.
• Intermediate concentration study:
  • HPIV-1: 56.7% agreement with positive result (expected due to concentration below LoD)
  • HPIV-2: 86.7% agreement with positive result (expected due to concentration below LoD)
  • HPIV-3: 30.0% agreement with positive result (expected due to concentration below LoD)

2. Sample Size and Data Provenance

• Prospective Study:
  • Sample Size (Test Set): 857 eligible nasopharyngeal (NP) swab samples.
  • Data Provenance: United States (4 U.S. clinical laboratories), prospective collection from symptomatic individuals suspected of respiratory infection.
• Retrospective Study (HPIV-1 only):
  • Sample Size (Test Set): 91 frozen NP swab samples.
  • Data Provenance: Not explicitly stated, but likely also US-based given the overall context. The data was retrospective, as samples were "previously tested by direct DFA."

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

The document does not specify the number or qualifications of experts for establishing the ground truth.

4. Adjudication Method for the Test Set

• Reference Method: Cell culture (rapid or traditional) followed by direct fluorescent antibody (DFA) screening and HPIV type identification was used as the primary reference method.
• Discrepant Analysis: For samples where the ProParaflu+ Assay and the reference method disagreed, RT-PCR with virus-specific primers (different from those used in ProParaflu+) followed by bi-directional sequencing was performed. This served as an adjudication method to re-evaluate the true status of discrepant samples.

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

No, a multi-reader multi-case (MRMC) comparative effectiveness study comparing human readers with AI assistance versus without AI assistance was not performed. This device is a molecular diagnostic assay, not an imaging AI system designed to aid human interpretation.

6. Standalone Performance Study

Yes, the clinical performance studies (prospective and retrospective) represent a standalone performance evaluation of the ProParaflu+ Assay. The reported sensitivity and specificity figures reflect the algorithm's (assay's) performance without human intervention in the result determination beyond running the assay and interpreting its output according to established rules.

7. Type of Ground Truth Used

The ground truth was established through a combination of:

• Reference Method: Cell culture (rapid or traditional) followed by direct fluorescent antibody (DFA) screening and HPIV type identification.
• Confirmatory Method for Discrepancies: RT-PCR with virus-specific primers followed by bi-directional sequencing. This suggests a form of expert consensus or highly reliable confirmatory testing to resolve ambiguities.

8. Sample Size for the Training Set

The document does not specify a separate "training set" sample size. For in vitro diagnostic assays like this, the development of the assay (primers, probes, conditions) inherently involves a form of "training" or optimization, but this is typically done using synthetic constructs, spiked samples, and smaller panels of clinical samples, not a formally defined "training set" in the machine learning sense. The clinical studies described (prospective and retrospective) are validation studies for the finalized device.

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

As no formal "training set" is described in the conventional sense for this type of device, the method for establishing ground truth for such a set is not detailed. The design of the assay (selection of conserved regions, primer/probe design) would have relied on existing genomic sequences and expert knowledge of the viruses.

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The ProFlu+™ Assay is a multiplex Real Time RT-PCR in vitro diagnostic test for the rapid and qualitative detection and discrimination of Influenza A Virus, Influenza B Virus, and Respiratory Syncytial Virus (RSV) nucleic acids isolated and purified from nasopharyngeal (NP) swab specimens obtained from symptomatic patients. This test is intended for use to aid in the differential diagnosis of Influenza A, Influenza B and RSV viral infections in humans and is not intended to detect Influenza C.

Negative results do not preclude influenza or RSV virus infection and should not be used as the sole basis for treatment or other management decisions. It is recommended that negative RSV results be confirmed by culture.

Performance characteristics for Influenza A Virus were established when Influenza A/H3 and A/H1 were the predominant Influenza A viruses in circulation. When other Influenza A viruses are emerging, performance characteristics may vary.

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

The ProFlu+ Assay enables detection and differentiation of Influenza A Virus, Influenza B Virus, Respiratory Syncytial Virus (RSV) (Types A and B), and Internal Control. Nasopharyngeal swab specimens are collected from symptomatic patients using a polyester, rayon or nylon tipped swab and place into viral transport medium.

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

The purified nucleic acids are added to ProFlu+ Supermix along with enzymes included in the ProFlu+ Detection Kit. The ProFlu+ Supermix contains oligonucleotide primers and target-specific oligonucleotide probes. The primers are complementary to highly conserved regions of genetic sequences for these respiratory viruses. The probes are dual-labeled with a reporter dye attached to the 5'-end and a quencher dye attached to the 3'-end.

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

This document describes the ProFlu+ Assay, a multiplex Real Time RT-PCR in vitro diagnostic test for the rapid and qualitative detection and discrimination of Influenza A Virus, Influenza B Virus, and Respiratory Syncytial Virus (RSV) nucleic acids. However, the provided text does not contain the information requested in your prompt regarding acceptance criteria, specific study details, sample sizes, ground truth establishment, expert qualifications, or comparative effectiveness study results.

The document primarily focuses on:

• Identification of the device: ProFlu+ Assay
• Contact information: Prodesse, Inc.
• Predicate device: K081030 - ProFlu+ Assay, Prodesse, Inc. and K091667 - ID-Tag Respiratory Virus Panel, Luminex Molecular Diagnostics, Inc.
• Intended Use: Aid in differential diagnosis of Influenza A, Influenza B, and RSV viral infections from nasopharyngeal swab specimens from symptomatic patients. Explicitly states it's not for Influenza C, and negative results for RSV should be confirmed by culture.
• Product Description: Details the methodology (multiplex Real Time RT-PCR, use of internal control, MagNA Pure LC Instrument or NucliSENS® easyMAGTM System for nucleic acid isolation, Cepheid SmartCycler® II for RT-PCR, Taqman chemistry).
• Substantial Equivalence: Notes that the intended use remains the same, and the device is reactive to the 2009 H1N1 Influenza Virus.
• FDA Clearance: Provides formal communication from the FDA regarding the 510(k) clearance (K092500).

Therefore, I cannot fulfill your request for the specific details outlined in your prompt based on the provided text. The document does not include a table of acceptance criteria, reported device performance metrics against such criteria, sample sizes for test/training sets, ground truth methods, expert details, or information on MRMC studies.

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The ProGastro™ Cd Assay is a Real Time PCR in vitro diagnostic test for the qualitative detection of toxigenic Clostridium difficile nucleic acids isolated and purified from liquid or soft stool specimens obtained from symptomatic patients. This test targets the Clostridium difficile toxin B gene (tcdB) and is intended for use to aid in the diagnosis of toxigenic Clostridium difficile infections.

The ProGastro Cd Assay detects toxigenic Clostridium difficile and an Internal Control by a process of nucleic acid extraction from patient specimens followed by PCR amplification and detection. Following collection of a soft or liquid stool sample from a symptomatic patient, a portion of the sample is diluted in Stool Transport and Recovery (S.T.A.R.) Buffer and the solids separated via centrifugation (Stool Clarification). The Internal Control is added to the sample prior to extraction to monitor for PCR inhibitors that may be present. The nucleic acids from the sample are extracted and purified using the bioMérieux NucliSENS easyMAG automated extractor. Nucleic acids are added to the C. diff Mix for subsequent PCR amplification and detection using the Cepheid SmartCycler II.

The C. diff Mix contains oligonucleotide primers and probes that target the tcdB gene of toxigenic strains of C. diff. The probes are dual-labeled with a reporter dye attached to the 5'-end and a quencher dye attached to the 3'-end (see table below). During PCR amplification the primers and probes anneal to the template (if present) followed by primer extension and template amplification. The 5'-3' exonuclease activity of the Taq polymerase cleaves the probe thus separating the reporter dye from the quencher and generating an increase in fluorescent signal. The amount of fluorescence at any given cycle is dependent on the amount of amplification product present. The SmartCycler II instrument and software monitors the process, interprets the data, and presents a report upon completion.

Here’s a breakdown of the acceptance criteria and the study proving the ProGastro Cd Assay meets them, based on the provided document:

1. Acceptance Criteria and Reported Device Performance

The document does not explicitly state pre-defined acceptance criteria for Sensitivity and Specificity in a table format. However, the reported performance against the reference method (Tissue Culture Cytotoxin Assay - CTA) implies an expectation of high diagnostic accuracy. For reproducibility, a high percentage of agreement with expected results across sites and operators is an implicit criterion.

Based on the provided data, we can infer the following:

2. Sample Size and Data Provenance

• Sample Size for Test Set: A total of 771 raw stool samples were tested in the clinical performance study.
• Data Provenance: The study was a prospective study conducted at 3 U.S. clinical laboratories from July through October 2008. The samples were "leftover raw stool specimens that were collected for routine Clostridium difficile testing from patients over two years of age by each site." This indicates real-world clinical samples from a U.S. patient population.

3. Number of Experts and Qualifications for Ground Truth

The document does not specify the number of experts used or their qualifications for establishing the initial ground truth (Tissue Culture Cytotoxin Assay - CTA). CTA is a laboratory-based method, and its interpretation would typically be performed by trained laboratory personnel.

4. Adjudication Method for the Test Set

A discrepant analysis was performed for samples where the ProGastro Cd Assay and CTA results disagreed. This involved a predetermined algorithm including:

• A molecular (PCR) test targeting a different region of the tcdB gene.
• Bidirectional genetic sequencing.
• Enzyme Immunoassay (EIA).
• Culture followed by PCR and bidirectional sequencing.

This constitutes a form of adjudication where additional, more definitive tests are used to resolve disagreements between the device and the primary reference method.

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

No MRMC comparative effectiveness study was done. This study evaluates an in vitro diagnostic (IVD) test, where the "reader" is essentially the instrument and the assay, not a human interpreter of an image or signal. Therefore, assessing how human readers improve with AI vs. without AI assistance is not applicable here.

6. Standalone (Algorithm-Only) Performance

The entire clinical performance study report (Sensitivity, Specificity) evaluates the standalone performance of the ProGastro Cd Assay. The assay is an in vitro diagnostic test for the qualitative detection of C. difficile nucleic acids, which runs entirely on the Cepheid SmartCycler II instrument and software for amplification, detection, and data interpretation. There is no human-in-the-loop component for the result generation itself.

7. Type of Ground Truth Used

The primary reference method used to establish ground truth for the clinical performance study was the Tissue Culture Cytotoxin Assay (CTA). For discrepant analysis, additional molecular tests (PCR, sequencing), EIA, and culture were used to refine the ground truth. This combines a gold standard (CTA) with a more comprehensive "adjudicated" gold standard for discordant results.

8. Sample Size for the Training Set

The document does not provide information about a specific training set or its sample size. This is typical for 510(k) submissions for diagnostic assays, where the focus is on the performance of the finalized device rather than the development process involving training data for algorithms. The "assay" itself, like most IVDs, is developed and validated, but does not typically undergo a 'training' phase with a specific dataset in the same way a machine learning algorithm would.

9. How Ground Truth for the Training Set Was Established

As no specific training set information is provided, the method for establishing its ground truth is also not described.

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The Pro hMPV+ Assay is a Real Time RT-PCR in vitro diagnostic test for the qualitative detection of human Metapneumovirus (hMPV) nucleic acid isolated and purified from nasopharyngeal swab (NP) specimens obtained from individuals exhibiting signs and symptoms of acute respiratory infection. This assay targets a highly conserved region of the Nucleocapsid gene of hMPV. The detection of hMPV nucleic acid from symptomatic patients aids in the diagnosis of human respiratory hMPV infection if used in conjunction with other clinical and laboratory findings. This test is not intended to differentiate the four genetic sub-lineages of hMPV.

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

The Pro hMPV+ Assay enables detection human Metapneumovirus and Internal Control nucleic acid. Nasopharyngeal swab specimens are collected from patients with signs and symptoms of a respiratory infection using a polyester, rayon or nylon tipped swab and placed into viral transport medium.

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

The purified nucleic acids are added to Pro hMPV+ Supermix along with enzymes included in the Pro hMPV+ Assay Kit. The Pro hMPV+ Supermix contains oligonucleotide primers and target-specific oligonucleotide probes. The primers are complementary to highly conserved regions of genetic sequences for these respiratory viruses. The probes are dual-labeled with a reporter dye attached to the 5'-end and a quencher dye attached to the 3'-end.

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

Here's an analysis of the acceptance criteria and study detailed in the provided 510(k) summary for the Pro hMPV+ Assay:

Acceptance Criteria and Device Performance for Pro hMPV+ Assay

This summary focuses on the clinical performance of the Pro hMPV+ Assay, which is a Real Time RT-PCR in vitro diagnostic test for the qualitative detection of human Metapneumovirus (hMPV).

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state pre-defined acceptance criteria for the clinical performance metrics (Percent Positive Agreement, Percent Negative Agreement). However, the reported performance is presented with 95% Confidence Intervals, which allows for an assessment of the assay's accuracy. For the purpose of this analysis, we will infer the desired performance to be high agreement with the composite reference methods.

2. Sample Size and Data Provenance for the Test Set

• Sample Size: A total of 1275 eligible nasopharyngeal (NP) swab samples were tested and included in the analysis.
• Data Provenance: The study was a prospective study conducted at 4 U.S. clinical laboratories during the 2008 respiratory virus season (January - March). The specimens represented excess NP swab specimens proactively collected from symptomatic individuals suspected of respiratory infection.

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

The ground truth was established using composite reference methods, which involved molecular testing and genetic sequencing, rather than direct expert interpretation of test results. Therefore, the concept of "experts" in the traditional sense (e.g., radiologists) for establishing ground truth doesn't directly apply here.

However, the "experts" involved would be the laboratory personnel performing the molecular (RT-PCR) tests and subsequent genetic sequencing, and those interpreting the sequencing data against the NCBI GenBank database. While no explicit qualifications are given, it can be inferred that these individuals are qualified laboratory professionals experienced in molecular diagnostics and bioinformatics, as they are performing highly specialized and technical analyses for clinical diagnostic purposes.

4. Adjudication Method for the Test Set

The adjudication method for establishing the ground truth was a composite reference standard approach:

• Two independent molecular (RT-PCR) tests for two separate gene targets of hMPV.
• Followed by bidirectional genetic sequencing of those targets.

True hMPV RNA positives were defined as any sample with bidirectional sequencing data meeting pre-defined quality acceptance criteria for one or both gene targets that matched hMPV sequences in the NCBI GenBank database.
True hMPV RNA negatives were defined as any sample tested negative by both comparator methods.

This effectively acts as an internal adjudication process based on multiple, high-specificity molecular methods. There is no mention of a human expert adjudication committee in the traditional sense (e.g., 2+1, 3+1).

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

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not conducted. This device is an in vitro diagnostic test, not an AI-assisted human reader interpretation tool. Therefore, the concept of measuring how much human readers improve with AI vs. without AI assistance is not applicable.

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

Yes, a standalone (algorithm only) performance study was conducted. The "Pro hMPV+ Assay" itself is the algorithm (or diagnostic method) being evaluated. Its performance was assessed directly against the composite reference methods. There is no human-in-the-loop component in its reported diagnostic performance.

7. Type of Ground Truth Used

The type of ground truth used was a composite reference standard based on:

• Molecular diagnostic testing (two independent RT-PCR tests) for different hMPV gene targets.
• Bi-directional genetic sequencing of those targets.
• Comparison of sequencing data to the National Center for Biotechnology Information (NCBI) GenBank database.

This is a highly reliable and objective form of ground truth for viral detection.

8. Sample Size for the Training Set

The document does not provide information regarding a distinct "training set" sample size. For in vitro diagnostic assays like the Pro hMPV+ Assay, the development process typically involves internal validation and optimization studies (which might be analogous to "training"), but specific sample sizes for these internal activities are usually not detailed in 510(k) summaries, which focus on the clinical validation (test set).

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

As no specific "training set" and its sample size were described, the method for establishing its ground truth is also not provided in this document. During assay development, ground truth for optimization and development samples would typically be established using similar highly sensitive and specific methods (e.g., highly characterized positive and negative controls, sequencing, or alternative validated molecular methods).

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The ProFlu+™ Assay is a multiplex Real Time RT-PCR in vitro diagnostic test for the rapid and qualitative detection and discrimination of Influenza A Virus, Influenza B Virus, and Respiratory Syncytial Virus (RSV) nucleic acids isolated and purified from nasopharyngeal (NP) swab specimens obtained from symptomatic patients. This test is intended for use to aid in the differential diagnosis of Influenza A, Influenza B and RSV viral infections in humans and is not intended to detect Influenza C.

Negative results do not preclude influenza or RSV virus infection and should not be used as the sole basis for treatment or other management decisions. It is recommended that negative RSV results be confirmed by culture.

Performance characteristics for Influenza A Virus were established when Influenza A/H3 and A/H1 were the predominant Influenza A viruses in circulation. When other Influenza A viruses are emerging, performance characteristics may vary.

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

The ProFlu+ Assay enables detection and differentiation of Influenza A Virus, Influenza B Virus, Respiratory Syncytial Virus (RSV) (Types A and B), and Internal Control. Nasopharyngeal swab specimens are collected from symptomatic patients using a polyester, rayon or nylon tipped swab and place into viral transport medium.

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

The purified nucleic acids are added to ProFlu+ Supermix along with enzymes included in the ProFlu+ Detection Kit. The ProFlu+ Supermix contains oligonucleotide primers and target-specific oligonucleotide probes. The primers are complementary to highly conserved regions of genetic sequences for these respiratory viruses. The probes are dual-labeled with a reporter dye attached to the 5'-end and a quencher dye attached to the 3'-end.

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

Here's an analysis of the ProFlu+ Assay's acceptance criteria and the study data provided, structured according to your request:

Acceptance Criteria and Device Performance

The acceptance criteria are not explicitly stated in numerical thresholds in the provided document. However, the intent is to demonstrate substantial equivalence to predicate devices, implying that the performance should be comparable or superior. The reported device performance is presented in terms of Sensitivity and Specificity with 95% Confidence Intervals.

Table 1: Acceptance Criteria (Implied) and Reported Device Performance (Prospective Study)

Table 2: Acceptance Criteria (Implied) and Reported Device Performance (Retrospective Study)

Study Details:

1. Sample Sizes Used for the Test Set and Data Provenance:

   • Prospective Study Test Set: 891 nasopharyngeal (NP) swab samples. After excluding 5 unresolved samples, 826 samples were used in the analysis (Note: The sum of total samples in individual analyte tables is 826).
   • Retrospective Study Test Set: Not explicitly stated as a total, but individual analyte tables sum to 60 samples for Influenza A, 60 samples for Influenza B, and 60 samples for RSV (likely the same set of 60 samples analyzed for all three).
   • Data Provenance:
     • Prospective Study: Conducted at 3 U.S. clinical laboratories during the 2006-2007 respiratory virus season (February - April).
     • Retrospective Study: Conducted at 1 U.S. site during the 2006-2007 respiratory virus season (February - April).
     • The samples were "collected for routine influenza or RSV testing by each site," indicating real-world clinical samples.

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

   • The document does not specify the number of experts or their qualifications for establishing the initial reference method (rapid culture/DFA).
   • The ground truth for discrepant analysis was established using "RT-PCR with virus specific primers obtained from literature followed by sequencing." This implies a molecular biology expert, but no specific number or qualifications are given.

3. Adjudication Method for the Test Set:

   • The primary reference method was rapid culture (shell vial) followed by direct fluorescent antibody (DFA) screening and identification.
   • For samples where the ProFlu+ Assay and the reference method (culture/DFA) disagreed, discrepant analysis using RT-PCR with virus-specific primers followed by sequencing was performed. This acts as a tie-breaker or a higher-tier reference method for discordant results.

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

   • No. The study described is a direct comparison of the ProFlu+ assay against a reference method (culture/DFA and sequencing), not a multi-reader multi-case study comparing human readers with and without AI assistance. The ProFlu+ Assay is an in vitro diagnostic test, not an AI-assisted interpretation tool for human readers.

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

   • Yes, the performance data presented (Sensitivity, Specificity) represent the standalone performance of the ProFlu+ Assay diagnostic test itself, processing the samples and providing results without human interpretation influencing the diagnostic outcome beyond standard laboratory procedures (e.g., sample handling, instrument operation).

6. The Type of Ground Truth Used:

   • The primary ground truth for the test set was established by a combination of methods:
     • Rapid culture (shell vial) and direct fluorescent antibody (DFA).
     • For discrepant results, the ground truth was re-established using RT-PCR with virus-specific primers followed by sequencing (which can be considered molecular pathology/genetic confirmation).

7. The Sample Size for the Training Set:

   • The document does not provide details about a specific "training set" for the ProFlu+ Assay. This assay is a diagnostic test based on molecular biology principles (RT-PCR), not a machine learning or AI model that typically requires a separate training set. The "design" and "optimization" of the primers and probes would be done during assay development, but not in the same sense as training a predictive algorithm on labeled datasets.

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

   • As noted above, the concept of a "training set" with ground truth in the context of an RT-PCR diagnostic assay like ProFlu+ is not directly applicable in the same way it would be for an AI algorithm. The development of the assay (e.g., selection of primer/probe sequences) would rely on known viral genetic sequences and established molecular biology techniques, rather than a "ground truth" derived from patient samples for algorithm training.

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The ProFlu+™ Assay is a multiplex Real Time RT-PCR in vitro diagnostic test for the rapid and qualitative detection and discrimination of Influenza A Virus, Influenza B Virus, and Respiratory Syncytial Virus (RSV) nucleic acids isolated and purified from nasopharyngeal (NP) swab specimens obtained from symptomatic patients. This test is intended for use to aid in the differential diagnosis of Influenza A, Influenza B and RSV viral infections in humans and is not intended to detect Influenza C.

A negative test is presumptive and it is recommended these results be confirmed by cell culture. Negative results do not preclude influenza or RSV virus infection and should not be used as the sole basis for treatment or other management decisions.

Performance characteristics for Influenza A Virus were established when Influenza A/H3 and A/H1 were the predominant Influenza A viruses in circulation. When other Influenza A viruses are emerging, performance characteristics may vary.

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

The ProFlu+ Assay enables detection and differentiation of Influenza A Virus, Influenza B Virus, Respiratory Syncytial Virus (RSV) (Types A and B), and Internal Control. Nasopharyngeal swab specimens are collected from symptomatic patients using a polyester, rayon or nylon tipped swab and placed into viral transport medium.

An Internal Control (IC) is added to each sample prior to nucleic acid isolation to monitor for inhibitors present in the specimens. The isolation and purification of the nucleic acids is performed using a MagNA Pure LC Instrument (Roche) and the MagNA Pure Total Nucleic Acid Isolation Kit (Roche).

The purified nucleic acids are added to ProFlu+ Supermix along with enzymes included in the ProFlu+ Detection Kit. The ProFlu+ Supermix contains oligonucleotide primers and target-specific oligonucleotide probes. The primers are complementary to highly conserved regions of genetic sequences for these respiratory viruses. The probes are dual-labeled with a reporter dye attached to the 5'-end and a quencher dye attached to the 3'-end (see table below).

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

Here's an analysis of the acceptance criteria and study details for the ProFlu+ Assay, based on the provided text:

Acceptance Criteria and Device Performance

The acceptance criteria are not explicitly stated in a formalized table within the provided text. However, they can be inferred from the reported sensitivity and specificity values. The study reports the following performance for the ProFlu+ Assay:

Table 1: ProFlu+ Assay Performance (Inferred Acceptance Criteria vs. Reported Performance)

Reproducibility (Overall Percent Agreement):

• Implied Acceptance Criteria: High (e.g., >95%)
• Reported Device Performance: 98% (96% - 99% CI)

Study Details

2. Sample Size and Data Provenance

• Sample Size for Test Set:
  • Prospective Study: 826 nasopharyngeal (NP) swab samples. A total of 891 samples were initially tested, but 5 unresolved samples were excluded.
  • Retrospective Study: 60 samples.
• Data Provenance:
  • Prospective Study: 3 U.S. clinical laboratories (prospective collection during February - April 2007 respiratory virus season).
  • Retrospective Study: 1 U.S. site.

3. Number of Experts and Qualifications for Ground Truth

The document does not explicitly state the number of "experts" (e.g., pathologists, radiologists) in the conventional sense for oncology or imaging studies. For this in vitro diagnostic (IVD) device:

• The "experts" can be considered the laboratory personnel performing the reference methods. The study was conducted at 3 U.S. clinical laboratories and 1 U.S. site, implying multiple trained technicians/scientists.
• Qualifications: While not explicitly detailed, it is assumed these individuals were qualified laboratory professionals experienced in performing rapid culture (shell vial) and direct fluorescent antibody (DFA) screening and identification for respiratory viruses, as these were the reference methods.

4. Adjudication Method for the Test Set

The adjudication method used for discrepant results was:

• Discrepant Analysis: For samples where ProFlu+ Assay results and culture results disagreed, RT-PCR with virus-specific primers (obtained from literature) followed by sequencing was performed. This served as the definitive arbiter for these cases.
• For the 23 DFA Respiratory Virus Screen positive samples that had too few cells for specific identification, genetic sequencing analysis was used to confirm the specific virus or to confirm negativity for Influenza A, Influenza B, and RSV.

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

• No, an MRMC comparative effectiveness study was not done. This is an in vitro diagnostic device for laboratory analysis, not an imaging device requiring human reader interpretation in a clinical setting in the way MRMC studies are typically performed. The "readers" here are the automated instruments and laboratory technicians, whose performance is assessed through reproducibility and accuracy against reference methods.

6. Standalone (Algorithm Only) Performance

• Yes, a standalone performance study was done. The entire clinical performance section (both prospective and retrospective studies) evaluates the ProFlu+ Assay as a standalone device, comparing its results directly against reference methods. There is no mention of a "human-in-the-loop" component for its primary diagnostic function.

7. Type of Ground Truth Used

The ground truth for the clinical performance studies was established using a combination of:

• Reference Methods:
  • Rapid culture (shell vial)
  • Direct fluorescent antibody (DFA) screening and identification
• Discrepant Analysis/Confirmation:
  • RT-PCR with virus-specific primers followed by sequencing (for resolving disagreements between ProFlu+ and reference methods, and for confirming DFA screen positives with insufficient cells).

Essentially, the ground truth was a combination of established laboratory methods, with molecular sequencing as the definitive arbiter for ambiguous or conflicting results.

8. Sample Size for the Training Set

• The document does not specify a sample size for a training set. This type of IVD (multiplex Real-Time RT-PCR assay) is developed based on pre-defined molecular targets and amplification conditions. While there would have been extensive assay development and optimization, the concept of a "training set" in the machine learning sense is not directly applicable or explicitly described for an RT-PCR assay in this context. The study focuses on clinical validation of the developed assay.

9. How Ground Truth for Training Set Was Established

• As a "training set" is not explicitly mentioned or relevant in the context of conventional machine learning for this specific device (a molecular diagnostic assay), the method for establishing ground truth for it is not applicable or described in the document. The assay's design relies on known genetic sequences of the target viruses, and its performance is validated clinically.

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