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The Prodesse® ProParaflu®+ Assay is a multiplex Real-Time PCR (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 nasopharvngeal (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-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 detection and differentiation of Parainfluenza 1 Virus. Parainfluenza 2 Virus, Parainfluenza 3 Virus 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.

A Universal Internal Control (UIC) 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 ProParaflu+ Supermix along with enzymes included in the ProParaflu+ Assay Kit. The ProParaflu+ 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 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 dve 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 II instrument.

The provided document describes a special 510(k) submission for the Gen-Probe Prodesse, Inc. Prodesse® ProParaflu®+ Assay (K132238). This submission focuses on modifications to an existing device (predicate device K091053, ProParaflu 101+ Assay) rather than a completely new device. Therefore, the details provided about acceptance criteria and study designs are predominantly related to demonstrating substantially equivalent performance with the modifications, rather than establishing initial performance for a novel diagnostic.

Here's an analysis of the provided information:

1. Table of Acceptance Criteria and Reported Device Performance

The document doesn't explicitly state numerical "acceptance criteria" in the format of a threshold to be met. Instead, it describes the objective of the verification/validation studies for the modified device: to ensure that the modifications did not negatively impact the device's ability to detect target organisms at the limit of detection or change its clinical performance. The reported performance is framed as meeting these objectives and demonstrating substantial equivalence to the previous device.

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

• Sample Size for Test Set: The document mentions a "retrospective clinical comparison study" for the UIC modification but does not specify the sample size for this study or any other test sets.
• Data Provenance: The document states "nasopharyngeal (NP) swab specimens obtained from individuals exhibiting signs and symptoms of respiratory tract infections." The country of origin is not specified but is implied to be within the scope of where Gen-Probe Prodesse, Inc. operates (Waukesha, WI, USA, suggests data from the USA). The clinical comparison study is explicitly stated to be retrospective.

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

This information is not provided in the document. As this is an in vitro diagnostic device, the ground truth is typically established by other laboratory methods rather than expert interpretation of images or other subjective data.

4. Adjudication Method for the Test Set

This information is not provided in the document. Given that it's an in vitro diagnostic test, the concept of expert adjudication in the same way it applies to image analysis might not be directly relevant. The "ground truth" would likely be determined by a different gold standard assay or cell culture, not a consensus of human reviewers of the device's output.

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

An MRMC study is not applicable and was not done. This device is an in vitro diagnostic for detecting viral nucleic acids, not an imaging device requiring human reader interpretation.

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

The device is an in vitro diagnostic Real-Time PCR assay. Its operation is inherently "standalone" in the sense that the assay itself generates a result (presence/absence of viral nucleic acid) based on the biochemical reaction and instrument detection. There isn't a "human-in-the-loop" component in the interpretation of the RT-PCR output itself, though a human performs the test and interprets the final qualitative result (positive/negative) from the instrument's readout. The performance studies (Analytical Sensitivity, IC Interference, Extractor Equivalency, Sample Stability, and the retrospective clinical comparison) demonstrate this standalone performance.

7. Type of Ground Truth Used

The document mentions that negative test results are presumptive and should be confirmed by cell culture. This indicates that cell culture is considered a gold standard or a primary method for confirming negative findings, and likely forms part of the "ground truth" for clinical evaluations. For positive results, the ground truth would typically be established by clinical diagnosis and/or comparison to a known highly sensitive and specific comparator assay or other reference methods in the clinical comparison study.

8. Sample Size for the Training Set

The document does not provide any information about a training set. As this is a molecular diagnostic assay using primers and probes targeting specific gene sequences, the "training" aspect is built into the assay design (selecting highly conserved regions) rather than a machine learning training paradigm with a specific dataset.

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

Since there is no mention of a "training set" in the context of machine learning, this question is not applicable. The "ground truth" for the assay's design (e.g., confirming the suitability of the chosen gene targets and primer/probe sequences) would have been established through bioinformatics analysis and empirical testing with characterized viral isolates.

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The Prodesse® ProFAST + Assay is a multiplex Real Time RT-PCR in vitro diagnostic test for the qualitative detection and discrimination of seasonal Influenza A/H1, seasonal Influenza A/H3 and 2009 H1N1 Influenza viral nucleic acids isolated and purified from nasopharyngeal (NP) swab specimens from human patients with signs and symptoms of respiratory infection in conjunction with clinical and epidemiological risk factors. This Assay targets conserved regions of the Hemagglutinin (HA) gene for seasonal Influenza A/H1, seasonal Influenza A/H3 and 2009 H1N1 Influenza Virus, respectively. This Assay is not intended to detect Influenza B or Influenza C Viruses.

A negative ProFAST+ Assay result is a presumptive negative result for Influenza A. These results should be confirmed by an FDA cleared nucleic acid-based test (NAT) detecting Influenza A.

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

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 Prodesse® ProFAST + Assay is a multiplex Real Time RT-PCR in vitro diagnostic test for the qualitative detection and discrimination of seasonal Influenza A/H1, seasonal Influenza A/H3 and 2009 H1N1 Influenza viral nucleic acids isolated and purified from nasopharyngeal (NP) swab specimens from human patients with signs and symptoms of respiratory infection in conjunction with clinical and epidemiological risk factors. This Assay targets conserved regions of the Hemagglutinin (HA) gene for seasonal Influenza A/H1, seasonal Influenza A/H3 and 2009 H1N1 Influenza Virus, respectively. This Assay is not intended to detect Influenza B or Influenza C Viruses.

The ProFAST+ Assay enables detection and discrimination of Influenza A Virus subtypes: seasonal A/H1, seasonal A/H3, and 2009 H1N1 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.

A Universal Internal Control (UIC) 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 ProFAST+ Supermix along with enzymes included in the ProFAST+ Assay Kit. The ProFAST+ Supermix contains oligonucleotide primers and targetspecific oligonucleotide probes. The primers are complementary to highly conserved regions of the Hemagglutinin (HA) gene for seasonal influenza A/H1, seasonal influenza A/H3 and 2009 H1N1 Influenza Virus. 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 ProFAST+ 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 II instrument.

The provided text describes a special 510(k) submission for the Prodesse® ProFAST®+ Assay, primarily focusing on modifications to the internal control and positive control, and an additional reactivity claim for H3N2v. The submission argues for substantial equivalence to a predicate device (K101855, ProFAST 101+ Assay).

Crucially, the document does not present acceptance criteria or detailed results from a study that "proves the device meets the acceptance criteria" in the format of a typical clinical validation study. Instead, it focuses on demonstrating that modifications did not negatively impact performance compared to the previously cleared predicate device.

Here's an attempt to extract the requested information, noting where details are missing based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state quantitative acceptance criteria (e.g., minimum sensitivity, specificity, or agreement percentages) for a clinical performance study of the modified device. Instead, it refers to the previous performance claims of the ProFAST+ Assay (the predicate device for the modifications) and states that the modified assay "continues to meet the performance claims."

The closest to "reported device performance" are the results of the verification/validation studies for the modifications:

Note: The document explicitly states that "the performance characteristics of this device with clinical specimens that are positive for H3N2v influenza virus have not been established." This means for H3N2v, only analytical reactivity was shown, not clinical performance.

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

• Sample Size for Test Set: The document mentions a "retrospective clinical comparison study" for the UIC modification but does not provide the sample size used in this study.
• Data Provenance: The document states "clinical comparison study," implying human patient samples were used. The term "retrospective" indicates that these samples were collected in the past. The country of origin is not specified but is implicitly the US given the FDA submission.

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

Not provided. The nature of the ground truth (e.g., a reference method like viral culture or another FDA-cleared NAT) is not detailed, nor is the number or qualifications of experts, if any, involved in establishing it. It's likely the "ground truth" for the clinical comparison study would have been established by the reference method against which the predicate device's original performance claims were made.

4. Adjudication Method for the Test Set

Not provided. Given that this appears to be a comparison study against a historical reference or predicate, an adjudication method might not have been
explicitly described in this type of submission.

5. If a Multi Reader Multi Case (MRMC) Comparative Effectiveness Study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance

Not applicable. This device is an in vitro diagnostic (IVD) RT-PCR assay for detecting viral nucleic acids, not an AI-assisted diagnostic tool that would be used by "human readers" in the sense of image interpretation. Therefore, an MRMC study with human readers and AI assistance is not relevant to this device. The "reader" here is the instrument interpreting PCR amplification curves.

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

Yes, the device is inherently a standalone algorithm/assay without human-in-the-loop performance influencing its primary result. It provides a qualitative (positive/negative) detection and discrimination of influenza A subtypes. The "retrospective clinical comparison study" would represent the standalone performance of the modified assay.

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

Not explicitly stated in the provided text. For RT-PCR assays, the ground truth for clinical studies is typically established by:

• A "gold standard" laboratory method (e.g., viral culture if available and sensitive enough, or a highly sensitive and specific FDA-cleared reference molecular test).
• A composite reference method combining multiple tests or clinical findings.

Given it's a "clinical comparison study," it implies comparison to established clinical diagnoses or reference lab results, but the specifics are absent.

8. The Sample Size for the Training Set

Not applicable/Not provided. This is an RT-PCR assay, not a machine learning or AI algorithm that requires a "training set" in the conventional sense. The "training" for such a device involves assay optimization and analytical validation using characterized samples (e.g., contrived samples with known viral concentrations, characterized clinical samples) to establish parameters like limit of detection, linearity, and specificity. The document refers to "Analytical Sensitivity, IC Interference, Extractor Equivalency, and Sample Stability studies," which utilize such characterized samples, but a specific "training set sample size" as per AI/ML terminology is not relevant here.

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

Not applicable/Not provided in the AI/ML context. For analytical studies, the "ground truth" (e.g., viral presence and concentration) is established by using characterized stocks, reference materials, or quantified clinical samples whose status is independently verified (e.g., by culture, sequencing, or quantitative PCR methods).

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The Prodesse ProFlu™+ Assay is a multiplex Real-Time PCR (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. Conversely, positive results do not rule-out bacterial infection or co-infection with other viruses. The agent detected may not be the definite cause of disease. The use of additional laboratory testing and clinical presentation must be considered in order to obtain the final diagnosis of respiratory viral infection.

Performance characteristics for Influenza A Virus were established when Influenza A/H3 and A/H1 were the predominant Influenza A viruses in circulation (2006 - 2007 respiratory season). Performance characteristics for Influenza A were confirmed when Influenza A/H1, Influenza A/H3, and Influenza A/2009 H1N1 were the predominant Influenza A viruses in circulation (2008 and 2009). 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 discrimination of Influenza A Virus, Influenza B Virus, RSV and universal 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. A Universal Internal Control (UIC) 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 Purc 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 Influenza B/RSV Mix along with enzymes included in the ProFlu+ Assay Kit. The Influenza A/Influenza B/RSV Mix 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 ProFity- 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 SmartCyclerII instrument.

The provided document describes a 510(k) premarket notification for a modified in vitro diagnostic device, the Prodesse ProFlu™+ Assay. As such, the information typically associated with acceptance criteria and a detailed study proving device performance against those criteria in the context of AI/ML or image processing devices is not present. This document focuses on demonstrating substantial equivalence to a predicate device, rather than proving performance against specific acceptance criteria with detailed statistical results.

However, I can extract the relevant information based on the prompt's request, interpreting "acceptance criteria" as the claimed performance or non-inferiority that the modification verification studies aimed to confirm.

Here's a breakdown of the requested information, acknowledging the limitations of a 510(k) submission for a molecular diagnostic device:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state numerical "acceptance criteria" in the format typically seen for sensitivity/specificity for algorithms. Instead, the modifications were verified to ensure the fundamental scientific technology and clinical performance remained unchanged from the predicate device. The "acceptance criteria" were met if these aspects were confirmed.

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

The document mentions a "retrospective clinical comparison study" for the UIC impact. However, the specific sample size used for this test set is not provided. The geographic provenance (e.g., country of origin) of the data is also not specified. The study is described as "retrospective."

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

For this type of molecular diagnostic device, ground truth is typically established by laboratory testing methods (e.g., gold standard PCR, sequencing, or culture) rather than expert human interpretation of images or clinical data, especially for a retrospective study focused on assay performance. Therefore, the concept of "number of experts" and their "qualifications" for establishing ground truth as one might consider for imaging devices does not directly apply in this context. The "ground truth" would be the result of a reference laboratory method.

4. Adjudication Method for the Test Set

Given that ground truth is likely based on objective laboratory methods, an "adjudication method" involving multiple human readers (e.g., 2+1, 3+1) is not applicable here.

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 type of study is relevant for evaluating the impact of AI assistance on human reader performance, which is not applicable to a non-AI molecular diagnostic assay validation.

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

The ProFlu™+ Assay is a molecular diagnostic test. The entire assay system (reagents, instrumentation, and protocol) constitutes the "algorithm" in a broad sense. The verification studies assess the performance of this system directly. Therefore, the "standalone" performance is what was evaluated in the analytical and clinical studies described, as there isn't a separate "human-in-the-loop" component in the direct interpretation of the PCR results for this device. The user performs the test and interprets the results based on predefined thresholds, but the core detection is algorithmic.

7. The Type of Ground Truth Used

The ground truth for the verification studies would likely be established through:

• Reference molecular methods: Such as a validated laboratory-developed test (LDT), sequencing, or other nucleic acid amplification tests (NAATs) that are considered the gold standard for detecting the target viruses.
• Viral culture: For confirmation of viable virus.
• Analytical spiking: For analytical sensitivity and reactivity studies, where known concentrations of target nucleic acids are used.

While the document doesn't explicitly state the exact "ground truth" method for the clinical comparison, for a molecular diagnostic, it would invariably involve a highly accurate reference laboratory test.

8. The Sample Size for the Training Set

This submission describes modifications to an existing device and its verification, not the development of a de novo algorithm requiring a "training set" in the context of machine learning. Therefore, the concept of a "training set" does not apply here. The initial development of the predicate ProFlu+ Assay would have involved studies to establish its design parameters.

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

As noted in point 8, the concept of a "training set" is not relevant to this type of device modification submission.

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The Pro hMPV™+ Assay is a Real-Time PCR (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 hMPVnucleic 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 easyMAGTM 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 51-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. 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 provided information, structured according to your request:

1. Table of Acceptance Criteria and Reported Device Performance

The document compares the "New Pro hMPV+ Assay" (reformulated) to the "Current Pro hMPV+ Assay" (predicate device). The acceptance criteria are implied by the "Percent Positive Agreement" and "Percent Negative Agreement" with confidence intervals. While specific numerical acceptance criteria (e.g., "must be >90%") are not explicitly stated, the reported performance is presented as demonstrating substantial equivalence.

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

• Sample Size (Clinical Comparison): 183 nasopharyngeal swab samples (one sample was excluded from the final analysis, resulting in 182).
• Data Provenance: Retrospective, collected during 2011-2012 from two sites: Milwaukee, WI, and Chicago, IL, USA.

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

The document does not mention the use of experts to establish the ground truth for the clinical comparison study. Instead, the "ground truth" was established by:

• "True" hMPV positives: Defined as any sample that tested positive by the original Pro hMPV+ Assay.
• "True" hMPV negatives: Defined as any sample that tested negative by the original Pro hMPV+ Assay.
• Discrepant Analysis: For samples where the new and original assays disagreed, RT-PCR with hMPV specific primers targeting the hMPV phosphoprotein gene followed by bi-directional genetic sequencing was performed. The document does not specify who performed this analysis or their qualifications, but this would be a laboratory-based method.

4. Adjudication Method for the Test Set

The primary comparison was against the predicate device's results. For discrepancies, a molecular method (RT-PCR followed by bi-directional genetic sequencing) was used to resolve disagreements. This acts as a form of "adjudication" based on a more definitive molecular test, rather than human expert consensus.

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 reported. This study evaluates human reader performance, with or without AI assistance. The described study is a comparison of two in vitro diagnostic (IVD) assays.

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

Yes, the described clinical comparison study is a standalone assessment of the new IVD assay's performance against the predicate IVD assay. There is no human-in-the-loop component mentioned; it evaluates the assay's ability to detect hMPV directly from samples.

7. The Type of Ground Truth Used

The ground truth for the clinical comparison study was multi-faceted:

• Reference standard (initial): The results of the predicate device (original Pro hMPV+ Assay).
• Adjudication/Confirmatory method: For discrepant results, RT-PCR with hMPV specific primers targeting the hMPV phosphoprotein gene followed by bi-directional genetic sequencing was used, which can be considered a more definitive molecular ground truth.

8. The Sample Size for the Training Set

The document does not specify a separate training set. The study describes the re-formulation of an existing assay and its performance evaluation. Diagnostic assays like this typically undergo development and optimization phases (which might involve various "training" or optimization samples), but the clinical comparison details the final performance validation using a test set.

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

As no specific training set is outlined in this document, the method for establishing its ground truth is not provided. The information focuses on the validation of the reformulated assay.

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The Prodesse® ProGastro SSCS Assav is a multiplex real time PCR in vitro diagnostic test for the qualitative detection and differentiation of Salmonella, Shigella, and Campylobacter (C. jejimi and C. coli only, undifferentiated) nucleic acids and Shiga Toxin 1 (stxl) and Shiga Toxin 2 (stx2) genes. Shiga toxin producing E. coli (STEC) typically harbor one or both genes that encode for Shiga Toxins I and 2. Nucleic acids are isolated and purified from preserved stool specimens obtained from symptomatic patients exhibiting signs and symptoms of gastroenteritis. This test is intended for use, in conjunction with clinical presentation and epidemiological risk factors, as an aid in the differential diagnosis of Salmonella, Shigella, Campylobacter jejuni/Campylobacter coli, and STEC infections in humans.

The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Positive results do not rule out co-infection with other organisms that are are not detected by this test, and may not be the sole or definitive cause of patient illness. Negative ProGastro SSCS Assay results in the setting of clinical illness compatible with gastroenteritis may be due to infection by pathogens that are not detected by this test or non-infectious causes such as ulcerative colitis, irritable bowel syndrome, or Crohn's disease.

The ProGastro SSCS Assay enables detection and differentiation of Salmonella, Shigella, Campylobacter (C. jejuni and C. coli only, undifferentiated) and an Internal Control in the SSC Mix and Shiga Toxin Producing E. coli (STEC, stx1 and stx2 differentiated) and an Internal Control in the STEC Mix.

An overview of the procedure is as follows:

• 1. Collect raw stool specimens from symptomatic patients and place into Cary Blair Transport Medium or ParaPak C&S (C&S) Transport Medium .
• 2. Add the Gastro RNA/DNA Internal Control (GIC) to every sample to monitor for inhibitors present in the specimens.
• 3. Perform isolation and purification of nucleic acids using a NucliSENS easyMAG System and the Automated Magnetic Extraction Reagents (bioMérieux).
• 4. Add purified nucleic acids to the SSC Mix included in the ProGastro SSCS Assay Kit. The SSC Mix contains target-specific oligonucleotide primers and probes for detection of Salmonella, Shigella, and Campylobacter (C. jejuni and C. coli only). The primers and probes are complementary to highly conserved regions of genetic sequences for these organisms. The probes are dual-labeled with a reporter dye and a quencher (see table below).
• 5. Add purified nucleic acids to the STEC Mix included in the ProGastro SSCS Assay Kit. The STEC Mix contains target-specific oligonucleotide primers and probes for detection of Shiga Toxin 1 and 2 genes (stxl and stx2). The primers and probes are complementary to highly conserved regions of these genes. The probes are dual-labeled with a reporter dye and a quencher (see table below).
• 6. Perform amplification of DNA in a Cepheid SmartCycler II instrument. In this process, the probe anneals specifically to the template followed by primer extension and amplification. The ProGastro SSCS Assay is based on Tagman reagent chemistry, which utilizes the 5' - 3' exonuclease activity of 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 real-time instrument.

Here's a breakdown of the acceptance criteria and study information for the ProGastro SSCS Assay:

The document provided details the clinical performance and analytical performance (reproducibility and precision) of the ProGastro SSCS Assay. The primary acceptance criteria for clinical performance are presented as Sensitivity, Specificity, Positive Percent Agreement (PPA), and Negative Percent Agreement (NPA). For analytical performance, it focuses on the agreement with expected results and the Coefficient of Variation (CV) for Ct values.

1. Table of Acceptance Criteria and Reported Device Performance

Clinical Performance (Prospective Study):

Clinical Performance (Retrospective Study):

Reproducibility (Across 3 Sites, 2 Operators/Site, 5 Days - Total 90 runs per condition):

Precision (Internal, 2 Operators, 12 Days - Total 72 runs per condition):

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

• Prospective Clinical Study:

  • Initial samples: 1214 patients.
  • Eligible samples for analysis: 1139 (after excluding 61 for protocol deviations and 14 for the SSC Mix and 14 for the STEC Mix due to "Unresolved" results and no successful retest).
  • Data Provenance: Prospective, collected from July 2011 - November 2011 and May 2012 - July 2012, and tested November 2011 - August 2012. Conducted at four U.S. clinical laboratories. Samples were "excess remnants of stool specimens that were prospectively collected from symptomatic individuals suspected of gastrointestinal infection, and were submitted for routine care or analysis by each site, and that otherwise would have been discarded."

• Retrospective Study:

  • Sample size: 105 stool samples.
  • Data Provenance: Retrospective, collected from 2007 - 2011. Conducted at two clinical sites. These samples were previously determined positive or negative by culture and/or Broth Enrichment/EIA.

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

The document does not explicitly state the number or qualifications of individual "experts" used to establish ground truth for the test set.

Instead, the ground truth was established by:

• Reference Methods:
  • Culture: For Campylobacter, Salmonella, and Shigella. This typically involves trained microbiologists following established laboratory protocols, but specific expert qualifications are not detailed.
  • Broth enrichment followed by FDA cleared EIA test: For Shiga Toxin producing E. coli (STEC). Similar to culture, this relies on standard laboratory procedures and trained personnel.
• Confirmatory Methods for Discrepancies:
  • PCR followed by bi-directional sequencing: Used to confirm the presence of stx1 and/or stx2 genes in samples positive for STEC by broth/EIA and/or the ProGastro SSCS Assay.
  • Analytically validated PCR/sequencing assays: Used to evaluate discrepant results between the ProGastro SSCS Assay and the initial reference methods. This suggests a rigorous molecular biology approach.

4. Adjudication Method for the Test Set

The adjudication method involved confirmatory testing for discrepant results:

• For STEC: Samples positive by either the broth/EIA method or the ProGastro SSCS Assay underwent PCR followed by bi-directional sequencing to confirm stx1 and/or stx2 genes. "True" STEC positives were considered any sample testing positive by broth/EIA, while "True" stx2 positives were defined as positive by broth/EIA and PCR/sequencing. This suggests a hierarchical adjudication focused on confirming the genes.
• For all targets: Discrepant results between the ProGastro SSCS Assay and the initial reference methods were evaluated using analytically validated PCR/sequencing assays. The results of these confirmatory assays are footnoted in the performance tables, indicating they informed the final determination of "true" status for discrepant samples.

There is no mention of a traditional expert panel consensus (e.g., 2+1, 3+1) for initial ground truth establishment; rather, it relies on established laboratory methodologies and subsequent molecular confirmation for 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 study evaluates the performance of an in vitro diagnostic test (a PCR assay), not a device that is read or interpreted by human readers in a diagnostic imaging or similar context. Therefore, the concept of "how much human readers improve with AI vs without AI assistance" is not applicable here. The assay provides a qualitative result (positive/negative) for specific nucleic acids.

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

Yes, the study primarily represents standalone performance.

The ProGastro SSCS Assay is an in vitro diagnostic test. Its performance (detection of nucleic acids) is assessed as an algorithm only, without direct human cognitive input for interpretation of the primary result (e.g., "reading" a PCR curve to decide positive/negative beyond a set threshold). The technical procedure is performed by trained laboratory personnel, but the "performance" data (Sensitivity, Specificity, PPA, NPA) presented are solely based on the assay's output compared to the ground truth, reflecting the diagnostic capability of the test itself. The intended use states it is "an aid in the differential diagnosis" and "should not be used as the sole basis for diagnosis," implying it contributes to a physician's overall assessment, but its technical performance is standalone.

7. The Type of Ground Truth Used

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

• Culture: For bacterial pathogens (Campylobacter, Salmonella, Shigella). This is a widely accepted gold standard for bacterial identification.
• FDA Cleared EIA Test (with broth enrichment): For Shiga Toxin producing E. coli (STEC). This is a recognized method for STEC detection.
• PCR followed by bi-directional sequencing: This molecular method served as a confirmatory gold standard for discrepant results and for the specific identification of stx1 and stx2 genes. This represents a highly specific and sensitive molecular confirmation.

So, the ground truth is a hybrid of established microbiological culture/EIA methods and molecular sequencing confirmation.

8. The Sample Size for the Training Set

The document does not report a separate training set or details on how the training of any underlying algorithm (if present, which is unlikely for a PCR assay) was performed.

For in vitro diagnostic assays like PCR tests, the "training" equivalent typically involves extensive analytical validation (e.g., limit of detection, inclusivity, exclusivity, interference studies) to establish the assay's performance characteristics, rather than a machine learning training set as understood for AI algorithms. The clinical studies (prospective and retrospective) are primarily for validation of performance.

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

As noted above, a distinct "training set" is not mentioned in the context of this PCR assay. The establishment of ground truth for the validation/test sets is detailed in point 7 (Culture, FDA cleared EIA, and PCR/sequencing).

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