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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 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 detection and differentiation of Parainfluenza 1 Virus, Parainfluenza 2 Virus, Parainfluenza 3 Virus and Universal Internal Control nucleic acids. Nasopharyngeal swab specimens are collected from symptomatic patients 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 and a quencher.

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 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.

This document describes the Prodesse® ProParaflu®+ Assay, a multiplex Real-Time PCR (RT-PCR) in vitro diagnostic test for the qualitative detection and discrimination of Parainfluenza 1, 2, and 3 Viruses.

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Acceptance Criteria and Device Performance

The provided document is a 510(k) summary, which focuses on demonstrating substantial equivalence to a predicate device. It highlights similarities and differences, and mentions limitations related to specific CAP samples. Crucially, this document does not explicitly state acceptance criteria in terms of performance metrics (e.g., sensitivity, specificity, accuracy) nor does it present detailed study results proving the device meets said acceptance criteria.

The "Substantial Equivalence" section primarily states that the Intended Use, Warnings and Precautions, Organisms Detected, Analyte, Technological Principles, Specimen Types, User Complexity, Sample Preparation Method, Instrumentation, Time to Result, and Controls are the same as the predicate device (K091053). This implies that the device is expected to perform at a similar level to the predicate, but specific performance criteria for the new device are not enumerated here.

The key difference noted is a "Limited reactivity with the 2014 CAP sample ID2-08 and the 2015 CAP sample ID2-02," attributed to a viral mutation in the probe binding region of HPIV3. This limitation implies a potential issue with sensitivity for specific HPIV3 variants, which could be considered a deviation from an implied acceptance criterion of detecting all clinically relevant HPIV3 strains.

Given the information, a table of implied acceptance criteria and reported "performance" (which is more a statement of similarity or a recognized limitation) would be:

The document does not provide the following information:

2. Sample sizes used for the test set and the data provenance: There is no specific data presented from a test set. The document refers to "the 2014 CAP sample ID2-08 and the 2015 CAP sample ID2-02" which indicates external proficiency testing samples, but not a full clinical test set used for performance validation in this submission.
3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts: Not applicable, as detailed performance data from a test set is not provided.
4. Adjudication method for the test set: Not applicable.
5. If a multi-reader multi-case (MRMC) comparative effectiveness study was done, and the effect size of how much human readers improve with AI vs without AI assistance: Not applicable. This is an in vitro diagnostic (IVD) assay, not an AI/imaging device requiring human reader analysis.
6. If a standalone (i.e. algorithm only without human-in-the-loop performance) was done: The device is a "standalone" in the sense that it's an assay. Performance is measured by its output based on the sample, not by an algorithm's interpretation for a human-in-the-loop. However, specific standalone performance study results (e.g., sensitivity, specificity clinical studies) are not provided in this 510(k) summary. The summary focuses on substantial equivalence based on the technological similarity to a predicate.
7. The type of ground truth used: Not explicitly stated for performance verification studies, as such studies are not detailed here. For the CAP samples mentioned, "sequencing analysis" was used to establish the genetic makeup of the viral strain, which served as a form of ground truth for assessing reactivity.
8. The sample size for the training set: Not applicable for this type of IVD assay. The assay utilizes PCR primers and probes designed based on conserved genetic regions, not a machine learning model trained on a dataset.
9. How the ground truth for the training set was established: Not applicable.

In summary, this 510(k) notification primarily argues for substantial equivalence based on the device's technical and analytical similarity to a previously cleared predicate and its intended use. It discloses a specific limitation identified through external proficiency samples but does not provide comprehensive clinical performance data or details of a validation study with defined acceptance criteria and corresponding results. Such data would typically be part of the full 510(k) submission, but not always summarized in this manner in the publicly available summary.

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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 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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