(82 days)
The 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 ruleout 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 differentiation of Influenza A Virus. Influenza B Virus, Respiratory Syncytial Virus (Types A and B), and Internal Control.
An overview of the procedure is as follows:
- Collect nasopharyngeal swab specimens from symptomatic patients using a polyester, rayon or nylon tipped swab and place into viral transport medium.
- Add an Internal Control (IC) to every sample to monitor for inhibitors present in the specimens.
- Perform isolation and purification of nucleic acids using a MagNA Pure LC System (Roche) and the MagNA Pure Total Nucleic Acid Isolation Kit (Roche) or a NucliSENS easyMAG System (bioMérieux) and the Automated Magnetic Extraction Reagents (bioMérieux).
- Add purified nucleic acids to Influenza A/Influenza B/RSV Mix along with enzymes included in the ProFlu+ Detection 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 and a quencher (see table below).
- Perform reverse transcription of RNA into complementary DNA (cDNA) and subsequent 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 ProFlu+ Assay is based on Taqman reagent 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 realtime instrument.
Here's a breakdown of the acceptance criteria and the study details for the Gen-Probe Prodesse, Inc. ProFlu+ Assay, based on the provided text:
1. Table of Acceptance Criteria and Reported Device Performance
The document does not explicitly state pre-defined acceptance criteria in terms of numerical thresholds for Agreement percentages. However, the study aims to demonstrate substantial equivalence by showing high agreement rates with the original ProFlu+ Assay. The reported performance is presented as Percent Positive Agreement (PPA) and Percent Negative Agreement (PNA) with 95% Confidence Intervals (CI).
| Analyte | Acceptance Criteria (Implied) | Reported Device Performance (New ProFlu+ Assay vs. Current ProFlu+ Assay) |
|---|---|---|
| Influenza A | High agreement with the current ProFlu+ Assay | Percent Positive Agreement: 100% (93.98%-100%) 95% CI |
| Percent Negative Agreement: 99.4% (96.80%-99.90%) 95% CI | ||
| Influenza B | High agreement with the current ProFlu+ Assay | Percent Positive Agreement: 100% (78.47% - 100%) 95% CI |
| Percent Negative Agreement: 100% (98.28% - 100%) 95% CI | ||
| RSV | High agreement with the current ProFlu+ Assay | Percent Positive Agreement: 100% (90.11% - 100%) 95% CI |
| Percent Negative Agreement: 99.0% (96.39%-99.72%) 95% CI |
2. Sample Size Used for the Test Set and Data Provenance
| Analyte | Sample Size (Total) | Positive Samples | Negative Samples | Data Provenance |
|---|---|---|---|---|
| Influenza A | 233 | 60 (determined by original ProFlu+) | 173 (determined by original ProFlu+) | Prospectively collected archived samples from respiratory season years 2008 and 2009, collected at two clinical study sites (Columbus, OH and Albuquerque, NM), USA. |
| Influenza B | 233 | 14 (determined by original ProFlu+) | 219 (determined by original ProFlu+) | Prospectively collected archived samples from respiratory season years 2008 and 2009, collected at two clinical study sites (Columbus, OH and Albuquerque, NM), USA. |
| RSV | 233 | 35 (determined by original ProFlu+) | 198 (determined by original ProFlu+) | Prospectively collected archived samples from respiratory season years 2008 and 2009, collected at two clinical study sites (Columbus, OH and Albuquerque, NM), USA. |
3. Number of Experts Used to Establish the Ground Truth for the Test Set and the Qualifications of Those Experts
The document does not explicitly mention "experts" in the traditional sense (e.g., radiologists, pathologists) for establishing ground truth for individual cases. The ground truth for the comparison study was based on a reference standard as described below.
4. Adjudication Method for the Test Set
The primary reference for the comparison study was the original ProFlu+ Assay.
- "True" influenza A, influenza B or RSV positives were considered as any sample that tested positive for the respective analyte by the original ProFlu+ Assay.
- "True" Influenza A, Influenza B or RSV negatives were considered as any sample that tested negative by the original ProFlu+ Assay.
For discordant results (where the "New" ProFlu+ Assay differed from the "Current" ProFlu+ Assay), bidirectional sequencing was used as an adjudicator:
- For Influenza A: One sample was negative by the current ProFlu+ but positive by the new ProFlu+. Sequencing confirmed it was positive for Influenza A.
- For RSV: Two samples were negative by the current ProFlu+ but positive by the new ProFlu+. Sequencing confirmed they were positive for RSV.
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
This is not applicable. The ProFlu+ Assay is an in vitro diagnostic test (a lab assay), not an AI-powered image analysis or diagnostic tool that assists human readers. Therefore, an MRMC study or an assessment of human reader improvement with AI assistance would not be conducted for this type of device.
6. If a Standalone (i.e. algorithm only without human-in-the-loop performance) was done
This is not applicable in the context of "algorithm only" as typically referred to for AI products. The ProFlu+ Assay is a PCR-based diagnostic test, and its performance is evaluated as a standalone test in the laboratory, without direct human cognitive input into the result generation process itself. The "New ProFlu+ Assay" was compared directly against the "Current ProFlu+ Assay" as standalone tests.
7. The Type of Ground Truth Used
The ground truth for the comparison study was established using a combination of a pre-existing validated assay (the original ProFlu+ Assay) as the primary reference, and bidirectional sequencing for resolving discordant results.
Specifically:
- Initial determination: The results from the original ProFlu+ Assay served as the provisional "true" positive or negative status.
- Adjudication for discrepant results: Bidirectional sequencing (targeting a different gene or region of the same gene) was used to confirm the presence of the virus in samples where the new and original assays disagreed. This sequencing acts as a higher-level confirmatory test.
8. The Sample Size for the Training Set
The document does not explicitly describe a "training set" in the context of machine learning (AI). This device is a diagnostic assay, not an AI model requiring a training phase with labeled data in the same way. The "reformulated supermix" implies a development and optimization process, but the specific data used for that internal development (if analogous to training data) is not detailed. The numbers provided (233 samples per analyte) refer to the clinical comparison study data.
9. How the Ground Truth for the Training Set Was Established
As noted above, the concept of a "training set" with ground truth in the AI/machine learning sense is not directly applicable here. The assay's development (including the reformulated supermix) would have involved extensive R&D and validation using characterized samples, but this information is not provided in a format that aligns with AI training set descriptions.
§ 866.3980 Respiratory viral panel multiplex nucleic acid assay.
(a)
Identification. A respiratory viral panel multiplex nucleic acid assay is a qualitative in vitro diagnostic device intended to simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or viral culture. The detection and identification of a specific viral nucleic acid from individuals exhibiting signs and symptoms of respiratory infection aids in the diagnosis of respiratory viral infection when used in conjunction with other clinical and laboratory findings. The device is intended for detection and identification of a combination of the following viruses:(1) Influenza A and Influenza B;
(2) Influenza A subtype H1 and Influenza A subtype H3;
(3) Respiratory Syncytial Virus subtype A and Respiratory Syncytial Virus subtype B;
(4) Parainfluenza 1, Parainfluenza 2, and Parainfluenza 3 virus;
(5) Human Metapneumovirus;
(6) Rhinovirus; and
(7) Adenovirus.
(b)
Classification. Class II (special controls). The special controls are:(1) FDA's guidance document entitled “Class II Special Controls Guidance Document: Respiratory Viral Panel Multiplex Nucleic Acid Assay;”
(2) For a device that detects and identifies Human Metapneumovirus, FDA's guidance document entitled “Class II Special Controls Guidance Document: Testing for Human Metapneumovirus (hMPV) Using Nucleic Acid Assays;” and
(3) For a device that detects and differentiates Influenza A subtype H1 and subtype H3, FDA's guidance document entitled “Class II Special Controls Guidance Document: Testing for Detection and Differentiation of Influenza A Virus Subtypes Using Multiplex Nucleic Acid Assays.” See § 866.1(e) for the availability of these guidance documents.