(21 days)
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)
| Analyte | Implied Acceptance Criteria (High Sensitivity & Specificity for Diagnosis) | Reported Sensitivity (95% CI) | Reported Specificity (95% CI) |
|---|---|---|---|
| Influenza A | Comparable to predicate devices and aiding in diagnosis. | 100% (97.1% - 100%) | 92.6% (90.4% - 94.3%) |
| Influenza B | Comparable to predicate devices and aiding in diagnosis. | 97.8% (88.7% - 99.6%) | 98.6% (97.5% - 99.2%) |
| RSV | Comparable to predicate devices and aiding in diagnosis. | 89.5% (75.9% - 95.8%) | 94.9% (93.2% - 96.2%) |
Table 2: Acceptance Criteria (Implied) and Reported Device Performance (Retrospective Study)
| Analyte | Implied Acceptance Criteria (High Sensitivity & Specificity for Diagnosis) | Reported Sensitivity (95% CI) | Reported Specificity (95% CI) |
|---|---|---|---|
| Influenza A | Comparable to predicate devices and aiding in diagnosis. | 100% (56.6% - 100%) | 96.4% (87.7% - 99.0%) |
| Influenza B | Comparable to predicate devices and aiding in diagnosis. | 89.5% (68.6% - 97.1%) | 100% (91.4% - 100%) |
| RSV | Comparable to predicate devices and aiding in diagnosis. | 100% (85.7% - 100%) | 97.3% (86.2% - 99.5%) |
Study Details:
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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.
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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.
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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.
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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.
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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).
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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).
- The primary ground truth for the test set was established by a combination of methods:
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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.
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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.
§ 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.