(67 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.
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)
| Analyte | Performance Metric | Implied Acceptance Criteria (Range/Threshold) | Reported Device Performance (95% CI) - Prospective Study | Reported Device Performance (95% CI) - Retrospective Study |
|---|---|---|---|---|
| Influenza A | Sensitivity | High (e.g., >95%) | 100% (97.1% - 100%) | 100% (56.6% - 100%) |
| Specificity | High (e.g., >90%) | 92.6% (90.4% - 94.3%) | 96.4% (87.7% - 99.0%) | |
| Influenza B | Sensitivity | High (e.g., >90%) | 97.8% (88.7% - 99.6%) | 89.5% (68.6% - 97.1%) |
| Specificity | High (e.g., >95%) | 98.6% (97.5% - 99.2%) | 100% (91.4% - 100%) | |
| RSV | Sensitivity | High (e.g., >85%) | 89.5% (75.9% - 95.8%) | 100% (85.7% - 100%) |
| Specificity | High (e.g., >90%) | 94.9% (93.2% - 96.2%) | 97.3% (86.2% - 99.5%) |
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.
§ 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.