(85 days)
The Quidel® Molecular Influenza A+B assay is a multiplex Real Time RT-PCR assay for the in vitro qualitative detection and differentiation of influenza A and influenza B viral RNA in nasal and nasopharyngeal swabs from patients with signs and symptoms of respiratory infection. This test is intended for use as an aid in the differential diagnosis of influenza A and influenza B viral infections in humans in conjunction with clinical and epidemiological risk factors. The assay does not detect the presence of influenza C virus.
Negative results do not preclude influenza virus infection and should not be used as the sole basis for diagnosis, treatment or other patient management decisions.
Performance characteristics for influenza A were established during the 2010 to 2011 influenza season when influenza A/H3 and 2009 H1N1 influenza were the predominant influenza A viruses in circulation. 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 Quidel Molecular Influenza A+B Assay detects viral RNA that have been extracted from a patient sample using the NucliSENS® easyMAG® automated extraction platform. A multiplex RT-PCR reaction is carried out under optimized conditions in a single tube generating amplicons for each of the target viruses present in the sample. This reaction is performed utilizing the Cepheid SmartCycler® II platform. Identification of influenza A occurs by the use of target specific primers and a fluorescent- labeled probe that hybridizes to a conserved influenza A sequence within the matrix protein gene. Identification of influenza B occurs by the use of target specific primers and fluorescentlabeled probes that will hybridize to a conserved influenza B sequence within the neuraminidase gene.
The following is a summary of the procedure:
- Sample Collection: Obtain nasal swabs and nasopharyngeal swabs specimens using standard techniques from symptomatic patients. These specimens are transported, stored, and processed according to established laboratory procedures.
- Nucleic Acid Extraction: Extract Nucleic Acids from the specimens with the NucliSENS easyMAG System following the manufacturer's instructions using the appropriate reagents.
Prior to the extraction procedure add 20 µL of the Process Control (PRC) to each 180 uL aliquot of specimen. The PRC serves to monitor inhibitors in the extracted specimen, assures that adequate amplification has taken place and that nucleic acid extraction was sufficient.
- Rehydration of Master Mix: Rehydrate the lyophilized Master Mix using 135uL of Rehydration Solution. The Master Mix contains oligonucleotide primers. fluorophore and quencher-labeled probes targeting highly conserved regions of the influenza A and influenza B viruses as well as the process control sequence. The primers are complementary to highly specific and conserved regions in the genome of these viruses. The probes are dual labeled with a reporter dye attached to the 5'end and a quencher attached to the 3' end. The rehydrated Master Mix is sufficient for eight reactions.
- Nucleic Acid Amplification and Detection: Add 15 µL of the rehydrated Master Mix to each reaction tube. SuL of extracted nucleic acids (specimen with PRC) is then added to the tube. Then place the tube into the Cepheid SmartCycler® II.
Once the reaction tubes are added to the instrument, the assay protocol is initiated. This protocol initiates reverse transcription of the RNA targets generating complementary DNA, and the subsequent amplification of the target amplicons occurs. The Quidel Molecular Influenza A+B assay is based on TaqMan® chemistry, and uses an enzyme with reverse transcriptase, DNA polymerase, and 5'-3' exonuclease activities. During DNA amplification, this enzyme cleaves the probe bound to the complementary DNA sequence, separating the quencher dye from the reporter dye. This step generates an increase in fluorescent signal upon excitation by a light source of the appropriate wavelength. With each cycle, additional dye molecules are separated from their quenchers resulting in additional signal. If sufficient fluorescence is achieved by 45 cycles, the sample is reported as positive for the detected nucleic acid.
Here's a breakdown of the acceptance criteria and the study results for the Quidel Molecular Influenza A+B Assay, based on the provided text:
1. Table of Acceptance Criteria and Reported Device Performance
The acceptance criteria are implicitly defined by the results of the comparative clinical study against an FDA-cleared predicate device. The performance characteristics are presented as Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA).
| Performance Metric | Acceptance Criteria (Implied) | Reported Device Performance (Influenza A) | Reported Device Performance (Influenza B) |
|---|---|---|---|
| Prospective Clinical Study (Nasal & Nasopharyngeal Swabs) | |||
| PPA (Positive Agreement) | High agreement with comparator | 100% (157/157) | 98.4% (123/125) |
| NPA (Negative Agreement) | High agreement with comparator | 98.7% (588/596) | 95.5% (600/628) |
| Retrospective Clinical Study (Nasopharyngeal Swabs) | |||
| PPA (Positive Agreement) | High agreement with comparator | 100% (37/37) | 97.4% (37/38) |
| NPA (Negative Agreement) | High agreement with comparator | 100% (315/315) | 98.4% (309/314) |
Note: The document explicitly states "good positive and negative percent agreement when compared to a high performance FDA Cleared Influenza A and B molecular test" in the conclusions, which serves as the general acceptance criterion. The precise numerical thresholds for "good" are not explicitly defined but are demonstrated by the presented results.
2. Sample Size Used for the Test Set and Data Provenance
- Prospective Clinical Study:
- Sample Size: 779 fresh specimens (427 nasal swabs and 352 nasopharyngeal swabs). After removing invalid specimens, 753 specimens were analyzed.
- Data Provenance: The study was conducted during the 2010-2011 respiratory virus season (January to March 2011) at thirteen sites across the United States. The specimens were collected for routine influenza testing and tested at one central location within 72 hours of collection. This is prospective data.
- Retrospective Clinical Study:
- Sample Size: 356 frozen nasopharyngeal swab specimens. After removing invalid specimens, 352 specimens were analyzed.
- Data Provenance: The study used frozen specimens collected during the 2010-2011 respiratory virus season (January to March of 2011). This is retrospective data. The country of origin is not explicitly stated but implied to be the United States, similar to the prospective study, as it's part of the same submission to the FDA in the US.
3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of those Experts
The ground truth for the clinical studies was established using a "high performance FDA Cleared Influenza A and B molecular test" as the comparator method. Thus, it was not established by human experts in the typical sense (e.g., radiologist consensus), but rather by the performance of an already-cleared diagnostic device.
For the discordant results in the prospective study, sequence analysis was used to resolve discrepancies for influenza A (8 specimens that were negative by comparator but positive by Quidel Molecular) and influenza B (26 specimens negative by comparator but positive by Quidel Molecular, and 2 specimens negative by comparator and negative by sequence analysis). The qualifications of those performing the sequence analysis are not detailed.
4. Adjudication Method for the Test Set
For the clinical studies, results were compared directly against the predicate FDA-cleared RT-PCR device. In cases of discordance between the subject device and the comparator device, sequence analysis was performed for some discrepant specimens (specifically, those where the comparator was negative but the Quidel Molecular assay was positive for Influenza A or B). This indicates a form of adjudication where a third, more definitive method (sequencing) was used to assess the comparator's accuracy in those specific cases, rather than an expert panel reviewing the results.
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
No, an MRMC comparative effectiveness study involving human readers and AI assistance was not done. This submission is for a molecular diagnostic assay (RT-PCR), not an AI-powered image analysis or diagnostic tool that would typically involve human "readers." The comparison is between two molecular tests.
6. If a Standalone (i.e. algorithm only without human-in-the-loop performance) was done
Yes, the device was evaluated in a standalone manner. The clinical performance data (Tables 5.8, 5.9, 5.10, 5.11) represent the Quidel Molecular Influenza A+B Assay's performance (algorithm only) compared to another FDA-cleared molecular test.
7. The Type of Ground Truth Used (expert consensus, pathology, outcomes data, etc.)
The primary "ground truth" for the clinical performance evaluation was the results of a "high performance FDA Cleared Influenza A and B molecular test" (the predicate comparator device). For specific discordant results, sequence analysis was used as a more definitive method to further evaluate the initial test results.
8. The Sample Size for the Training Set
The document does not explicitly state a sample size for a "training set" in the context of machine learning or AI. This is a molecular diagnostic assay that functions through specific primer and probe binding, not a learning algorithm that requires a distinct training and test set in the AI sense.
However, the analytical performance studies (Limit of Detection, Analytical Reactivity, Analytical Specificity) involve testing various strains and concentrations, which could be considered akin to "training" or "development" data in a broader sense for optimizing assay parameters. For example:
- LoD study: Replicates of 20 per concentration for 3 influenza A strains and 3 influenza B strains.
- Analytical Reactivity: 38 influenza A strains and 15 influenza B strains, tested in triplicate.
- Analytical Specificity: Panels of 26 viral, 24 bacterial, and 1 yeast strain, tested in triplicate.
9. How the Ground Truth for the Training Set was Established
As this is not an AI/ML device with a conventional "training set," the concept of "ground truth for the training set" as it applies to AI is not directly applicable.
Instead, for the analytical studies:
- Limit of Detection (LoD): Ground truth was established by using quantified (TCID50/mL) cultures of known influenza strains serially diluted in negative matrix. The known concentration was the "truth."
- Analytical Reactivity (Inclusivity): Ground truth was established by using known, well-characterized viral strains (Influenza A subtypes and Influenza B strains) at specified TCID50 levels. The knowledge of the specific viral strain and its presence was the "truth."
- Analytical Specificity (Cross-reactivity): Ground truth was established by using known concentrations of specific viral, bacterial, and yeast strains. The knowledge of which organism was present (or not present, if testing for Influenza A/B) was the "truth."
§ 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.