(29 days)
The Influenza A Subtyping Kit contains reagents and controls of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel and is intended for use in real-time RT-PCR (rRT-PCR) assays on an in vitro diagnostic real-time PCR instrument that has been FDA-cleared for use with this kit in conjunction with clinical and epidemiological information:
· For determination of the subtype of seasonal human influenza A viruses as seasonal A(H3) and/or A(H1)pdm09 from viral RNA in upper respiratory tract clinical specimens (including nasopharyngeal swabs [NS], throat swabs [TS], nasal aspirates [NA], nasal washes [NW] and dual nasopharyngeal/throat swabs [NPS/TS]) and lower respiratory tract specimens (including bronchoalveolar lavage [BAL], bronchial wash [BW], tracheal aspirate [TA], sputum, and lung tissue) from human patients with signs and symptoms of respiratory infection and/or from viral culture;
· To provide epidemiologic information for surveillance of circulating influenza viruses.
The Influenza A Subtyping Kit contains components of the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel that is used in rRT-PCR assays on an FDA-cleared in vitro diagnostic real-time PCR instrument. The primer and probe sets contained in the Influenza A Subtyping Kit are designed for the detection and characterization of influenza type A viruses that infect humans.
The Influenza A Subtyping Kit consists of oligonucleotide primers and dual-labeled hydrolysis (TagMan®) probes and controls. which may be used in rRT-PCR assays for the in vitro qualitative detection and characterization of the human influenza virus RNA in respiratory specimens from patients presenting with influenza-like illness (IL). The oligonucleotide primers and probes for detection of Influenza A and 2009 Influenza A (swine origin) were selected from highly conserved regions of the matrix (M), and the nucleoprotein (NP), respectively. Oligonucleotide primers and probes for characterization and differentiation of seasonal influenza A(H3) and A(H1)pdm09 viruses were selected from highly conserved regions of their respective HA genes. Detection of viral RNA not only aids in the diagnosis of illness caused by seasonal. newly emerging, and novel influenza viruses in patients with ILI, but also provides epidemiological and surveillance information on influenza and aids in the presumptive laboratory identification of specific novel influenza A viruses.
Here's a breakdown of the acceptance criteria and the study proving the device meets them, based on the provided text:
1. Table of Acceptance Criteria and Reported Device Performance
The acceptance criteria are implied through the performance metrics evaluated and compared to the predicate device and NGS as a gold standard. The core idea is that the modified assay should perform equivalently or better than the previous version, especially for strains with the 3' mutation.
| Acceptance Criterion (Implied) | Reported Device Performance (H3_v2 ZEN & BHQ) |
|---|---|
| Analytical Sensitivity (Limit of Detection - LoD) equivalent to or better than predicate. | A/Darwin/09/2021 (with 3' mutation): LoD was 10^(1.66) EID50/mL for both H3_v2 (ZEN) and H3_v2 (BHQ) with Invitrogen Platinum III SuperScript™. For Quanta qScript™, LoD was 10^(1.18) EID50/mL for H3_v2 (ZEN) and 10^(1.66) EID50/mL for H3_v2 (BHQ). |
| A/HongKong/4801/2014 (without 3' mutation): LoD was 10^(2.12) EID50/mL for both H3_v2 (ZEN) and H3_v2 (BHQ) with Invitrogen Platinum III SuperScript™. For Quanta qScript™, LoD was 10^(2.12) EID50/mL for both H3_v2 (ZEN) and H3_v2 (BHQ). | |
| Overall confirmed LoD of the H3 v2 assay for both ZEN and BHQ quenchers was equivalent to the current H3 IVD assay (10^(-12) or 1.23x10^(2-04) EID50/mL). | |
| Inclusivity for diverse A(H3) strains equivalent to or better than predicate. | All influenza A(H3) strains tested, representing temporal, geographic, and genetic diversity, were detected by the modified H3 v2 assay (both ZEN and BHQ) at low and high titers. |
| Inclusivity of influenza A(H3) strains was not impacted. | |
| Analytical Specificity (Cross-Reactivity with other influenza subtypes) – No cross-reactivity with non-target influenza. | No cross-reactivity was seen with the H3 v2 assay with either ZEN or BHQ quenchers when tested against various other influenza A subtypes (H1N1pdm09, H1N2v, H1N1v, H3N8, H5N8, H7N9, H9N2) and influenza B and C viruses. |
| Analytical Specificity (Cross-Reactivity with non-influenza respiratory pathogens) – No cross-reactivity. | None of the tested non-influenza human respiratory viruses, bacteria, or yeast were detected with either the H3 v2 ZEN or BHQ assays. |
| Positive Percent Agreement (PPA) with NGS for A(H3) strains equivalent to or better than predicate, especially with 3' mutation. | Invitrogen Superscript™ III: H3 v2 (ZEN) 100% (93.4-100), H3 v2 (BHQ) 100% (93.4-100) vs. H3 IVD 100% (93.4-100). |
| Quanta qScript™: H3 v2 (ZEN) 96.67% (89-99), H3 v2 (BHQ) 96.67% (89-99) vs. H3 IVD 95% (86-98). | |
| For both PPA and NPA, the modified H3 v2 assay performed equivalent or better than the current H3 IVD assay. | |
| Negative Percent Agreement (NPA) for negative specimens equivalent to or better than predicate. | Invitrogen Superscript™ III: H3 v2 (ZEN) 100% (93.4-100), H3 v2 (BHQ) 100% (93.4-100) vs. H3 IVD 100% (93.4-100). |
| Quanta qScript™: H3 v2 (ZEN) 100% (93.4-100), H3 v2 (BHQ) 100% (93.4-100) vs. H3 IVD 100% (93.4-100). | |
| For both PPA and NPA, the modified H3 v2 assay performed equivalent or better than the current H3 IVD assay. | |
| No significant shift in Ct values compared to predicate in common positive specimens. | No significant shift in Ct values was seen with the modified H3 v2 assay when comparing average Ct values for positive specimens generating a positive result with both sets of primers and probes. |
| Improved sensitivity for strains with the 3’ mutation. | Illustrative example: One positive specimen with a double mutation showed a shift in Ct value from an average of 31.1 for H3 IVD to 22.94 (H3 v2 ZEN) and 22.77 (H3 v2 BHQ), indicating improved detection. |
2. Sample Size Used for the Test Set and Data Provenance
- Sample Size (Clinical Evaluation):
- Positive Specimen Panel: 60 influenza A(H3) specimens (30 with 3' mutation, 30 without 3' mutation).
- Negative Specimen Panel: 60 negative specimens (from symptomatic patients known to be positive for influenza H1N1).
- Total Clinical Test Set: 120 specimens (60 positive, 60 negative).
- Data Provenance: Retrospective study. Clinical specimens were collected from patients during previous influenza seasons in the United States.
3. Number of Experts Used to Establish the Ground Truth for the Test Set and Their Qualifications
Not applicable. The ground truth for the clinical test set was established using Next Generation Sequencing (NGS) and direct clinical specimen classification, not through expert consensus of visual or diagnostic interpretation.
4. Adjudication Method for the Test Set
Not applicable. The ground truth for the clinical test set was established via Next Generation Sequencing (NGS), which directly determines the genetic identity of the virus, rather than a consensus among human reviewers.
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
Not applicable. This device is an in vitro diagnostic real-time RT-PCR diagnostic panel, not an AI-assisted diagnostic tool that would involve human readers interpreting AI output.
6. If a Standalone (i.e. algorithm only without human-in-the loop performance) was done
Yes, the performance evaluation in this submission is of the standalone device (algorithm only). The RT-PCR assay itself provides the result, without human interpretation of the assay's primary output (e.g., a visual scan of a reaction). The results are considered definitive from the machine output.
7. The Type of Ground Truth Used
- Analytical Performance: The ground truth for analytical sensitivity (LoD) and inclusivity studies was based on known, quantified viral stocks (EID50/mL or ID50/mL) of specific influenza strains.
- Clinical Performance: The ground truth for positive and negative clinical specimens was established using Next Generation Sequencing (NGS) as the comparator assay. This method directly confirms the identity and specific genetic characteristics (e.g., presence of 3' mutation) of the influenza virus in the samples.
8. The Sample Size for the Training Set
The document does not explicitly state a "training set" in the context of device development. This is a modification to an existing RT-PCR assay, and the "development" or "training" of such a device primarily involves bioinformatic analysis, primer/probe design adjustments, and analytical testing with known isolates. The "in-silico analysis" described (Process 1: assessment of primers against global H3N2 sequence information from GISAID EpiFlu database; Process 2: BLAST against NCBI nr/nt database) serves a similar function to a training set for algorithm-based devices by informing the optimal primer and probe sequences. No numerical sample size is provided for these bioinformatic databases.
9. How the Ground Truth for the Training Set Was Established
As noted above, for an RT-PCR assay, the "training set" concept is different from an AI/ML context. The ground truth for informing the primer/probe design (which is effectively the "training" data for the assay's specificity and sensitivity for detecting target sequences) was established through:
- GISAID EpiFlu database: This database contains comprehensive, publicly shared influenza sequence information, used to assess potential primer and probe sets against known H3N2 sequences and calculate nucleotide mismatches. The sequences in this database are derived from laboratories globally undergoing influenza surveillance.
- NCBI BLAST+ against the nr/nt database: This is a vast database of non-redundant nucleotide sequences, against which primer sequences were compared to confirm inclusivity for H3 Influenza virus HA segments and exclusivity against non-target sequences.
This information is based on established genetic sequences, which serve as the fundamental "ground truth" for designing molecular diagnostic assays.
§ 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.