(18 days)
The CDC Influenza 2009 A(H1N1)pdm Real-Time RT-PCR Panel (CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel) is intended for use in real-time RT-PCR assays on an Applied Biosystems (ABI) 7500 Fast Dx Real-Time PCR Instrument in conjunction with clinical and epidemiological information:
- For the qualitative detection of influenza virus type A viral RNA from nasopharyngeal swabs (NPS), nasal swabs (NS), nasal aspirates (NA), nasal washes (NW), dual nasopharyngeal / throat swabs (NPS/TS), broncheoalveolar lavage (BAL), tracheal aspirate (TA), and bronchial wash (BW), collected from the respiratory tract of human patients with signs and symptoms of respiratory infection and/or from viral culture.
- For differentiation of 2009 H1N1 influenza virus RNA from nasopharyngeal swabs (NPS), nasal swabs (NS), nasal aspirates (NA), nasal washes (NW) dual nasopharyngeal / throat swabs (NPS/TS), broncheoalveolar lavage (BAL), tracheal aspirate (TA), and bronchial wash (BW) collected from the respiratory tract of human patients with signs and symptoms of respiratory infection and/or from viral culture.
- To provide epidemiologic information for surveillance of the 2009 H1N1 influenza virus.
The CDC Influenza 2009 A(H1N1)pdm Real-time RT-PCR Panel (CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel) is a panel of oligonucleotide primers and dual-labeled hydrolysis (TaqMan®) probes which may be used in real-time RT-PCR (rRT-PCR) assays for the in vitro qualitative detection and characterization of human influenza viruses (RNA) in respiratory specimens from patients presenting with influenza-like illness (ILI). Detection of viral RNA not only aids in the diagnosis of illness caused by seasonal and novel influenza viruses in patients with ILI, but also provides epidemiologic information on influenza and aids in the presumptive laboratory identification of specific novel influenza A viruses. The influenza A primer and probe sets are designed for universal detection of type A influenza viruses, Influenza A subtyping primer and probe sets are designed to specifically detect 2009 A (H1N1) influenza virus. The CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel is an updated version of the assay distributed by CDC. during the 2009 A(H1N1) pandemic strain to qualified laboratories (CDC rRT-PCR Swine Flu Panel G090072) under Emergency Use Authorization (EUA) from the FDA in that it has different sequences of primers and probes that are more specific to the currently circulating 2009 A(H1N1) influenza virus. The CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel includes the following primer and probe sets and positive control: InfA detects all influenza A strains, but does not detect influenza B strains. pdm InfA is specific for 2009 influenza A. pdm H1 is specific for 2009 influenza A, subtype H1. RNase P (RP) detects human RNase P and is used with human clinical specimens to indicate that adequate isolation of nucleic acid resulted from the extraction of the clinical specimen. Inactivated Influenza Typing Panel Real-Time RT-PCR Positive Control is a positive control designed to react with all the primer and probe sets including RNase P.
Device Acceptance Criteria and Study Details: CDC Influenza 2009 A(H1N1)pdm Real-Time RT-PCR Panel
1. Table of Acceptance Criteria and Reported Device Performance
| Criteria Category | Specific Criterion | Acceptance Criteria | Reported Device Performance |
|---|---|---|---|
| Analytical Performance | |||
| Limit of Detection (LoD) | Lowest detectable concentration (EID50/mL) | 95.0% or greater positivity for each primer and probe set. | - A/California/07/2009: InfA (10^1.6), pdm InfA (10^1.6), pdm H1 (10^1.6). Final LoD = 10^1.6 (EID50/mL). |
- A/New York/18/2009: InfA (10^1.3), pdm InfA (10^0.6), pdm H1 (10^1.3). Final LoD = 10^1.3 (EID50/mL). |
| Inclusivity | Concordance with diverse 2009 A(H1N1) influenza viruses | Not explicitly stated as a numerical threshold, implied to be high for specific detection. | 100% concordance with all primer and probe sets when testing ten diverse 2009 A(H1N1) influenza viruses at low concentrations. |
| Exclusivity (Seasonal) | Non-detection of seasonal influenza viruses by pdm InfA and pdm H1 | Complete non-reactivity (negative results) of pdm InfA and pdm H1 with seasonal Inf A and Inf B strains, while Inf A should detect seasonal A. | - Seasonal H1N1: InfA detected all 16 tested strains; pdm InfA and pdm H1 were negative for all. - Seasonal H3N2: InfA detected all 7 tested strains; pdm InfA and pdm H1 were negative for all.
- Influenza B: InfA, pdm InfA, and pdm H1 were negative for all 12 tested strains. |
| Exclusivity (H5N1) | Non-detection of H5N1 by pdm InfA and pdm H1, while InfA should detect H5N1 | Complete non-reactivity (negative results) of pdm InfA and pdm H1 with H5N1 strains, while Inf A should detect H5N1. | InfA detected all 10 tested H5N1 strains; pdm InfA and pdm H1 were negative for all, except for one H5N1 strain (A/Chicken/Vietnam/NCVD-016/2008) where pdm H1 result was not provided. |
| Non-Influenza Pathogens | Non-cross reactivity with common respiratory pathogens and flora | 100% agreement with expected negative results. | 100% concordance with expected results for all primer and probe sets (InfA, pdm InfA, pdm H1) when tested against 34 non-influenza organisms (commensal bacteria, yeast, and non-influenza respiratory viruses). This means all markers were negative. |
| Reproducibility | Agreement with expected results and %CV for Ct values across sites and operators | Demonstrated reproducibility across six sites, two operators, and five days. Percentage of agreement for moderate, low, "high negative", and negative samples. Low %CV for average Ct values, particularly for moderate and low viral RNA concentrations. | - No Template Control, RP, HSC, Positive Control: 100% agreement (60/60) across all sites. - Sample 1 (moderate viral RNA): 100% agreement (60/60) for InfA, pdm InfA, pdm H1, and RP. Low %CV for all markers (e.g., InfA 2.59-4.23%, pdm InfA 2.10-3.10%, pdm H1 1.48-3.17%, RP 1.33-2.65%).
- Sample 2 (low viral RNA): High agreement (54/60 to 60/60 total). Some sites showed lower agreement (e.g., 7/10 for pdm H1 at Roche MagNA Pure LC and Compact NA). %CV generally higher than moderate samples (e.g., InfA 1.04-35.61%, pdm InfA 2.48-35.27%, pdm H1 1.27-52.94%, RP 1.34-5.11%).
- Sample 3 ("high negative"): Agreement ranged from 54/60 to 58/60, with some individual site variations. All samples were expected to be negative for InfA, pdm InfA, and pdm H1. |
| Clinical Performance | | | |
| Clinical Sensitivity | Greater than a specified percentage | >96% for all markers (upper respiratory specimens).
83% for all markers (lower respiratory specimens). | - Upper Respiratory (NPS/NS): InfA (96.8%), 2009 H1N1 (100%).
- Upper Respiratory (NA/NW): InfA (100%), 2009 H1N1 (100%).
- Upper Respiratory (NPS/TS): InfA (100%), 2009 H1N1 (100%).
- Lower Respiratory (BAL, TA, BW): InfA (83.3%), 2009 H1N1 (100%). Results meet or exceed criteria. |
| Clinical Specificity | Greater than a specified percentage | >96% for all markers (upper respiratory specimens).
83% for all markers (lower respiratory specimens). | - Upper Respiratory (NPS/NS): InfA (96.2%), 2009 H1N1 (96.7%).
- Upper Respiratory (NA/NW): InfA (99.3%), 2009 H1N1 (98.6%).
- Upper Respiratory (NPS/TS): InfA (100%), 2009 H1N1 (98.3%).
- Lower Respiratory (BAL, TA, BW): InfA (83.3%), 2009 H1N1 (84.0%). Results meet or exceed criteria. |
| Positive Percent Agreement | Greater than a specified percentage | >96% for all markers (upper respiratory specimens).
100% for all markers (lower respiratory specimens). | - Upper Respiratory (NPS/NS): InfA (99.4%), 2009 H1N1 (99.7%). - Upper Respiratory (NA/NW): InfA (97.7%), 2009 H1N1 (99.0%).
- Upper Respiratory (NPS/TS): InfA (100%), 2009 H1N1 (100%).
- Lower Respiratory (BAL, TA, BW): InfA (100%), 2009 H1N1 (100%). Results meet or exceed criteria. |
| Overall Performance Call Rate | (Clinical Evaluation) | >99% | >99% |
2. Sample Size and Data Provenance (Clinical Performance Test Set)
- Sample Size for Test Set: 1901 total patient specimens.
- 1191 nasopharyngeal swabs (NPS) and nasal swabs (NS)
- 50 throat swabs (TS)
- 519 nasal washes (NW) and nasal aspirates (NA)
- 99 dual nasopharyngeal / throat swabs (NPS/TS)
- 42 lower respiratory specimens (broncheoalveolar lavage (BAL), tracheal aspirate (TA), and bronchial wash (BW))
- Data Provenance: Prospective study, supplemented with retrospective specimens. Data collected during the 2009-2010 respiratory virus season (February-April) from 8 U.S. public health laboratories and a Department of Defense (DoD) laboratory. Samples were from individuals symptomatic with influenza-like illness (ILI). Retrospective specimens were used to supplement due to low prevalence of influenza.
3. Number of Experts and Qualifications for Test Set Ground Truth
The document does not explicitly state the "number of experts" used to establish the ground truth in the same way one would delineate expert radiologists for image annotation.
Instead, the ground truth for the clinical performance study was established by standard laboratory methods:
- For InfA (Influenza A detection): Virus culture with Immunofluorescent Antibody (IFA) or Direct Fluorescent Antibody (DFA) was used for screening and identification of influenza type.
- For 2009 H1N1 (subtype detection): Bi-directional sequencing was used for confirmation of 2009 influenza A (H1N1) subtype. (Sequencing was performed only on specimens already identified as positive for influenza A by virus culture).
These methods represent established diagnostic procedures typically performed by trained laboratory personnel, rather than interpretation by "experts" in the context of a consensus reading.
4. Adjudication Method for the Test Set
The document does not describe an explicit "adjudication method" in the context of multiple human readers reconciling differences. The ground truth was established by laboratory methods as described above. If there were discrepancies between initial screening (IFA/DFA) and confirmatory sequencing, the sequencing result would have been the definitive ground truth for subtyping.
5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study
No, a multi-reader multi-case (MRMC) comparative effectiveness study was not performed. The study evaluates the standalone performance of the RT-PCR panel against laboratory reference methods, not improvement in human reader performance with AI assistance.
6. Standalone (Algorithm Only) Performance Study
Yes, a standalone performance study was done. The entire document focuses on evaluating the analytical and clinical performance of the CDC Influenza 2009 A(H1N1)pdm Real-Time RT-PCR Panel (an algorithm/assay) independent of human interpretation or assistance, against established laboratory reference methods. The results presented for sensitivity, specificity, and agreement are for the device's performance directly.
7. Type of Ground Truth Used
- Clinical Performance Ground Truth:
- Virus Culture (with IFA/DFA): For the detection of influenza type A (against which the InfA analyte of the device was compared).
- Bi-directional Sequencing: For the confirmation of 2009 influenza A (H1N1) subtype (against which the pdm InfA and pdm H1 analytes of the device were compared).
- Analytical Performance Ground Truth:
- Known viral concentrations (EID50/mL, TCID50/mL): For Limit of Detection, Inclusivity, and Exclusivity studies.
- Known identity of specific influenza and non-influenza organisms: For Inclusivity, Exclusivity, and Reactivity of Non-Influenza Respiratory Viral and Bacterial Pathogens studies.
8. Sample Size for the Training Set
The document does not provide information about a "training set" or its sample size. This type of device (RT-PCR panel) is a diagnostic kit where the primers and probes are designed based on known viral sequences, rather than an AI/ML algorithm that is "trained" on a dataset in the conventional sense. The "development" and "optimization" of such a panel would involve bioinformatic design and iterative testing, not "training" on a sample set like an AI model.
9. How the Ground Truth for the Training Set (if applicable) was Established
As mentioned above, the concept of a "training set" and "ground truth establishment for a training set" as it applies to AI/ML devices is not applicable here. The CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel is a molecular diagnostic assay where the "design" is based on established scientific principles of molecular biology and virology, including viral genome sequencing and primer/probe design. The specificity and sensitivity are intrinsically built into the primer and probe sequences themselves, which are designed to target specific genetic regions of the influenza virus.
§ 866.3332 Reagents for detection of specific novel influenza A viruses.
(a)
Identification. Reagents for detection of specific novel influenza A viruses are devices that are intended for use in a nucleic acid amplification test to directly detect specific virus RNA in human respiratory specimens or viral cultures. Detection of specific virus RNA aids in the diagnosis of influenza caused by specific novel influenza A viruses in patients with clinical risk of infection with these viruses, and also aids in the presumptive laboratory identification of specific novel influenza A viruses to provide epidemiological information on influenza. These reagents include primers, probes, and specific influenza A virus controls.(b)
Classification. Class II (special controls). The special controls are:(1) FDA's guidance document entitled “Class II Special Controls Guidance Document: Reagents for Detection of Specific Novel Influenza A Viruses.” See § 866.1(e) for information on obtaining this document.
(2) The distribution of these devices is limited to laboratories with experienced personnel who have training in standardized molecular testing procedures and expertise in viral diagnosis, and appropriate biosafety equipment and containment.