(210 days)
The Cepheid Xpert Flu-RSV Xpress Assay, performed on the GeneXpert Xpress System, is an automated, multiplex realtime, reverse transcriptase polymerase chain reaction (RT-PCR) assay intended for the in vitro qualitative detection and differentiation of influenza B, and respiratory syncytial virus (RSV) viral RNA. The Xpert Flu+RSV Xpress Assay uses nasopharyngeal swab specimens collected from patients with signs and symptoms of respiratory infection. The Xpert Flu+RSV Xpress Assay is intended as an aid in the diagnosis of influenza and respiratory syncytial virus infections in conjunction with clinical and epidemiological risk factors.
Negative results do not preclude influenza virus or respiratory syncytial virus infection and should not be used as the sole basis for treatment or other patient management decisions.
Performance characteristics for influenza A were established during the 2014-2015 influenza season. When other novel 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 Xpert® Nasopharyngeal Sample Collection Kit is designed to collect, preserve, and transport nasopharyngeal swab specimens and to preserve and transport nasal aspirate/wash specimens containing viruses from patients with signs and symptoms of respiratory infection prior to analysis with the Xpert Flu Assay or the Xpert Flu/RSV XC Assay. The Xpert® Nasopharyngeal Sample Collection Kit is designed to collect, preserve, and transport nasopharyngeal swab specimens containing viruses from patients with signs and symptoms of respiratory infection prior to analysis with the Xpert Flu+RSV Xpress Assay.
The Xpert Flu+RSV Xpress Assay is an automated in vitro diagnostic test for qualitative detection and differentiation of influenza A. influenza B, and respiratory syncytial virus (RSV). The assay is performed on the Cepheid GeneXpert Xpress System (GeneXpert Dx System, GX-I). The GeneXpert Xpress System platform automates and integrates sample extraction, purification, amplification, and detection of the target sequence in simple or complex samples using real-time PCR and reverse transcriptase PCR (RT-PCR) assays. The systems require the use of single-use disposable cartridges (the Xpert Flu+RSV Xpress cartridges) that hold the PCR reagents and host the PCR process. Because the cartridges are self-contained and specimens never come into contact with working parts of the instrument modules, cross-contamination between samples is minimized.
The Xpert Flu+RSV Xpress Assay includes reagents for the detection and differentiation of influenza A, influenza B, and RSV viral RNA directly from nasopharyngeal (NP) swab specimens collected from patients with signs and symptoms of respiratory infection. A Sample Processing Control (SPC) and a Probe Check Control (PCC) are also included in the cartridge. The SPC is present to control for adequate processing of the target viruses and to monitor the presence of inhibitors in the PCR reaction. The Probe Check Control (PCC) verifies reagent rehydration, PCR tube filling in the cartridge, probe integrity, and dye stability.
The single-use, multi-chambered fluidic cartridges are designed to complete sample preparation and real-time RT-PCR for detection and differentiation of influenza B and RSV viral RNA in approximately 60 minutes. The GeneXpert Xpress System, comprised of the GeneXpert Dx System GX-I, has one module that is capable of performing separate sample preparation and real-time PCR and RT-PCR tests. Each module contains a syringe drive for dispensing fluids (i.e., the syringe drive activates the plunger that works in concert with the rotary valve in the cartridge to move fluids between chambers), an ultrasonic horn for lysing cells or spores, and a proprietary I-CORE® thermocycler for performing realtime PCR and RT-PCR and detection.
Specimens are collected following the instructions for collecting NP swab specimens provided in Xpert Flu+RSV Xpress Assay package insert for influenza and RSV testing. The Cepheid Xpert Nasopharyngeal Sample Collection Kit (Cepheid catalog #SWAB/B-100) is required but not provided for use with the assay. The NP swab specimen is placed in the Xpert viral transport medium and sent to the GeneXpert® Xpress testing area for processing. When stored in the transport medium, the NP swab specimen is stable for up to 24 hours at 2-30 °C or up to seven days at 2-8 °C. When ready to test the specimen, the user briefly mixes the specimen by inverting the tube five times, transfers the eluted material to the sample chamber in the top of the disposable fluidic cartridge. The user initiates a test from the system user interface and places the cartridge into the GeneXpert Xpress instrument platform, which performs hands-off real-time, multiplex polymerase chain reaction (PCR) for detection of RNA. The results are automatically generated at the end of the process in a report that can be viewed and printed.
1. Table of Acceptance Criteria and Reported Device Performance
| Device Performance Metric | Acceptance Criteria (Implicit) | Reported Device Performance |
|---|---|---|
| Analytical Sensitivity (LoD) | Reproducibly distinguish negative samples with 95% confidence or at least 19/20 replicates positive at the lowest concentration. | Influenza A 2009 H1N1: A/California/7/2009: 0.3 TCID50/mL (20/20 positive); A/Florida/27/2011: 16.0 TCID50/mL (20/20 positive). |
| Influenza A H3N2: A/Perth/16/2009: 0.3 TCID50/mL (20/20 positive); A/Victoria/361/2011: 0.8 TCID50/mL (20/20 positive). | ||
| Influenza B: B/Massachusetts/2/2012: 0.5 TCID50/mL (20/20 positive); B/Wisconsin/01/2011: 0.6 TCID50/mL (20/20 positive). | ||
| RSV A: RSV A/2/Australia/61: 1.2 TCID50/mL (20/20 positive); RSV A/Long/MD/56: 1.0 TCID50/mL (20/20 positive). | ||
| RSV B: RSV B/Washington/18537/62: 1.8 TCID50/mL (20/20 positive); RSV B/9320/Massachusetts/77: 2.0 TCID50/mL (20/20 positive). | ||
| Influenza A H7N9: A/Anhui/1/2013: 0.8 TCID50/mL (19/20 positive). | ||
| Analytical Specificity | 100% specificity against common respiratory pathogens and potentially encountered organisms in the nasopharynx. | 100% analytical specificity against a panel of 44 cultures (16 viral, 26 bacterial, 2 yeast strains). No false positives observed. |
| Analytical Reactivity | Detection of multiple strains of influenza A (H1N1, H3N2, avian), influenza B (Victoria and Yamagata lineages), and RSV (A and B subgroups) at levels near the LoD. | Successfully detected all 64 tested strains (54 influenza and 10 RSV) at levels near the analytical LoD. Results consistently showed Flu A POSITIVE, Flu B POSITIVE, or RSV POSITIVE as expected, with negative results for non-target analytes. |
| Potentially Interfering Substances | No assay interference in the presence of various substances (e.g., blood, nasal secretions, medications, vaccines) at tested concentrations. | No assay interference observed in the presence of 11 different potentially interfering substances (e.g., Albuterol Sulfate, Human Blood, Mucin, Mupirocin, Phenylephrine, Zanamivir, Tobramycin, FluMist, Fluticasone Propionate, saline nasal spray, various transport media). All positive and negative replicates were correctly identified. FluMist was correctly reported as Flu A POSITIVE; FLU B POSITIVE; RSV NEGATIVE. |
| Carry-Over Contamination | Single-use, self-contained cartridges prevent carry-over contamination in negative samples following very high positive samples. | All 40 positive samples correctly reported, and all 42 negative samples directly following very high positive samples were correctly reported as negative. |
| Fresh vs. Frozen Sample Equivalency | No statistically significant effect in performance between fresh and freeze-thawed samples for positive and negative specimens. | All positive and negative replicates were correctly identified after fresh testing, one freeze-thaw cycle, and two freeze-thaw cycles. No statistically significant effect on performance was observed. |
| Clinical Comparison (PPA/NPA) | Acceptable PPA and NPA relative to an FDA-cleared molecular comparator assay for influenza A, influenza B, and RSV. | Fresh NP Swab Specimens: |
- Flu A: PPA 100% (98.5-100), NPA 94.8% (93.7-95.7)
- Flu B: PPA 100% (94.3-100), NPA 99.5% (99.1-99.8)
- RSV: PPA 96.9% (92.3-99.1), NPA 99.6% (99.2-99.8)
Pre-selected Frozen NP Swab Specimens: - Flu A: NPA 98.5% (96.1-99.6) (PPA not applicable as no positive collected)
- Flu B: PPA 100% (96.4-100), NPA 98.7% (95.5-99.8)
- RSV: PPA 97.6% (87.1-99.9), NPA 99.5% (97.5-100) |
| Reproducibility (Total Agreement) | High percentage of samples yielding expected results across sites, days, and operators, especially for positive samples. | Negative: 100% (88/88)
Flu A-Low Pos: 98.9% (89/90)
Flu A-Mod Pos: 100% (90/90)
Flu B-Low Pos: 98.9% (89/90)
Flu B-Mod Pos: 100% (90/90)
RSV-Low Pos: 97.8% (87/89)
RSV-Mod Pos: 100% (90/90) (Note: High Neg samples show lower agreement, consistent with near cutoff concentrations). |
| Reproducibility (Ct Variation) | Low variability (SD, CV) in Ct values across sites, days, and operators for detected targets. | SPC: Total Std Dev 1.5, Total CV 4.8%
Flu A1 (Low Pos): Total Std Dev 1.6, Total CV 4.5%
Flu A1 (Mod Pos): Total Std Dev 1.4, Total CV 4.2%
Flu B (Low Pos): Total Std Dev 1.9, Total CV 5.9%
Flu B (Mod Pos): Total Std Dev 1.0, Total CV 3.3%
RSV (Low Pos): Total Std Dev 1.7, Total CV 5.0%
RSV (Mod Pos): Total Std Dev 1.0, Total CV 3.0% (Similar values for corresponding High Neg samples). |
2. Sample Size Used for the Test Set and Data Provenance
- Clinical Comparison Study (Test Set):
- Total NP Swab Specimens: 2435
- Fresh, Prospectively Collected: 2176
- Pre-selected Frozen, Archived Specimens: 259
- Data Provenance: United States (12 institutions in the U.S.), retrospective for archived samples and prospective for fresh samples. The study was conducted during the 2014-2015 influenza season.
3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of those Experts
The document does not explicitly state the number of experts used to establish the ground truth for the test set or their specific qualifications (e.g., radiologist with 10 years of experience).
Instead, the ground truth for the clinical comparison study was established using a FDA-cleared molecular comparator assay. For discrepant results between the Xpert Flu+RSV Xpress Assay and the comparator assay, bidirectional sequencing was performed. While sequencing is a definitive molecular method, it's not described as an "expert" review in the traditional sense of a clinical expert.
4. Adjudication Method for the Test Set
- Initial Comparison: Xpert Flu+RSV Xpress Assay results were compared to an FDA-cleared molecular comparator assay.
- Discrepancy Resolution/Adjudication: For specimens where the Xpert Flu+RSV Xpress Assay and the comparator assay were discrepant, bidirectional sequencing was performed. The results from sequencing were provided for informational purposes only. This implies that while sequencing helped understand discrepancies, the primary comparison was with the FDA-cleared molecular assay.
5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was done
- No, a multi-reader multi-case (MRMC) comparative effectiveness study was not explicitly mentioned or described in the provided text.
- The study focuses on the diagnostic performance of the device itself (algorithm only) against a comparator assay, and its reproducibility under different operator conditions, not on human reader performance with or without AI assistance.
- The statement "The study results showed the Xpert Flu+RSV Xpress Assay is acceptable for its intended use with inexperienced lab users" refers to the device's performance when used by such users, not an improvement in human reader performance aided by AI.
6. If a Standalone (i.e. algorithm only without human-in-the loop performance) was done
- Yes, a standalone study (algorithm-only performance) was effectively done. The clinical comparison study evaluates the performance of the Xpert Flu+RSV Xpress Assay (the device/algorithm) against an FDA-cleared molecular comparator assay.
- While operators load samples and initiate the test, the entire process of sample extraction, purification, amplification, and detection, and interpretation of the fluorescent signals to yield a positive/negative result, is automated by the device ("GeneXpert Xpress System platform automates and integrates sample extraction, purification, amplification, and detection..."). The output is a "report that can be viewed and printed," indicating an algorithm-generated result.
7. The Type of Ground Truth Used
- Clinical Comparison Study: The primary ground truth was established by an FDA-cleared molecular comparator assay. For discordant results, bidirectional sequencing was used as an additional, more definitive form of molecular ground truth for informational purposes.
- Non-Clinical Studies (Analytical Sensitivity, Specificity, Reactivity): The ground truth was based on known concentrations of purified viral cultures (TCID50/mL or pg/µL) or bacterial/yeast cultures (CFU/mL), a laboratory-defined ground truth.
8. The Sample Size for the Training Set
The document does not explicitly specify a sample size for a "training set." This type of in-vitro diagnostic device (PCR assay) typically relies on extensive analytical validation (LoD, specificity, inclusivity, interference) and clinical validation with prospectively and retrospectively collected samples, rather than a distinct "training set" in the machine learning sense. The device's underlying algorithm is based on established molecular biology principles (RT-PCR) calibrated during development, not specifically "trained" on a dataset in the way an AI model would be.
9. How the Ground Truth for the Training Set Was Established
As noted above, the concept of a clear "training set" with ground truth in the context of machine learning is not directly applicable here. For the development and establishment of the assay's performance characteristics, ground truth would have been established through:
- Analytical studies: Using known concentrations and strains of target and non-target pathogens to define sensitivity (LoD), specificity, and inclusivity.
- Internal validation/optimization: During the assay development phase, characterized clinical samples or contrived samples with confirmed presence/absence of targets via established reference methods (e.g., PCR, sequencing, culture) would have been used to optimize assay parameters (e.g., primer/probe design, cycle thresholds).
§ 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.