(119 days)
The Cepheid® Xpert Flu Assay is an automated, multiplex real-time RT-PCR assay intended for the in vitro qualitative detection and differentiation of influenza B and 2009 H1N1 influenza viral RNA. The Xpert Flu Assay uses nasal aspirates/washes and nasopharyngeal swab specimens collected from patients with signs and symptoms of respiratory infection in conjunction with clinical and epidemiological risk factors. The Xpert Flu Assay is intended as an aid in the diagnosis of influenza.
Negative results do not preclude influenza 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 2009-2010 influenza season when 2009 H1N1 influenza was the predominant influenza A virus 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 Xpert Flu Assay is a rapid, automated in vitro diagnostic test for qualitative detection and differentiation of influenza A, influenza B and influenza A, subtype 2009 HINI. The assay is performed on the Cepheid GeneXpert Instrument Systems. The GeneXpert Instrument Systems automate and integrate sample purification, nucleic acid amplification, and detection of the target sequence in simple or complex samples using real-time PCR and RT-PCR assays. The systems require the use of single-use disposable 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 eliminated.
The Xpert Flu Assay includes reagents for the detection and differentiation of influenza A, influenza B and influenza A, subtype 2009 H1N1 directly from nasal aspirates/washes (NA/W) and nasopharyngeal (NP) swab specimens from patients with signs and symptoms of respiratory infection. A Sample Processing Control (SPC) and a Probe Check Control (PCC) are also included. 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 liquid specimen (NA/W) or swab specimen (NP) is collected according to the institution's standard procedures and placed into Universal Transport Medium (3mL UTM tubes). Following a brief mixing by inverting the UTM tube five times, the eluted material and one single-use reagent (Reagent 1), that is provided with the assay, are transferred to different, uniquely-labeled chambers of the disposable fluidic cartridge (the Xpert Flu cartridge). The user initiates a test from the system user interface and places the cartridge into the GeneXpert instrument platform, which performs hands-off reverse transcription and real-time, multiplex polymerase chain reaction (PCR) for detection of DNA. In this platform, additional sample preparation, amplification, and real-time detection are all fully-automated and completely integrated.
The single-use, multi-chambered fluidic cartridges are designed to complete sample preparation and real-time RT-PCR for detection and differentiation of influenza A, influenza B and influenza A, subtype 2009 H1N1 in 75 minutes. The GeneXpert Instrument Systems have 1 to 48 randomly accessible modules that are each capable of performing separate sample preparation and real-time 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 real-time PCR and detection.
Acceptance Criteria and Device Performance Study for Xpert Flu Assay
This report details the acceptance criteria and the study proving the Cepheid Xpert Flu Assay meets these criteria, based on the provided 510(k) summary (K103766).
1. Table of Acceptance Criteria and Reported Device Performance
The 510(k) summary does not explicitly state pre-defined quantitative acceptance criteria for sensitivity and specificity. Instead, the clinical study results are presented as the "performance characteristics" and are compared against a reference method. It's implied that achieving high agreement with the reference method across various influenza types and specimen types constitutes acceptable performance for substantial equivalence.
Based on the clinical performance study, the device performance is reported as follows:
Clinical Performance on Prospective Nasal Aspirates/Washes (NA/W)
| Target | Performance Measure | Acceptance/Reference Method Performance |
|---|---|---|
| Influenza A | Sensitivity | 85.7% (95% CI: 42.1-99.6) |
| Specificity | 99.1% (95% CI: 97.4-99.8) | |
| 2009 H1N1 | Sensitivity | 100% (95% CI: 39.8-100) |
| Specificity | 98.8% (95% CI: 97.0-99.7) | |
| Influenza B | Sensitivity | 100% (95% CI: 65.2-100) |
| Specificity | 99.4% (95% CI: 98.1-99.9) |
Clinical Performance on Prospective Nasopharyngeal (NP) Swabs
| Target | Performance Measure | Acceptance/Reference Method Performance |
|---|---|---|
| Influenza A | Sensitivity | 100% (95% CI: 59.0-100) |
| Specificity | 98.3% (95% CI: 96.0-99.4) | |
| 2009 H1N1 | Sensitivity | 100% (95% CI: 47.8-100) |
| Specificity | 99.0% (95% CI: 97.0-99.8) | |
| Influenza B | Sensitivity | 87.5% (95% CI: 47.3-99.7) |
| Specificity | 99.7% (95% CI: 98.1-100) |
Clinical Performance on Archived NA/W Specimens (vs. FDA Cleared Molecular Comparator)
| Target | Performance Measure | Accepatance/Reference Method Performance |
|---|---|---|
| Influenza A | Positive Agreement | 99.4% (95% CI: 96.6-100) |
| Negative Agreement | 100% (95% CI: 98.6-100) | |
| 2009 H1N1 | Positive Agreement | 98.4% (95% CI: 94.4-99.8) |
| Negative Agreement | 99.7% (95% CI: 98.1-100) | |
| Influenza B | Positive Agreement | 100% (95% CI: 91.2-100) |
| Negative Agreement | 100% (95% CI: 99.0-100) |
Clinical Performance on Archived NP Swabs (vs. Viral Culture + DFA)
| Target | Performance Measure | Acceptance/Reference Method Performance |
|---|---|---|
| Influenza A | Positive Agreement | 97.5% (95% CI: 92.7-99.5) |
| Negative Agreement | 100% (95% CI: 89.1-100) | |
| 2009 H1N1 | Positive Agreement | 100% (95% CI: 95.7-100) |
| Negative Agreement | 100% (95% CI: 94.5-100) | |
| Influenza B | Positive Agreement | 93.8% (95% CI: 79.2-99.2) |
| Negative Agreement | 99.2% (95% CI: 95.4-100) |
Clinical Performance on Archived NP Swabs (vs. FDA Cleared Molecular Comparator)
| Target | Performance Measure | Acceptance/Reference Method Performance |
|---|---|---|
| Influenza A | Positive Agreement | 98.1% (95% CI: 89.7-100) |
| Negative Agreement | 99.2% (95% CI: 95.5-100) | |
| 2009 H1N1 | Positive Agreement | 100% (95% CI: 88.1-100) |
| Negative Agreement | 99.3% (95% CI: 96.2-100) | |
| Influenza B | Positive Agreement | 93.8% (95% CI: 69.8-99.8) |
| Negative Agreement | 100% (95% CI: 97.7-100) |
2. Sample Size Used for the Test Set and Data Provenance
Prospective Specimens:
- NA/W specimens: 342
- NP swab specimens: 297
Archived Specimens:
- NA/W specimens: 425
- NP swab specimens: 150 (compared to viral culture + DFA), 177 (compared to FDA cleared molecular assay)
Data Provenance: The clinical study was conducted at six institutions in the U.S. and Australia. The study included both prospective and archived specimens.
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 or their specific qualifications (e.g., radiologist with X years of experience) used to establish the ground truth. However, the ground truth for "viral culture followed by direct fluorescent assay (DFA)" is a standard laboratory method, implying trained laboratory personnel perform these tests, which typically require specific certifications and experience. Sequencing results for influenza A positive specimens were also used as part of the ground truth.
4. Adjudication Method for the Test Set
The concept of an "adjudication method" (like 2+1, 3+1) is typically associated with studies where multiple human readers interpret results, and disagreement is resolved by an adjudicator. This is not directly applicable to a molecular diagnostic assay where results are objectively determined by instrumentation.
The document describes sequencing being performed for all influenza A positive specimens (identified by viral culture/DFA or the FDA cleared molecular assay) to differentiate subtypes. This acts as a confirmatory "adjudication" step for influenza A subtyping.
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 done. This study is for a diagnostic assay, not an AI-assisted human reading task.
6. If a Standalone (i.e. algorithm only without human-in-the loop performance) was done
Yes, a standalone performance study was done. The Xpert Flu Assay is an automated, multiplex real-time RT-PCR assay intended for in vitro qualitative detection and differentiation of influenza viral RNA. The performance characteristics described in the "Clinical Performance Study" sections (Pgs. 14-21) are for the device (Xpert Flu Assay) operating independently, generating its own results. There is no human interpretation of imaging or other complex data involved in generating the primary test result from the assay itself.
7. The Type of Ground Truth Used
The ground truth used for the clinical performance study varied based on the specimen type and whether it was prospective or archived:
- Prospective specimens: Viral culture followed by direct fluorescent assay (DFA) was the primary comparator. This is a recognized laboratory standard.
- Archived specimens (where viral culture was not performed prior to freezing): An FDA cleared molecular assay was performed as the comparator assay.
- For all influenza A positive specimens (from both prospective and archived sets): Sequencing was used to differentiate influenza A subtypes (e.g., 2009 H1N1 from other influenza A). This can be considered as a highly specific confirmatory method.
8. The Sample Size for the Training Set
The document describes analytical and clinical performance studies but does not detail a separate "training set" or its size for an algorithm development since this is a molecular diagnostic assay, not a machine learning model in the typical sense. The assay is based on predefined biological reactions and detection thresholds, not trainable parameters derived from a large dataset. The analytical studies (Analytical Sensitivity, LoD, Analytical Specificity) and reproducibility studies define the assay's fundamental performance characteristics.
9. How the Ground Truth for the Training Set Was Established
As noted above, there isn't a traditional "training set" as understood in machine learning. The assay's design and operating parameters would have been established through extensive laboratory work and optimization, including:
- Analytical Reactivity (Inclusivity): Testing against known influenza strains at specific concentrations (Table 5.2).
- Limit of Detection (LoD): Empirically determined as the lowest concentration (TCID50/mL) where 19/20 or 20/20 replicates were positive (Tables 5.3-5.6).
- Analytical Specificity (Exclusivity): Testing against potentially interfering viral, bacterial, and yeast strains at specified concentrations (Table 5.7).
These studies use well-defined, characterized strains and concentrations as their "ground truth" to ensure the assay performs as expected.
§ 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.