(164 days)
The Cepheid Xpert® Xpress Flu Assay, performed on the GeneXpert® Instrument Systems, is an automated, multiplex real-time, reverse transcriptase polymerase chain reaction (RT-PCR) assay intended for the in vitro qualitative detection and differentiation of influenza A and influenza B viral RNA. The Xpert Xpress Flu Assay uses nasopharyngeal (NP) swab specimens collected from patients with signs and symptoms of respiratory infection. The Xpert Xpress Flu Assay is intended as an aid in the diagnosis of influenza infections in conjunction with clinical and epidemiological risk factors.
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 2015-2016 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 departments 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 Xpress Flu Assay is a rapid, automated in vitro diagnostic test for qualitative detection and differentiation of influenza A (Flu A) and influenza B (Flu B) viral RNA directly from nasopharyngeal (NP) swab specimens. The assay is performed on the Cepheid GeneXpert® Instrument Systems.
The Xpert Xpress Flu Assay includes reagents for the simultaneous detection and differentiation of the target viruses. The primers and probes in the Xpert Xpress Flu Assay detect the presence of nucleic acid sequences for Flu A and Flu B directly from 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 internal controls utilized by the GeneXpert Instrument System platform. The SPC is present in every assay to control for adequate processing of the target viruses and to monitor for the presence of inhibitor(s) in the PCR assay to avoid false-negative results. The PCC verifies reagent rehydration, real-time PCR tube filling in the cartridge, probe integrity, and dye stability.
The specimens are collected in viral transport medium and transported to the GeneXpert area. The specimen is prepared according to package insert instructions and transferred to the sample chamber (large opening) of the Xpert Xpress Flu Assay cartridge. The GeneXpert cartridge is loaded onto the GeneXpert Instrument System platform, which performs hands-off automated sample processing and real-time RT-PCR for detection of Flu viral RNA. Summary and detailed test results are obtained in approximately 30 minutes or less. The results are automatically generated at the end of the process in a report that can be viewed and printed.
The provided document describes the Cepheid Xpert Xpress Flu Assay, an automated, multiplex real-time, reverse transcriptase polymerase chain reaction (RT-PCR) assay for the qualitative detection and differentiation of influenza A and influenza B viral RNA. The study referenced aims to demonstrate the substantial equivalence of this device to a legally marketed predicate device, the Xpert Flu/RSV XC Assay (K142045).
Here's a breakdown of the requested information based on the provided text:
1. Table of Acceptance Criteria and Reported Device Performance
The document does not explicitly state pre-defined acceptance criteria in terms of specific thresholds for PPA (Positive Percent Agreement) and NPA (Negative Percent Agreement) that the device must meet. However, the reported performance is compared to an FDA-cleared molecular comparator assay to demonstrate substantial equivalence. The reported performance can be interpreted as implicitly meeting the expectation for substantial equivalence.
| Metric (Target) | Acceptance Criteria (Implicit) | Reported Device Performance (Combined Dataset) |
|---|---|---|
| PPA (Influenza A) | Performance comparable to FDA-cleared molecular comparator assay | 98.1% (CI: 93.4-99.5) |
| NPA (Influenza A) | Performance comparable to FDA-cleared molecular comparator assay | 98.4% (CI: 97.7-98.8) |
| PPA (Influenza B) | Performance comparable to FDA-cleared molecular comparator assay | 100.0% (CI: 95.3-100.0) |
| NPA (Influenza B) | Performance comparable to FDA-cleared molecular comparator assay | 98.7% (CI: 98.1-99.1) |
| Analytical Specificity | 100% (No cross-reactivity with common respiratory pathogens) | 100% |
| Analytical Reactivity (Inclusivity) | All tested influenza strains to be detected | Positive in all three replicates for all but one strain (which was 2/3 at 0.1 TCID50/mL) |
| Carry-Over Contamination | No contamination of negative samples | 100% correct reporting for all negative samples (Flu A NEGATIVE; Flu B NEGATIVE) |
| Fresh vs. Frozen Sample Equivalency | No difference in performance between fresh and freeze-thaw samples | No difference; all positive and negative replicates correctly identified |
| Competitive Interference | Identification of potential inhibitory effects | Competitive inhibitory effects observed under specific conditions |
2. Sample Size Used for the Test Set and Data Provenance
-
Sample Size (Clinical Comparison Study - NP Swab Specimens):
- Total: 2065 NP swab specimens
- Fresh, prospectively collected: 1142 specimens
- Consistently collected, frozen: 923 specimens
- Pre-selected frozen NP swab specimens (analyzed separately): 102 specimens
-
Data Provenance: The study was conducted at eleven institutions in the U.S. during the 2015-2016 influenza season. The data includes both prospectively collected fresh specimens and retrospectively (consecutively) collected frozen specimens.
3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Those Experts
The document does not specify the number or qualifications of experts used to establish the ground truth for the clinical test set. The ground truth for the clinical comparison study was established by an "FDA-cleared molecular comparator assay."
4. Adjudication Method for the Test Set
- For discordant results between the Xpert Xpress Flu Assay and the comparator assay, bi-directional sequencing was performed. The text states this was "for informational purposes only," implying it was used to investigate discrepancies rather than for primary adjudication that would re-classify initial ground truth results.
5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study
- No, a multi-reader multi-case (MRMC) comparative effectiveness study was not conducted as described in the document. This study focuses on the performance of the assay itself compared to a reference method, not on human reader performance with or without AI assistance.
6. Standalone (Algorithm Only Without Human-in-the-Loop Performance) Study
- Yes, the clinical comparison study directly evaluates the standalone performance of the Xpert Xpress Flu Assay (the algorithm/device) against an FDA-cleared molecular comparator assay. The results provided in Table 8-9 (PPA, NPA) are for the device alone.
7. Type of Ground Truth Used
- Clinical Comparison Study: The ground truth for the clinical comparison study was established using an FDA-cleared molecular comparator assay. For discordant results, bi-directional sequencing was used for informational purposes.
- Analytical Studies (LoD, Specificity, Reactivity, Interference): Ground truth was established by known concentrations of purified viruses or bacterial/yeast strains in simulated matrices.
8. Sample Size for the Training Set
- The document describes a submission for a medical device (assay) and its performance evaluation. It does not refer to an "AI algorithm" in the common sense of machine learning that would have a separate "training set" of data. The assay's design and optimization would have involved internal development and validation, but this is not presented as a distinct "training set" in the context of typical AI device submissions. The analytical and clinical studies describe the test set used to validate the final device.
9. How the Ground Truth for the Training Set Was Established
- As mentioned above, the concept of a "training set" in the context of an AI algorithm is not explicitly applicable here. The device is a molecular diagnostic assay. Its "training" (development and optimization) would involve standard laboratory methods, reagent formulation, and protocol refinement to achieve optimal detection and differentiation of target nucleic acid sequences. The ground truth for this development phase would be established through controlled experiments using known positive and negative controls, spiked samples, and characterized clinical samples, as outlined in the non-clinical studies (e.g., LoD, inclusivity, specificity studies).
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Image /page/0/Picture/1 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo consists of a circular seal with the text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" around the perimeter. Inside the circle is a stylized symbol that resembles three human profiles facing to the right, stacked on top of each other.
February 13, 2017
Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002
Cepheid Jim Kellv. PhD Executive Director, Regulatory Affairs 904 Caribbean Drive Sunnyvale, CA 94089
Re: K162456
Trade/Device Name: Xpert® Xpress Flu Assay Regulation Number: 21 CFR 866.3980 Regulation Name: Respiratory viral panel multiplex nucleic acid assay Regulatory Class: II Product Code: OCC. OOI. JSM Dated: January 10, 2017 Received: January 11, 2017
Dear Dr. Kelly:
We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food. Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.
If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.
Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.
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If you desire specific advice for your device on our labeling regulations (21 CFR Parts 801 and 809), please contact the Division of Industry and Consumer Education at its toll-free number (800) 638 2041 or (301) 796-7100 or at its Internet address
http://www.fda.gov/MedicalDevices/Resourcesfor You/Industry/default.htm. Also, please note the regulation entitled. "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to
http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.
You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address
http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.
Sincerely yours,
Steven R. Gitterman -S
Uwe Scherf, M.Sc., Ph.D. Director Division of Microbiology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health
Enclosure
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Indications for Use
510(k) Number (if known)
Device Name
Xpert Xpress Flu
Indications for Use (Describe)
The Cepheid Xpert Xpress Flu Assay, performed on the GeneXpert® Instrument Systems, is an automated, multiplex real-time, reverse transcriptase polymerase chain reaction (RT-PCR) assay intended for the in virro qualitative detection and differentiation of influenza B viral RNA. The Xpert Xpress Flu Assay uses nasopharyngeal (NP) swab specimens collected from patients with signs and symptoms of respiratory infection. The Xpert Xpress Flu Assay is intended as an aid in the diagnosis of influenza infections in clinical and epidemiological risk factors.
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 2015-2016 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 departments for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.
Ancillary Collection Kit Indications for Use:
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, Xpert Xpress Flu/RSV Assay or the Xpert Xpress Flu Assay.
2 Prescription Use (Part 21 CFR 801 Subpart D)
Over-The-Counter Use (21 CFR 801 Subpart C)
PLEASE DO NOT WRITE BELOW THIS LINE - CONTINUE ON A SEPARATE PAGE IF NEEDED.
FOR FDA USE ONLY
Concurrence of Center for Devices and Radiological Health (CDRH) (Signature)
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8.0 510(k) Summary
As required by 21 CFR Section 807.92(c).
| Submitted by: | Cepheid904 Caribbean DriveSunnyvale, CA 90489Phone number: (847) 228-3299Fax number: (847) 890-6589 |
|---|---|
| Contact: | Scott A. Campbell, PhD, MBA |
| Date of Preparation: | September 01, 2016 |
| Device: | |
| Trade name: | Xpert® Xpress Flu |
| Common name: | Xpert Xpress Flu Assay |
| Type of Test: | Automated, multiplex real-time reverse transcription-polymerase chain reaction (RT-PCR) assay intended for the invitro qualitative detection and differentiation of influenza Aand influenza B viral RNA. |
| Regulation number/Classification name/ | 866.3980/Respiratory viral panel multiplex nucleic acidassay/OCC |
| Product code: | 866.2570/Instrumentation for clinical multiplex testsystems/OOI |
| 866.2390/Culture Media, Non-PropagatingTransport | |
| Classification | Class II |
| Advisory Panel | Microbiology (83) |
| Prescription Use | Yes |
| Predicate DevicesAssay: | 1) For the detection and differentiation of influenza A,influenza B, and RSV A/B viral RNA in nasopharyngealswab specimens:Xpert® Flu/RSV XC Assay [510(k) #K142045] |
| 2) For the Sample Collection Kits:Cepheid Xpert® Nasopharyngeal Sample Collection Kit[510(k) # K151226] |
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Device Description:
The Xpert Xpress Flu Assay is a rapid, automated in vitro diagnostic test for qualitative detection and differentiation of influenza A (Flu A) and influenza B (Flu B) viral RNA directly from nasopharyngeal (NP) swab specimens. The assay is performed on the Cepheid GeneXpert® Instrument Systems.
The Xpert Xpress Flu Assay includes reagents for the simultaneous detection and differentiation of the target viruses. The primers and probes in the Xpert Xpress Flu Assay detect the presence of nucleic acid sequences for Flu A and Flu B directly from 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 internal controls utilized by the GeneXpert Instrument System platform. The SPC is present in every assay to control for adequate processing of the target viruses and to monitor for the presence of inhibitor(s) in the PCR assay to avoid false-negative results. The PCC verifies reagent rehydration, real-time PCR tube filling in the cartridge, probe integrity, and dye stability.
The specimens are collected in viral transport medium and transported to the GeneXpert area. The specimen is prepared according to package insert instructions and transferred to the sample chamber (large opening) of the Xpert Xpress Flu Assay cartridge. The GeneXpert cartridge is loaded onto the GeneXpert Instrument System platform, which performs hands-off automated sample processing and real-time RT-PCR for detection of Flu viral RNA. Summary and detailed test results are obtained in approximately 30 minutes or less. The results are automatically generated at the end of the process in a report that can be viewed and printed.
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Device Intended Use:
The Cepheid Xpert® Xpress Flu Assay, performed on the GeneXpert® Instrument Systems, is an automated, multiplex real-time, reverse transcriptase polymerase chain reaction (RT-PCR) assay intended for the in vitro qualitative detection and differentiation of influenza A and influenza B viral RNA. The Xpert Xpress Flu Assay uses nasopharyngeal (NP) swab specimens collected from patients with signs and symptoms of respiratory infection. The Xpert Xpress Flu Assay is intended as an aid in the diagnosis of influenza infections in conjunction with clinical and epidemiological risk factors.
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 2015-2016 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 departments for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.
Ancillary Specimen Collection Kit
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
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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, Xpert Xpress Flu/RSV Assay or the Xpert Xpress Flu Assay.
Substantial Equivalence:
The Xpert Xpress Flu Assay is substantially equivalent to the current Xpert® Flu/RSV XC Assay [510(k) #K142045]. The Xpert Xpress Flu Assay detects influenza A and influenza B from nasopharyngeal (NP) swab specimens and the Xpert® Flu/RSV XC Assay detects influenza A, influenza B, and RSV from both NP swab specimens and nasal aspirate/wash (NA/W) specimens. Both assays utilize the same technology by determining the presence of the target organisms through real-time RT-PCR amplification and fluorogenic target-specific hybridization detection. A multi-center clinical study was conducted and obtained data using the Xpert Xpress Flu/RSV Assay which was then reanalyzed with the Xpert Xpress Flu Assay Definition File (ADF). The reanalyzed data was used to determine the performance characteristics of the Xpert Xpress Flu Assay relative to the predicate device, which has been FDA cleared for NP swab and NA/W specimens. Discordant results between the Xpert Xpress Flu Assay and the reference method Prodesse ProFlu+ Assay [510(k) #K132129] were analyzed by bidirectional sequencing using primers different from those used in the Xpert Xpress Flu Assay. The study results showed that the Xpert Xpress Flu Assay is substantially equivalent to the predicate device.
Table 8-1 shows the similarities and differences between the Xpert Xpress Flu Assay and the predicate device.
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| Similarities | ||
|---|---|---|
| Device | Predicate | |
| Item | Cepheid Xpert® Xpress Flu | Cepheid Xpert® Flu/RSV XC510(k)# K142045 |
| Regulation | 866.3980 | Same |
| Product Code | OCC, OOI | Same |
| Device Class | II | Same |
| TechnologyPrinciple ofOperation | Multiplex real time RT-PCR | Same |
| Intended Use | The Cepheid Xpert® XpressFlu Assay, performed on theGeneXpert® InstrumentSystems, is an automated,multiplex real-time, reversetranscriptase polymerasechain reaction (RT-PCR)assay intended for the in vitroqualitative detection anddifferentiation of influenza Aand influenza B viral RNA.The Xpert Xpress Flu Assayuses nasopharyngeal (NP)swab specimens collectedfrom patients with signs andsymptoms of respiratoryinfection. The Xpert XpressFlu Assay is intended as anaid in the diagnosis ofinfluenza infections inconjunction with clinical andepidemiological risk factors.Negative results do notpreclude influenza virusinfection and should not be | The Cepheid Xpert® Flu/RSVXC Assay is an automated,multiplex real-time, reversetranscriptase polymerase chainreaction (RT-PCR) assayintended for the in vitroqualitative detection anddifferentiation ofinfluenza A, influenza B, andrespiratory syncytial virus(RSV) viral RNA. The XpertFlu/RSV XC Assay usesnasopharyngeal swab andnasal aspirate/wash specimenscollected from patients withsigns and symptoms ofrespiratory infection. TheXpert Flu/RSV XC Assay isintended as an aid in thediagnosis of influenza andrespiratory syncytial virusinfections in conjunction withclinical and epidemiologicalrisk factors.Negative results do not |
| Similarities | ||
| Device | Predicate | |
| Item | Cepheid Xpert® Xpress Flu | Cepheid Xpert® Flu/RSV XC |
| 510(k)# K142045 | ||
| used as the sole basis fortreatment or other patientmanagement decisions.Performance characteristicsfor influenza A wereestablished during the 2015-2016 influenza season. Whenother novel influenza Aviruses are emerging,performance characteristicsmay vary.If infection with a novelinfluenza A virus is suspectedbased on current clinical andepidemiological screeningcriteria recommended bypublic health authorities,specimens should be collectedwith appropriate infectioncontrol precautions for novelvirulent influenza viruses andsent to state or local healthdepartments for testing. Viralculture should not beattempted in these casesunless a BSL 3+ facility isavailable to receive andculture specimens. | preclude influenza virus orrespiratory syncytial virusinfection and should not beused as the sole basis fortreatment or other patientmanagement decisions.Performance characteristics forinfluenza A were establishedduring the 2013-2014influenza season. When othernovel influenza A viruses areemerging, performancecharacteristics may vary.If infection with a novelinfluenza A virus is suspectedbased on current clinical andepidemiological screeningcriteria recommended bypublic health authorities,specimens should be collectedwith appropriate infectioncontrol precautions for novelvirulent influenza viruses andsent to state or local healthdepartments for testing. Viralculture should not beattempted in these cases unlessa BSL 3+ facility is availableto receive and culturespecimens. | |
| Similarities | ||
| Item | DeviceCepheid Xpert® Xpress Flu | PredicateCepheid Xpert® Flu/RSV XC510(k)# K142045 |
| Indications for Use | Patients with signs and symptoms of respiratory infection in conjunction with clinical and epidemiological risk factors | Same |
| Nucleic Acid Extraction | Yes | Same |
| Extraction Methods | Sample preparation integrated in GeneXpert Cartridge and GeneXpert Instrumentation System | Same |
| Assay Results | Qualitative | Same |
| Instrument System | Cepheid GeneXpert Instrument Systems; same Cepheid I-core technology | Same |
| Primers and probes | Primers and probes to detect the presence of nucleic acid sequences of influenza A, influenza B, and RSV. Only results for influenza A and influenza B are reported. | Primers and probes to detect the presence of influenza A, influenza A subtype H7N9, influenza B and RSV. Results for influenza A, influenza B and RSV analytes are reported. |
| Laboratory Users | Laboratory users in moderate and high complexity laboratory settings. | Same |
| Sample Preparation | Self-contained and automated after mixed specimen is added to cartridge. All other reagents are contained in the cartridge. | Same |
| Similarities | ||
| Item | DeviceCepheid Xpert® Xpress Flu | PredicateCepheid Xpert® Flu/RSV XC510(k)# K142045 |
| Primers andprobes forinfluenza A,influenza B | Primers and probes to detectthe presence of nucleic acidsequences of influenza A,influenza B, and RSV A/B.The Xpert Xpress Flu Assaycontains primers and probes todetect additional RNAsegments in order to protectthe assay sensitivity andspecificity from mutations inthe influenza genome due toantigenic drifts and shifts.Only results for influenza Aand influenza B are reported. | Primers and probes to detectthe presence of nucleic acidsequences of influenza A,influenza B, and RSV A/B.The Xpert Flu/RSV XC Assaycontains primers and probes todetect additional RNAsegments in order to protectthe assay sensitivity andspecificity from mutations inthe influenza genome due toantigenic drifts and shifts. |
| TargetSequences | Influenza A: Matrix protein(MP),basic polymerase (PB2)and acidic protein (PA)Influenza B: Matrix protein(MP) and Non-structuralproteins (NS 1 and NS 2)RSV A and RSV B:Nucleocapsid proteinOnly results for influenza Aand influenza B are reported. | Influenza A: Matrix protein(MP),basic polymerase (PB2)and acidic protein (PA)Influenza B: Matrix protein(MP) and Non-structuralproteins (NS 1 and NS 2)RSV A and RSV B:Nucleocapsid protein |
| InternalControls | Sample processing control(SPC) and probe checkcontrol (PCC). | Same |
| Early assayterminationfunction | Yes | Yes |
| Differences | ||
| Device | Predicate | |
| Item | Cepheid Xpert® Xpress Flu | Cepheid Xpert® Flu/RSV XC |
| Assay Targets | Influenza A Virus andInfluenza B Virus viral RNA | Influenza A Virus, Influenza BVirus, and RSV viral RNA |
| SpecimenTypes | Nasopharyngeal (NP) swabspecimens | Nasal aspirate/wash (NA/W)specimens andNasopharyngeal (NP) swabspecimens |
| Assay Controls | Encapsulated (armored) RNApseudovirus as a sampleprocessing control. | Encapsulated (armored) RNApseudovirus as a sampleprocessing control. |
| Available but not provided areinactivated virus controls forinfluenza A/B as externalpositive controls, andCoxsackie virus as an externalnegative control. | Available but not provided areinactivated virus controls forinfluenza A/B and RSV asexternal positive controls, andCoxsackie virus as an externalnegative control. | |
| Time to obtaintest results | Approximately 30 minutes orless for sample preparationand RT-PCR | Approximately 60 minutes orless for sample preparation andRT-PCR |
| CombinatorialAssaySelections | Not applicable | Yes, user may select combinedassay with all targets or a Fluonly assay or a RSV onlyassay. |
Table 8-1: Comparison of Similarities and Differences of the Xpert Xpress Flu Assay with the Predicate Device
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The Xpert Xpress Flu Assay and the predicate device have the same general intended use and technological characteristics, and both detect influenza A and influenza B viral RNA from NP swab specimens. The clinical study demonstrates that the Xpert Xpress Flu Assay is substantially equivalent to the predicate device.
The predicate device for the ancillary specimen collection kit, the Xpert® Nasopharyngeal Sample Collection Kit is the Cepheid Nasopharyngeal Sample Collection Kit, [510(k) # K151226]. The similarities and differences are shown in Table 8-2.
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| Similarities | ||
|---|---|---|
| Device | Predicate | |
| Item | Xpert® NasopharyngealSample Collection Kit | Xpert® NasopharyngealSample Collection Kit510(k)# K151226 |
| Intended Use | The Xpert® NasopharyngealSample Collection Kit isdesigned to collect, preserve,and transport nasopharyngealswab specimens and to preserveand transport nasalaspirate/wash specimenscontaining viruses from patientswith signs and symptoms ofrespiratory infection prior toanalysis with the Xpert FluAssay or the Xpert Flu/RSV XCAssay. The Xpert®Nasopharyngeal SampleCollection Kit is designed tocollect, preserve, and transportnasopharyngeal swab specimenscontaining viruses from patientswith signs and symptoms ofrespiratory infection prior toanalysis with the XpertFlu+RSV Xpress Assay, XpertXpress Flu/RSV Assay or theXpert Xpress Flu Assay. | The Xpert® NasopharyngealSample Collection Kit is designedto collect, preserve, and transportnasopharyngeal swab specimensand to preserve and transportnasal aspirate/wash specimenscontaining viruses from patientswith signs and symptoms ofrespiratory infection prior toanalysis with the Xpert Flu Assayor the Xpert Flu/RSV XC Assay.The Xpert® NasopharyngealSample Collection Kit is designedto collect, preserve, and transportnasopharyngeal swab specimenscontaining viruses from patientswith signs and symptoms ofrespiratory infection prior toanalysis with the Xpert Flu+RSVXpress Assay. |
| Similarities | ||
| Device | Predicate | |
| Item | Xpert® NasopharyngealSample Collection Kit | Xpert® NasopharyngealSample Collection Kit510(k)# K151226 |
| Transport MediumFormulation | Hank's Balanced Salt SolutionBovine Serum AlbuminL-cysteineGelatinSucroseL-glutamic acidHEPES bufferVancomycinAmphotericin BColistinPhenol red | Same |
| pH | 7.3 ± 0.2 | Same |
| Storage Temperature | 2 - 25°C (refrigeratedand room temperature) | Same |
| Volume | 3 ml | Same |
| Glass Beads | 3 x 3 mm | Same |
| Container | Plastic (medical-gradepolypropylene) | Same |
| Product Configuration | Medium Tube in Kit withindividually-wrapped sterileswab. | Same |
Table 8-2: Comparison of Similarities and Differences of the Xpert Nasopharyngeal Sample Collection Kit with the Predicate Device
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| Differences | ||
|---|---|---|
| Device | Predicate | |
| Item | Xpert® NasopharyngealSample Collection Kit | Xpert® NasopharyngealSampleCollection Kit510(k)# K151226 |
| Intended Use | For collection, preservationand transport ofnasopharyngeal swabspecimens and to preserveand transport nasalaspirate/wash specimenscontaining viruses frompatients with signs andsymptoms of respiratoryinfection prior to analysiswith the Xpert Flu Assay,Xpert Flu/RSV XC Assay,Xpert Flu+RSV XpressAssay, Xpert XpressFlu/RSV Assay and XpertXpress Flu Assay. | For collection, preservationand transport ofnasopharyngeal swabspecimens and to preserveand transport nasalaspirate/wash specimenscontaining viruses frompatients with signs andsymptoms of respiratoryinfection prior to analysiswith the Xpert Flu Assay,Xpert Flu/RSV XC Assay andXpert Flu+RSV XpressAssay. |
The proposed collection kit and predicate collection kit have the same general intended use and the same technology to collect, store and transport clinical specimens, including viruses, to the laboratory for further testing. The prospective component of the multicenter clinical study of the Xpert Xpress Flu Assay was conducted using Xpert Nasopharyngeal Sample Collection Kit [510(k) # K151226] demonstrating that the Xpert Nasopharyngeal Sample Collection Kit is substantially equivalent to the predicate device.
Non-Clinical Studies:
Analytical Sensitivity (Limit of Detection)
Studies were performed to determine the analytical limit of detection (LoD) of the Xpert Xpress Flu Assay with two lots of reagents across three testing days. The higher LoD
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observed per strain and per lot as determined by probit analysis was selected for verification. Verification of the estimated LoD claim was performed on one reagent lot across a minimum of three testing days. LoD was established using two influenza A H3N2 strains, two influenza A 2009 H1N1 strains and two influenza B strains. Viruses were diluted into negative pooled NP swab clinical matrix for testing. The LoD is defined as the lowest concentration (tissue culture infective dose, TCID50/mL) per sample that can be reproducibly distinguished from negative samples with 95% confidence or the lowest concentration at which 19 of 20 replicates were positive. Each strain was tested in replicates of 20 per concentration of virus. The LoD values for each strain tested are summarized in Tables 8-3 – 8-5.
| Table 8-3 Confirmed LoD (TCID50/mL):Influenza A 2009 H1N1 | |
|---|---|
| T 7 ------- | Confirmed |
| VirusStrain | ConfirmedLoD(TCID50/mL) |
|---|---|
| Influenza A/California/7/2009 | 0.02 |
| Influenza A/Florida/27/2011 | 0.04 |
Table 8-4 Confirmed LoD (TCID50/mL): Influenza A H3N2
| VirusStrain | Confirmed LoD(TCID50/mL) |
|---|---|
| Influenza A/Perth/16/2009 | 0.01 |
| Influenza A/Victoria/361/2011 | 0.75 |
Table 8-5 Confirmed LoD (TCID50/mL): Influenza B
| VirusStrain | ConfirmedLoD(TCID50/mL) |
|---|---|
| Influenza B/Mass/2/2012 | 0.40 |
| Influenza B/Wisconsin/01/2011 | 0.19 |
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Analytical Specificity (Exclusivity)
The analytical specificity of the Xpert Xpress Flu Assay was evaluated by testing a panel of 44 cultures consisting of 16 viral, 26 bacterial, and two yeast strains representing common respiratory pathogens or those potentially encountered in the nasopharynx. Three replicates of each bacterial and yeast strain were tested at concentrations of ≥ 1 x 106 CFU/mL with the exception of one strain that was tested at 1 x 105 CFU/mL (Chlamydia pneumoniae). Three replicates of each virus were tested at concentrations of ≥ 1 x 105 TCID50/mL. The analytical specificity was 100%. Results are shown in Table 8-6.
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| Result | |||
|---|---|---|---|
| Organism | Concentration(per cartridge) | InfluenzaA | InfluenzaB |
| No Template Control | N/A | NEG | NEG |
| Adenovirus Type 1 | 1.12E+06 TCID50/mL | NEG | NEG |
| Adenovirus Type 7 | 1.87E+05 TCID50/mL | NEG | NEG |
| Human coronavirus OC43 | 2.85E+05 TCID50/mL | NEG | NEG |
| Human coronavirus 229E | 1.00E+05 TCID50/mL | NEG | NEG |
| Cytomegalovirus | 1.00E+05 TCID50/mL | NEG | NEG |
| Echovirus | 3.31E+07 TCID50/mL | NEG | NEG |
| Enterovirus | 3.55E+05 TCID50/mL | NEG | NEG |
| Epstein Barr Virus | 7.16E+07 TCID50/mL | NEG | NEG |
| HSV | 8.90E+05 TCID50/mL | NEG | NEG |
| Measles | 6.31E+05 TCID50/mL | NEG | NEG |
| Human metapneumovirus | 1.00E+05 TCID50/mL | NEG | NEG |
| Mumps virus | 6.31E+06 TCID50/mL | NEG | NEG |
| Human parainfluenza Type 1 | 1.15E+06 TCID50/mL | NEG | NEG |
| Human parainfluenza Type 2 | 6.31E+05 TCID50/mL | NEG | NEG |
| Human parainfluenza Type 3 | 3.55E+06 TCID50/mL | NEG | NEG |
| Rhinovirus Type 1A | 1.26E+05 TCID50/mL | NEG | NEG |
| Acinetobacter baumannii | 1.00E+06 CFU/mL | NEG | NEG |
| Burkholderia cepacia | 3.30E+06 CFU/mL | NEG | NEG |
| Candida albicans | 3.20E+06 CFU/mL | NEG | NEG |
| Candida parapsilosis | 3.00E+06 CFU/mL | NEG | NEG |
| Bordetella pertussis | 3.30E+06 CFU/mL | NEG | NEG |
| Chlamydia pneumoniae | 1.00E+05 CFU/mL | NEG | NEG |
| Citrobacter freundii | 3.30E+06 CFU/mL | NEG | NEG |
| Corynebacterium sp. | 3.30E+06 CFU/mL | NEG | NEG |
| Escherichia coli | 1.00E+07 CFU/mL | NEG | NEG |
| Enterococcus faecalis | 1.30E+06 CFU/mL | NEG | NEG |
| Haemophilus influenzae | 1.00E+06 CFU/mL | NEG | NEG |
| Lactobacillus reuteri | 1.00E+06 CFU/mL | NEG | NEG |
| Legionella spp. | 1.00E+06 CFU/mL | NEG | NEG |
| Moraxella catarrhalis | 1.00E+07 CFU/mL | NEG | NEG |
| Organism | Concentration(per cartridge) | Result | |
| InfluenzaA | InfluenzaB | ||
| Mycobacterium tuberculosis(avirulent) | 1.00E+06 CFU/mL | NEG | NEG |
| Mycoplasma pneumoniae | 1.00E+06 CFU/mL | NEG | NEG |
| Neisseria meningitidis | 2.15E+06 CFU/mL | NEG | NEG |
| Neisseria mucosa | 1.00E+07 CFU/mL | NEG | NEG |
| Propionibacterium acnes | 2.40E+07 CFU/mL | NEG | NEG |
| Pseudomonas aeruginosa | 3.70E+06 CFU/mL | NEG | NEG |
| Staphylococcus aureus (protein Aproducer) | 2.20E+06 CFU/mL | NEG | NEG |
| Staphylococcus epidermidis | 3.40E+06 CFU/mL | NEG | NEG |
| Staphylococcus haemolyticus | 4.00E+06 CFU/mL | NEG | NEG |
| Streptococcus agalactiae | 3.50E+06 CFU/mL | NEG | NEG |
| Streptococcus pneumoniae | 1.00E+06 CFU/mL | NEG | NEG |
| Streptococcus pyogenes | 1.00E+07 CFU/mL | NEG | NEG |
| Streptococcus salivarius | 1.00E+07 CFU/mL | NEG | NEG |
| Streptococcus sanguinis | 3.10E+06 CFU/mL | NEG | NEG |
Table 8-6 Analytical Specificity of the Xpert Xpress Flu Assay
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{21}------------------------------------------------
Analytical Reactivity (Inclusivity)
The analytical reactivity of the Xpert Xpress Flu Assay was evaluated against multiple strains of influenza A H1N1 (seasonal pre-2009), influenza A H1N1 (pandemic 2009), influenza A H3N2 (seasonal), avian influenza A (H5N1, H5N2, H6N2, H7N2, H7N3, H2N2, H7N9, and H9N2) and influenza B (representing strains from both Victoria and Yamagata lineages) at levels near the analytical LoD. A total of 48 strains comprised of 35 influenza A and 13 Influenza B strains were tested in this study with the Xpert Xpress Flu Assay. Three replicates were tested for each strain. All Flu strains tested positive in all three replicates, except for one Flu A H1N1 strain (A/New Jersey/8/76), which tested positive in 2 of 3 replicates at 0.1 TCID50/mL. Results are shown in Table 8-7.
Further in silico analysis was conducted to determine the predicted cross reactivity of additional influenza A 2009 H1N1-like strains. The results showed 100% sequence homology for all primer target nucleotide sequences analyzed.
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| Virus | Strain | TargetConcentration | Result | |
|---|---|---|---|---|
| No Template Control | n/a | NEG | NEG | |
| A/swine/Iowa/15/30 | 0.1 TCID50/mL | POS | NEG | |
| A/WS/33 | 0.1 TCID50/mL | POS | NEG | |
| A/PR/8/34 | 0.1 TCID50/mL | POS | NEG | |
| A/Mal/302/54 | 0.1 TCID50/mL | POS | NEG | |
| A/Denver/1/57 | 0.1 TCID50/mL | POS | NEG | |
| A/New Jersey/8/76 | 0.1 TCID50/mL | POS | NEG | |
| InfluenzaA H1N1(pre-2009) | A/New Caledonia/20/1999 | 0.1 TCID50/mL | POS | NEG |
| A/New York/55/2004 | 0.1 TCID50/mL | POS | NEG | |
| A/Soloman Island/3/2006 | 0.1 TCID50/mL | POS | NEG | |
| A/Taiwan/42/06 | 0.1 TCID50/mL | POS | NEG | |
| A/Brisbane/59/2007 | 0.1 TCID50/mL | POS | NEG | |
| A/swine/NY/02/2009 | 0.1 TCID50/mL | POS | NEG | |
| InfluenzaA H1N1(pdm2009) | A/Colorado/14/2012 | 0.1 TCID50/mL | POS | |
| A/Washington/24/2012 | 0.1 TCID50/mL | POS | ||
| A/Aichi/2/68 | 2.0 TCID50/mL | POS | ||
| A/HongKong/8/68 | 2.0 TCID50/mL | POS | NEG | |
| A/Port Chalmers/1/73 | 2.0 TCID50/mL | POS | NEG | |
| A/Hawaii/15/2001 | 2.0 TCID50/mL | POS | NEG | |
| InfluenzaA H3N2(Seasonal) | A/Wisconsin/67/05 | 2.0 TCID50/mL | POS | NEG |
| A/Brisbane/10/2007 | 2.0 TCID50/mL | POS | NEG | |
| A/Minnesota/11/2010 (H3N2)v | 2.0 TCID50/mL | POS | NEG | |
| A/Indiana/08/2011 (H3N2)v | 2.0 TCID50/mL | POS | NEG | |
| A/Texas/50/2012 | 2.0 TCID50/mL | POS | NEG |
Table 8-7 Analytical Reactivity (Inclusivity) of the Xpert Xpress Flu Assay
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Result Target Flu A Flu B Virus Strain Concentration ≤ 1pg/μLª A/duck/Hunan/795/2002 (H5N1) POS NEG A/chicken/Hubei/327/2004 POS NEG < 1pg/μLª (HSN1) A/Anhui/01/2005 (H5N1) POS NEG < 1pg/μLa A/Japanese white eye/ POS NEG ≤ 1pg/μLª HongKong/ 1038/2006 (H5N1) A/mallard/WI/34/75 (H5N2) POS NEG ≤ 1pg/μLª A/chicken/CA431/00 (H6N2) POS NEG ≤ 1pg/μLª Avian influenza A A/duck/LTC-10-82743/1943 POS NEG < 1pg/μLa (H7N2) A/chicken/NJ/15086-3/94 (H7N3) POS NEG ≤ 1pg/μLª N/Ab A/Anhui/1/2013 (H7N9) POS NEG N/Ab POS A/Shanghai/1/2013 (H7N9) NEG A/chicken/Korea/38349-p96323/ POS NEG < 10g/μLª 1996 (H9N2) A/Mallard/NY/6750/78 (H2N2) POS NEG ≤ 1pg/μLª 1.0 TCID50/mL B/Lee/40 POS NEG 1.0 TCID50/mL B/Allen/45 NEG POS 1.0 TCID50/mL B/GL/1739/54 POS NEG 1.0 TCID50/mL B/Maryland/1/59 NEG POS 1.0 TCID50/mL B/Panama/45/90C NEG POS B/Florida/07/2004d 1.0 TCID50/mL NEG POS 1.0 TCID50/mL B/Florida/02/06C NEG POS 1.0 TCID50/mL B/Florida/04/06d NEG POS 1.0 TCID50/mL B/Hong Kong/5/72 NEG POS 1.0 TCID50/mL B/Wisconsin/01/2010d POS NEG Influenza B 1.0 TCID50/mL B/Malaysia/2506/04C NEG POS 1.0 TCID50/mL B/Taiwan/2/62 NEG POS
1.0 TCID50/mL
B/Brisbane/60/2008C
CONFIDENTIAL
POS
NEG
{24}------------------------------------------------
- Purified viral RNA in simulated background matrix was used for avian influenza A viruses due to a. biosafety regulations.
- Inactivated avian influenza A (H7N9) viruses without viral titer was diluted 100,000 fold in b. simulated background matrix and tested due to biosafety regulations.
- ﻥ Known Victoria lineage.
- Known Yamagata lineage. d.
Potentially Interfering Substances
In a non-clinical study, potentially interfering substances that may be present in the nasopharynx were evaluated directly relative to the performance of the Xpert Xpress Flu Assay. Potentially interfering substances in the nasopharynx may include, but are not limited to: blood, nasal secretions or mucus, and nasal and throat medications used to relieve congestion, nasal dryness, irritation, or asthma and allergy symptoms, as well as antibiotics and antivirals. Negative samples (n = 8) were tested per each substance to determine the effect on the performance of the sample processing control (SPC).
Positive samples (n = 8) were tested per substance with six influenza (four influenza A and two influenza B) strains spiked at 3X the analytical LoD determined for each strain. All results were compared to positive and negative simulated nasal matrix controls. The simulated nasal matrix consisted of 2.5% (w/v) porcine mucin, 1% (v/v) human whole blood in 0.85% sodium chloride (NaCl) formulated in 1x PBS solution with 15% glycerol, which was then diluted 1:5 in UTM.
The evaluated substances are listed in Table 8-8 with active ingredients and concentrations tested shown. None of the substances caused interference of the assay at the concentrations tested in this study. All positive and negative replicates were identified correctly using the Xpert Xpress Flu Assay.
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| Substance/Class | Description/ActiveIngredient | ConcentrationTested |
|---|---|---|
| Control | Simulated nasalmatrix | 100% (v/v) |
| Beta-adrenergic bronchodilator | Albuterol Sulfate | 0.83 mg/mL(equivalent to 1dose per day) |
| Blood | Blood (Human) | 2% (v/v) |
| BDTM Universal ViralTransport System | Transport Media | 100% (v/v) |
| Remel M4® | Transport Media | 100% (v/v) |
| Remel M4RT® | Transport Media | 100% (v/v) |
| Remel M5® | Transport Media | 100% (v/v) |
| Remel M6® | Transport Media | 100% (v/v) |
| Throat lozenges, oralanesthetic and analgesic | Benzocaine, Menthol | 1.7 mg/mL |
| Mucin | Purified Mucin protein(Bovine or porcinesubmaxillary gland) | 2.5% (w/v) |
| Antibiotic, nasal ointment | Mupirocin | 10 mg/mL |
| Saline Nasal Spray | Sodium Chloride (0.65%) | 15% (v/v) |
| Anefrin Nasal Spray | Oxymetazoline, 0.05% | 15% (v/v) |
| PHNY Nasal Drops | Phenylephrine, 0.5% | 15% (v/v) |
| Tamiflu Anti-viral drugs | Zanamivir | 7.5 mg/mL |
| Antibacterial, systemic | Tobramycin | 4 µg/mL |
| Zicam Nasal Gel | Luffa opperculata,Galphimia glauca,Histaminumhydrochloricum Sulfur | 15% (w/v) |
| Nasal corticosteroid | Fluticasone Propionate | 5 µg/mL |
Table 8-8 Potentially Interfering Substances in the Xpert Xpress Flu Assay
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Carry-Over Contamination
A study was conducted to demonstrate that single-use, self-contained GeneXpert cartridges prevent carry-over contamination of negative samples when followed by very high positive samples in the same GeneXpert module. The study consisted of a negative sample processed in the same GeneXpert module immediately followed by a very high influenza A sample (A/Victoria/361/2011, 2x107 TCID50/mL) spiked into a simulated nasal matrix. This testing scheme was repeated 20 times for a total of 41 runs resulting in 20 positive and 21 negative specimens for each virus type. All 20 positive samples were correctly reported as Flu A POSITIVE; Flu B NEGATIVE. All 21 negative samples were correctly reported as Flu A NEGATIVE; Flu B NEGATIVE.
Fresh vs. Frozen Sample Equivalency Study
Fresh and frozen specimen equivalency in the Xpert Xpress Flu Assay was evaluated by testing individual influenza strains at three different concentrations representing low positives (2X LoD), moderate positives (5X LoD), and high positives (10X LoD) in pooled negative NP swab clinical matrix. Negative samples consisted of pooled negative NP swab clinical matrix only. Fresh and frozen specimen equivalency was determined using one seasonal Flu A H3N2 strain (A/Victoria/361/2011) and one Flu B strain (B/Mass/2/2012). Replicates of 20 were tested for each specimen type and concentration. All positive and negative specimens were tested fresh, after one freezethaw cycle, and after two freeze-thaw cycles. There was no difference in the performance of the Xpert Xpress Flu Assay between fresh virus dilutions and two sequential freeze thaw cycles for positive and negative samples. All positive and negative replicates were correctly identified using the Xpert Xpress Flu Assay.
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Competitive Interference Study
Competitive interference of the assay caused by the presence of two targets in the Xpert Xpress Flu Assay was evaluated by testing individual influenza strains near the LoD in the presence of different influenza strains at a higher concentration in a simulated nasal matrix. Analytical competitive interference was assessed using one (1) seasonal Flu A H3 strain (H3/Victoria/361/2011) at 0.8 TCID50/mL and one (1) Flu B strain (B/Mass/2/2012) at 0.45 TCID50/mL; the strains were tested at in the presence of competing strains at either 1 x 102 TCID50mL or 1 x 103 TCID50mL. Replicates of 20 were tested for each target strain and each competitive strain combination. The normal binomial distribution with 20 replicate samples at LoD is between 17 and 20 positive results based on the binomial distribution with N=20, p=0.95 (X~Bin(20,0.95)). Therefore, sets of 20 with 16 or less positives would be rare and an indication of a competitive inhibitory effect due to high levels of a competing analyte.
With Flu A/Victoria/361/2011 at a concentration of 0.8 TCID50/mL no competitive inhibitory effects were observed in the presence of 1x103 TCID50/mL of Flu B/Mass/2/2012.
With Flu B/Mass/2/2012 at a concentration of 0.45 TCID50/mL competitive inhibitory effects were observed in the presence of 1x103 TCIDs0mL of Flu A/Victoria/361/2011. No competitive inhibitory effects were observed in the presence of 1x102 TCID50/mL of Flu A/Victoria/361/2011.
Under the conditions of this study, internal competitive inhibitory effects were observed on the targets (Flu A and Flu B) in the presence of two targets for the Xpert Xpress Flu Assay. The competitive inhibitory effect on the Xpert Xpress Flu targets is addressed in the Limitations section of the package insert.
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Clinical Studies
Clinical Comparison Study
Performance characteristics of the Xpert Xpress Flu Assay were evaluated at eleven institutions in the U.S. during the 2015-2016 influenza season. Due to the low prevalence of influenza viruses and the difficulty in obtaining fresh influenza specimens, the specimen population for this study was supplemented with consecutively collected, frozen specimens.
Specimens were collected from the following:
- Individuals exhibiting signs and symptoms of respiratory infection who provided . informed consent for the collection of a NP swab specimen.
- Individuals with signs and symptoms of respiratory infection and whose routine . care called for collection of NP swab specimens for influenza testing. For eligible subjects, aliquots of leftover specimens were obtained for testing with the Xpert Xpress Flu Assay and reference testing, and patient management continued at the site per their standard practice.
The Xpert Xpress Flu Assay performance was compared to FDA-cleared molecular comparator assay. Bi- directional sequencing was performed on specimens where the Xpert Xpress Flu Assay and the comparator assay were discrepant, and is provided for informational purposes only.
Overall Results – NP Swab Specimens
A total of 2065 NP swab specimens were tested for influenza A and influenza B by the Xpert Xpress Flu Assay and the comparator assay. Of the 2065 NP swab specimens, 1142 were fresh, prospectively collected and 923 were consecutively collected, frozen specimens.
For the fresh, prospectively collected NP swab specimens, the Xpert Xpress Flu Assay demonstrated a PPA and NPA of 94.6% and 99.3% for the detection of influenza A and 100% and 99.2% for influenza B, respectively (Table 8-9).
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For the consecutively collected, frozen NP swab specimens, the Xpert Xpress Flu Assay demonstrated a PPA and NPA of 100% and 97.2% for the detection of influenza A, respectively; 100% and 98.2% for influenza B, respectively (Table 8-9).
For the combined dataset, the Xpert Xpress Flu Assay demonstrated a PPA and NPA of 98.1% and 98.4% for the detection of influenza A and 100% and 98.7% for influenza B, respectively (Table 8-9).
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| Specimen Type | Target | n | TP | FP | TN | FN | PPA(95% CI) | NPA(95% CI) |
|---|---|---|---|---|---|---|---|---|
| Fresh, prospectivelycollected | Flu A | 1142 | 35 | 8a | 1097 | 2b | 94.6%(CI:82.3-98.5) | 99.3%(CI:98.6-99.6) |
| Flu B | 1142 | 42 | 9c | 1091 | 0 | 100.0%(CI:91.6-100.0) | 99.2%(CI:98.5-99.6) | |
| Frozen,consecutivelycollected | Flu A | 923 | 69 | 24d | 830 | 0 | 100.0%(CI:94.7-100.0) | 97.2%(CI:95.9-98.1) |
| Flu B | 923 | 36 | 16e | 871 | 0 | 100.0%(CI:90.4-100.0) | 98.2%(CI:97.1-98.9) | |
| Combined1 | Flu A | 2065 | 104 | 32f | 1927 | 2b | 98.1%(CI:93.4-99.5) | 98.4%(CI:97.7-98.8) |
| Flu B | 2065 | 78 | 25g | 1962 | 0 | 100.0%(CI:95.3-100.0) | 98.7%(CI:98.1-99.1) |
Table 8-9 Xpert Xpress Flu Assay Performance on NP Swab Specimens
Testing results by sequencing: 3 of 8 were Flu A Positive; 4 of 8 were Flu A Negative; 1 of 8 insufficient specimen. ದ.
b. Testing results by sequencing: 2 of 2 were Flu A Negative.
Testing results by sequencing: 6 of 9 were Flu B Positive; 2 of 9 were Flu B Negative; 1 of 9 insufficient specimen. C.
d. Testing results by sequencing: 8 of 24 were Flu A Positive; 11 of 24 were Flu A Negative; 5 of 24 insufficient specimen.
Testing results by sequencing: 7 of 16 were Flu B Positive; 3 of 16 were Flu B Negative; 6 of 16 insufficient specimen. e.
f. Testing results by sequencing: 11 of 32 were Flu A Positive; 15 of 32 insufficient specimen.
Testing results by sequencing: 13 of 25 were Flu B Positive; 7 of 25 insufficient specimen. പ്ര
Seven specimens (6 Flu A FP; 7 Flu B FP) were positive for all three targets. 1.
Of the Xpert Xpress Flu Assay runs performed with eligible specimens, 98.4% (2038/2071) of these specimens were successful on the first attempt. The remaining 33 gave indeterminate results on the first attempt (20 ERROR, 10 INVALID, and 3 NO RESULT). The initial indeterminate rate was 1.59% (33/2071) with the 95% CI 1.14-2.23%. Thirty of the 33 indeterminate cases were retested, of which 27 yielded valid results upon repeat testing; three specimens were not retested.
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The overall rate of assay success was 99.7% (2065/2071). The overall indeterminate rate after retesting was 0.3% (6/2071) with 95% CI 0.13- 0.63%.
In addition, there were 102 pre-selected frozen NP swab specimens tested. The results of this testing were analyzed separately and are as follows; the Xpert Xpress Flu Assay demonstrated a PPA and NPA of 100% and 95.8%, for influenza A, respectively; and 100% and 94.5% for influenza B, respectively.
Reproducibility Study
Reproducibility was established in a multi-center, blinded study using a 5-member specimen panel consisting of a negative control and two each of simulated nasal matrix spiked with influenza A, or influenza B at 1X (low pos) and 2-3X (mod pos) the respective LODs. Testing was performed at three sites (one internal, two external) using the GeneXpert Dx system, the Infinity-48 system, and the Infinity-80 system. Two operators at each site tested one panel in duplicate two times per day (equivalent to four replicates per day) over six, not necessarily consecutive days. Three lots of Xpert Xpress Flu cartridges were used, with each lot representing approximately two days of testing. Results are summarized in Table 8-10.
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| Sample ID | Site 1/Infinity-80 | Site 2/DX | Site 3/Infinity-48 | % TotalAgreement bySamplea | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Op 1 | Op 2 | Site | Op 1 | Op 2 | Site | Op 1 | Op 2 | Site | ||
| Negative | 100%(24/24) | 100%(24/24) | 100%(48/48) | 100%(24/24) | 100%(24/24) | 100%(48/48) | 100%(24/24) | 100%(24/24) | 100%(48/48) | 100%(144/144) |
| Flu A-Low Pos | 87.5%(21/24) | 95.8%(23/24) | 91.7%(44/48) | 95.7%(22/23) | 91.7%(22/24) | 93.6%(44/47) | 100%(24/24) | 91.7%(22/24) | 95.8%(46/48) | 93.7%(134/143)b |
| Flu A-Mod Pos | 100%(24/24) | 100%(24/24) | 100%(48/48) | 100%(23/23) | 100%(23/23) | 100%(46/46) | 100%(24/24) | 100%(24/24) | 100%(48/48) | 100%(142/142)b |
| Flu B-Low Pos | 95.8%(23/24) | 95.8%(23/24) | 95.8%(46/48) | 95.8%(23/24) | 95.8%(23/24) | 95.8%(46/48) | 95.8%(23/24) | 91.7%(22/24) | 93.8%(45/48) | 95.1%(137/144) |
| Flu B-Mod Pos | 100%(24/24) | 100%(24/24) | 100%(48/48) | 100%(24/24) | 100%(24/24) | 100%(48/48) | 100%(24/24) | 95.8%(23/24) | 97.9%(47/48) | 99.3%(143/144)c |
Table 8-10 Summary of Reproducibility Results
Agreement calculated based on expected result: Negative (targeted positivity: 0%); Positive for Low Pos (targeted positivity: 95%) and a. Mod Pos (targeted positivity: 100% ) samples.
b. Three samples 2x indeterminate [Flu A Low Pos (1); Flu A Mod Pos (2)]
One Flu B Mod Pos sample was positive for both targets C.
The reproducibility of the Xpert Xpress Flu Assay was also evaluated in terms of the fluorescence signal expressed in Ct values for each target detected. The mean, standard deviation (SD), and coefficient of variation (CV) between-sites, between- days, between-lots and between-operators for each panel member are presented in Table 8-11.
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| Sample | AssayChannel(Analyte) | Na | MeanCt | Between-Site | Between-Lot | Between-Day | Between-Operator | Within-Assay | Total | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| SD | CV(%) | SD | CV(%) | SD | CV(%) | SD | CV(%) | SD | CV(%) | SD | CV(%) | ||||
| Negative | SPC | 144 | 32.3 | 0 | 0 | 0.7 | 2.1 | 0 | 0 | 0.2 | 0.5 | 0.6 | 1.8 | 0.9 | 2.8 |
| Flu A-Low Pos | FluA1 | 134 | 35.3 | 0 | 0 | 0.4 | 1.1 | 0.6 | 1.8 | 0.1 | 0.4 | 0.9 | 2.5 | 1.2 | 3.3 |
| Flu A-Mod Pos | FluA1 | 142 | 33.1 | 0 | 0 | 0.1 | 0.4 | 0.1 | 0.4 | 0 | 0 | 0.6 | 1.9 | 0.7 | 2.0 |
| Flu B-Low Pos | FluB | 137 | 34.6 | 0 | 0 | 0 | 0 | 0.4 | 1.3 | 0 | 0 | 1.4 | 4.1 | 1.5 | 4.3 |
| Flu B-Mod Pos | FluB | 144 | 32.2 | 0.2 | 0.5 | 0.2 | 0.7 | 0 | 0 | 0.2 | 0.7 | 1.0 | 3.1 | 1.1 | 3.3 |
Table 8-11 Summary of Reproducibility Data
Results with non-zero Ct values of 144. a.
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Conclusions
The results of the nonclinical analytical and clinical performance studies summarized above demonstrate that the Xpert Xpress Flu Assay is substantially equivalent to the predicate device.
§ 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.