Xpert Xpress Flu/RSV, Xpert Nasopharyngeal Sample Collection Kit, GeneXpert Dx Systems (GX-I, GX-II, GX-IV, GX-XVI), GeneXpert Infinity-48 System and GeneXpert Infinity-48s System, GeneXpert Infinity-80 System

K162331 · Cepheid · OCC · Feb 9, 2017 · Microbiology

Device Facts

• Record ID: K162331
• Device Name: Xpert Xpress Flu/RSV, Xpert Nasopharyngeal Sample Collection Kit, GeneXpert Dx Systems (GX-I, GX-II, GX-IV, GX-XVI), GeneXpert Infinity-48 System and GeneXpert Infinity-48s System, GeneXpert Infinity-80 System
• Applicant: Cepheid
• Product Code: OCC · Microbiology
• Decision Date: Feb 9, 2017
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3980
• Device Class: Class 2

Indications for Use

The Cepheid Xpert® Xpress Flu/RSV 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, influenza B, and respiratory syncytial virus (RSV) viral RNA. The Xpert Xpress Flu/RSV Assay uses nasopharyngeal (NP) swab specimens collected from patients with signs and symptoms of respiratory infection. The Xpert Xpress Flu/RSV 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 RSV 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.

Device Story

Device performs automated, multiplex real-time RT-PCR for qualitative detection/differentiation of influenza A, influenza B, and RSV viral RNA from nasopharyngeal (NP) swab specimens; utilizes self-contained, single-use cartridges containing all necessary reagents for sample lysis, RNA extraction, and amplification; operated on GeneXpert Dx or Infinity Instrument Systems; system uses syringe drive for fluidics, ultrasonic horn for cell lysis, and I-CORE® thermocycler for amplification/detection; results generated in ~30 minutes; intended for use by laboratory personnel in moderate/high complexity settings; output provided as tabular/graphic results via software; aids clinical decision-making for respiratory infection management.

Clinical Evidence

Prospective clinical study (n=2065) conducted during 2015-2016 influenza season comparing Xpert Xpress Flu/RSV Assay to FDA-cleared molecular comparator. Combined dataset PPA/NPA: Flu A (98.1%/98.4%), Flu B (100%/98.7%), RSV (98.5%/98.8%). Study included fresh prospective and consecutive frozen specimens. Assay success rate 99.7%.

Technological Characteristics

Multiplex real-time RT-PCR; targets conserved RNA sequences (M, PB2, PA for Flu A; M, NS1, NS2 for Flu B; nucleocapsid for RSV). Uses GeneXpert cartridge with integrated reagents, ultrasonic lysis, and I-CORE® thermocycler. Connectivity: Networked/standalone GeneXpert systems. Software: Embedded firmware/PC-based control. Materials: Medical-grade polypropylene cartridge.

Indications for Use

Indicated for patients with signs and symptoms of respiratory infection to aid in the diagnosis of influenza A, influenza B, and RSV infections in conjunction with clinical and epidemiological risk factors.

Regulatory Classification

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.

Special Controls

*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.

Predicate Devices

• Xpert® Flu/RSV XC Assay (K142045)
• Cepheid Xpert Nasopharyngeal Sample Collection Kit (K151226)

Reference Devices

• Xpert® Nasopharyngeal Sample Collection Kit (K151226)

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION MEMORANDUM

A. 510(k) Number:
K162331

B. Purpose for Submission:
To obtain 510(k) clearance for the Xpert® Xpress Flu/RSV Assay for use on the GeneXpert® Dx and Infinity Instrument Systems.

C. Measurand:
Conserved RNA sequences within the genes encoding the matrix protein (M), basic polymerase protein 2 (PB2), and polymerase acidic protein (PA) of influenza A viruses; the matrix protein (M) and non-structural proteins (NS1 and NS2) of influenza B viruses; and the nucleocapsid protein of respiratory syncytial virus (RSV) A and B viruses.

D. Type of Test:
A multiplexed, real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay for the qualitative detection and differentiation of influenza A, influenza B, and RSV viral RNA from nasopharyngeal swab (NP) specimens using the GeneXpert Instrument Systems platform.

E. Applicant:
Cepheid

F. Proprietary and Established Names:
Xpert® Xpress Flu/RSV

G. Regulatory Information:
1. Regulation Section:
21 CFR 866.3980, Respiratory viral panel multiplex nucleic acid assay
2. Classification:
Class II

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3. Product Code(s):

OCC - Respiratory virus panel nucleic acid assay system
OOI - Real time nucleic acid amplification system
JSM - Culture media, non-propagating transport

4. Panel:

Microbiology (83)

H. Intended Use:

1. Intended Use(s):

The Cepheid Xpert® Xpress Flu/RSV 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, influenza B, and respiratory syncytial virus (RSV) viral RNA. The Xpert Xpress Flu/RSV Assay uses nasopharyngeal (NP) swab specimens collected from patients with signs and symptoms of respiratory infection. The Xpert Xpress Flu/RSV 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 RSV 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

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symptoms of respiratory infection prior to analysis with the Xpert Flu+RSV Xpress Assay or the Xpert Xpress Flu/RSV Assay.

2. Indication(s) for Use:

Same as intended use

3. Special Conditions for Use Statement(s):

For prescription use only

4. Special Instrument Requirements:

GeneXpert Instrument Systems

- GeneXpert Dx Systems (GX-I, GX-II, GX-IV, GX-XVI)
- GeneXpert Infinity Systems (Infinity-48, Infinity-80, Infinity-48s)

I. Device Description:

1. Overview

The Xpert® Xpress Flu/RSV Assay, performed on the GeneXpert® Instrument (Dx and Infinity) Systems, is a rapid, multiplex nucleic acid amplification test for the qualitative detection and differentiation of influenza A, influenza B, and RSV viral RNA from NP swab specimens.

To perform the Xpert® Xpress Flu/RSV Assay, NP swab specimens are collected from patients and placed into viral transport medium using the Xpert® Xpress Nasopharyngeal Sample Collection Kit for Viruses. With a transfer pipette, eluted NP swab specimens are loaded into the sample chamber of a single-use, self-contained Xpert Xpress Flu/RSV Assay cartridge. Each assay cartridge contains separate chambers for sample loading, sample processing and target amplification by real-time RT-PCR, and contains all the reagents necessary to carry out these processes. Because the cartridges are self-contained, and specimens never contact working parts of the instrument modules, cross-contamination between samples is minimized.

The assay cartridge containing the patient sample is inserted into the GeneXpert Instrument System, which performs fully automated and integrated sample preparation and real-time RT-PCR for the Xpert Xpress Flu/RSV Assay in approximately 30 minutes or less. The GeneXpert Instrument Systems, comprised of the GeneXpert Dx and GeneXpert Infinity Systems, have one to 80 randomly accessible modules that are each 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

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fluids between chambers), an ultrasonic horn for lysing cells or spores, and a proprietary I-CORE® thermocycler for performing real-time PCR and RT-PCR detection.

After completion of the test, the assay results are interpreted by the GeneXpert software from measured fluorescent signals and embedded calculation algorithms, and are shown in the “View Results” window in tabular and graphic formats.

## 2. Materials Provided

The Xpert® Xpress Flu/RSV Assay (Cat # XPRSFLU/RSV-10) contains sufficient reagents to process 10 NP swab specimens or quality control samples. The assay kit includes 10 individual cartridges with integrated reaction tubes that hold all of the reagents necessary for sample lysis, RNA extraction, and nucleic acid amplification. Additionally, each kit includes a set of 12 transfer pipettes for loading samples into the assay cartridge and a CD containing the Assay Definition Files (ADFs) with instructions for importing ADFs into the GeneXpert software.

## 3. Materials Required but Not Provided

### Validated Specimen Collection and Transport Kit

- Cepheid Xpert® Nasopharyngeal Sample Collection Kit for Viruses (Cat # SWAB/B-100) containing one individually wrapped, sterile nasopharyngeal flocked nylon swab and one Xpert Viral Transport Medium tube with three mL of Xpert Viral Transport Medium.

### GeneXpert® Instrument System

- GeneXpert Dx System (GX-I, GX-II, GX-IV, GX-XVI)
- Gene Xpert Infinity System (Infinity-48, Infinity-48s, Infinity-80)

### External Controls

- Positive inactivated influenza A and B virus control from ZeptoMetrix (Cat # NATFLUA/B-6C)
- Positive inactivated respiratory syncytial virus control from ZeptoMetrix (Cat # NATRSV-6C)
- Negative inactivated coxsackievirus control from ZeptoMetrix (Cat # NATCXVA9-6C)

## 4. Quality Control

The Xpert® Xpress Flu/RSV Assay includes two internal controls: a sample processing control, and a probe check control.

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# Sample Processing Control

The sample processing control (SPC) is a non-infectious armored RNA pseudovirus that ensures adequate processing of target viruses, monitors the presence of PCR inhibitors, and verifies the use of proper PCR conditions. The SPC should be POSITIVE in a sample that is negative for all three influenza A, influenza B and RSV target analytes, and can be NEGATIVE or POSITIVE in a sample containing detectable levels of one or more of the target analytes.

# Probe Check Control

The probe check control (PCC) is present to control for sufficient reagent rehydration, PCR tube filling, probe integrity and dye stability. All assay reagents must be present and intact for the PCC to pass the validated acceptance criteria. If any of the PCC conditions fail, the result is reported as an ERROR and the test must be repeated using a new assay cartridge.

# 5. Results Interpretation

POSITIVE or NEGATIVE test results for influenza A (Flu A), influenza B (Flu B), and RSV viral RNA are automatically generated by the GeneXpert software and presented as text in the "View Results" window of the GeneXpert Instrument Systems following completion of the test. The GeneXpert software is equipped with embedded algorithms to determine the result of the test based on the calculated cycle threshold (Ct) values obtained for each of the RNA target-specific optical curves generated by fluorescent probes during the real-time PCR cycling process. Optical curves for each of the RNA targets are also displayed in the "View Results" window but do not require manual data interpretation or reviewing.

The Xpert Xpress Flu/RSV Assay uses two separate channels for the detection of influenza A viral RNA (Flu A 1 and Flu A 2). The primers and probes in the Flu A 1 channel have 100% homology to human influenza A strains. The primers and probes in the Flu A 2 channel have >95% homology to avian influenza A strains and approximately 80% homology to human influenza A strains. Detection of influenza A strains in either the Flu A 1 or Flu A 2 channel is reported as Flu A POSITIVE.

The GeneXpert Instrument Systems software also reports if the test is INVALID, encounters an ERROR, or produces NO RESULT.

All possible test results for the Xpert Xpress Flu/RSV assay are shown in Table 1.

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Table 1: All Possible Final Test Results for the Xpert Xpress Flu/RSV Assay

| | RNA target | | | | |
| --- | --- | --- | --- | --- | --- |
| Result | Flu A 1 | Flu A 2 | Flu B | RSV | SPC |
| Flu A POSITIVE; Flu B NEGATIVE; RSV NEGATIVE | POS | POS/NEG | NEG | NEG | POS/NEG |
| | POS/NEG | POS | | | |
| Flu A POSITIVE; Flu B POSITIVE; RSV NEGATIVE | POS | POS/NEG | POS | NEG | POS/NEG |
| | POS/NEG | POS | | | |
| Flu A POSITIVE; Flu B NEGATIVE; RSV POSITIVE | POS | POS/NEG | NEG | POS | POS/NEG |
| | POS/NEG | POS | | | |
| Flu A POSITIVE; Flu B POSITIVE; RSV POSITIVE | POS | POS/NEG | POS | POS | POS/NEG |
| | POS/NEG | POS | | | |
| Flu A NEGATIVE; Flu B POSITIVE; RSV NEGATIVE | NEG | NEG | POS | NEG | POS/NEG |
| Flu A NEGATIVE; Flu B NEGATIVE; RSV POSITIVE | NEG | NEG | NEG | POS | POS/NEG |
| Flu A NEGATIVE; Flu B POSITIVE; RSV POSITIVE | NEG | NEG | POS | POS | POS/NEG |
| Flu A NEGATIVE; Flu B NEGATIVE; RSV NEGATIVE | NEG | NEG | NEG | NEG | POS |
| INVALID | NEG | NEG | NEG | NEG | NEG |
| ERROR | NO RESULT | NO RESULT | NO RESULT | NO RESULT | NO RESULT |
| NO RESULT | NO RESULT | NO RESULT | NO RESULT | NO RESULT | NO RESULT |

If any of the test results mentioned below occur, the test should be repeated using leftover specimen from the original transport medium tube and a new assay cartridge.

- INVALID: indicates that the control SPC failed. The sample was not properly processed, PCR was inhibited, or the sample was not properly collected.
- ERROR: could be a result of PCC failure or that the maximum pressure limits were exceeded. PCR does not initiate when there is a PCC failure.
- NO RESULT: indicates that insufficient data were collected. For example, the operator stopped a test that was in progress or a power failure occurred.
- Co-infection with two or more viruses: the incidence of co-infection with influenza A, influenza B and RSV is low. Specimens should undergo repeat testing if nucleic acids from two or more analytes are detected in a single specimen.

# J. Substantial Equivalence Information:

1. Predicate Device Name(s):

Cepheid Xpert® Flu/RSV XC Assay

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2. Predicate 510(k) Number:

K142045

3. Comparison with Predicate:

Table 2: Xpert Xpress Flu/RSV Assay Comparison with Predicate Device

| | Similarities and Differences | |
| --- | --- | --- |
| | Device | Predicate device |
| Item | Cepheid Xpert Xpress Flu/RSV Assay | Cepheid Xpert Flu/RSV XC Assay |
| 510(k) number | K162331 | K142045 |
| Regulation | 866.3980 | Same |
| Product code | OCC, OOI | Same |
| Device class | II | Same |
| Technology principle of operation | Multiplex real-time RT-PCR | Same |
| Assay targets | Influenza A, Influenza B, and RSV viral RNA | Same |
| Assay results | Qualitative | Same |
| Intended Use | The Cepheid Xpert® Xpress Flu/RSV 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, influenza B, and respiratory syncytial virus (RSV) viral RNA. The Xpert Xpress Flu/RSV Assay uses nasopharyngeal (NP) swab specimens collected from patients with signs and symptoms of respiratory infection. The Xpert Xpress Flu/RSV Assay is intended as an aid in the diagnosis of influenza and respiratory syncytial virus infections in conjunction with clinical and epidemiological risk factors. | The Cepheid Xpert Flu/RSV XC Assay 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, influenza B, and respiratory syncytial virus (RSV) viral RNA. The Xpert Flu/RSV XC Assay uses nasopharyngeal swab and nasal aspirate/wash specimens collected from patients with signs and symptoms of respiratory infection. The Xpert Flu/RSV XC Assay is intended as an aid in the diagnosis of influenza and respiratory syncytial virus infections in conjunction with clinical and epidemiological risk factors. |

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| Similarities and Differences | | |
| --- | --- | --- |
| | Device | Predicate device |
| Item | Cepheid Xpert Xpress Flu/RSV Assay | Cepheid Xpert Flu/RSV XC Assay |
| | Negative results do not preclude influenza virus or RSV 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. | 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 2013-2014 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. |
| Indications for Use | Patients with signs and symptoms of respiratory infection in conjunction with clinical and epidemiological risk factors | Same |
| Specimen types | NP swab specimens | Nasal aspirate/wash (NA/W) specimens and NP swab specimens |

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| | Similarities and Differences | |
| --- | --- | --- |
| | Device | Predicate device |
| Item | Cepheid Xpert Xpress Flu/RSV Assay | Cepheid Xpert Flu/RSV XC Assay |
| Sample preparation | Self-contained and automated after mixed specimen is added to cartridge | Same |
| Nucleic acid extraction | Yes | Same |
| Extraction methods | Sample preparation integrated in GeneXpert cartridge and GeneXpert Instrument Systems | Same |
| Instrument system | Cepheid GeneXpert Instrument Systems' 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 | Primers and probes to detect the presence of nucleic acid sequences of influenza A (including subtype H7N9), influenza B, and RSV |
| Target sequences | Influenza A: M, PB2 and PA proteinsInfluenza B: M, NS1 and NS2 proteinsRSV A and RSV B: nucleocapsid protein | Same |
| Internal assay controls | Encapsulated RNA pseudovirus SPC and PCC | Same |
| External assay controls | Available but not provided are inactivated virus controls for influenza A/B and RSV as positive controls, and Coxsackie virus as a negative control | Same |
| Time to obtain results | ~ 30 minutes or less for sample preparation and RT-PCR | ~60 minutes or less for sample preparation and RT-PCR |
| Combinatorial assay selections | User may select combined assay with all targets or a Flu only assay or an RSV only assay | Same |
| Early assay termination function | On Flu only or RSV only assay selections | Same |
| Intended users | Laboratory users in moderate and high complexity laboratory settings | Same |

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Table 3: Collection Kit Comparison with Predicate Device

| | Similarities and differences | |
| --- | --- | --- |
| | Device | Predicate |
| Item | Xpert® Nasopharyngeal Sample Collection Kit | Xpert® Nasopharyngeal Sample Collection Kit (K151226) |
| Intended Use | The Xpert® Nasopharyngeal Sample Collection Kit is designed to collect, preserve, and transport nasopharyngeal (NP) 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 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 and the Xpert Xpress Flu/RSV 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 Nasopharyngeal Sample Collection Kit has been cleared for use only with the Xpert Flu Assay, Xpert Flu/RSV XC Assay, Xpert Flu+RSV Xpress Assay and Xpert Xpress Flu/RSV Assay. | The Xpert Nasopharyngeal Sample Collection Kit has been cleared for use only with the Xpert Flu Assay, Xpert Flu/RSV XC Assay, and Xpert Flu+RSV Xpress Assay. |
| Single-use device | Yes | Same |
| Transport medium formulation | Hank's Balanced Salt Solution Bovine Serum Albumin L-cysteine Gelatin Sucrose L-glutamic acid HEPES buffer | Same |

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| | Similarities and differences | |
| --- | --- | --- |
| | Device | Predicate |
| Item | Xpert® Nasopharyngeal Sample Collection Kit | Xpert® Nasopharyngeal Sample Collection Kit (K151226) |
| | Vancomycin Amphotericin B Colistin Phenol red | |
| pH | 7.3 ± 0.2 | Same |
| Storage temperature | 2 - 25°C (refrigerated and room temperature) | Same |
| Volume | 3 mL | Same |
| Glass beads | 3 x 3 mm | Same |
| Container | Plastic (medical-grade polypropylene) | Same |
| Product configuration | Medium Tube in kit with individually-wrapped sterile swab | Same |

# K. Standard/Guidance Document Referenced (if applicable):

1. Guidance for Industry and FDA Staff: Format for Traditional and Abbreviated 510(k)s. Document issued on August 12, 2005.
2. Class II Special Controls Guidance Document: Instrumentation for Clinical Multiplex Test Systems-Guidance for Industry and Staff. Document issued on March 10, 2005.
3. Guidance for the Content of Premarket Submissions for Software Contained in Medical Devices. Document issued on May 11, 2005.
4. EP17-A2: Protocols for Determination of Limit of Detection and Limits of Quantitation; Approved Guideline-Second Edition 2006.
5. Class II Special Controls Guidance Document: Testing for Detection and Differentiation of Influenza A Virus Subtypes Using Multiplex Nucleic Acid Assays. Document issued on October 9, 2009.
6. Class II Special Controls Guidance Document: Respiratory Viral Panel Multiplex Nucleic Acid Assay. Document issued October 9, 2009.

# L. Test Principle:

The Xpert Xpress Flu/RSV Assay performed on the GeneXpert Instrument platform utilizes automated real-time RT-PCR for unique gene-specific sequence amplification and $5^{\prime}$ end cleavage of hybridized, fluorogenic target-specific probe for the detection of influenza A, influenza B and RSV viral RNA. Specific gene targets of the Xpert Xpress Flu/RSV Assay primers and probes include the matrix protein (M), basic polymerase protein 2 (PB2), and polymerase acidic protein (PA) of influenza A viruses; the matrix protein (M) and nonstructural proteins (NS1 and NS2) of influenza B viruses; and the nucleocapsid protein of

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RSV A and B viruses. Additional assay primers generate amplicons for the SPC. The GeneXpert Instrument Systems automates sample preparation, amplification and real-time detection of target-specific cDNA from clinical specimens. Each system utilizes a syringe pump drive to dispense fluids to and from the different cartridge chambers and ultrasonic lysis to release nucleic acids from both the target organisms and integrated SPC. During the amplification process, the I-CORE^{®} module contained within the GeneXpert Instrument systems heats and cools the reaction tube contents, and detects emitted fluorescence generated in the presence of target sequences. Embedded data analysis algorithms within the GeneXpert Instrument software generate test results based on the calculated cycle threshold (Ct) values obtained for each of the RNA target-specific optical curves generated by fluorescent probes during the real-time PCR cycling process. The time to results is 30 minutes or less.

### IV.2 Performance Characteristics

#### 1. Analytical Performance:

##### Precision/Reproducibility

A blinded, multi-center reproducibility study was conducted to assess the total variability of the Xpert Xpress Flu/RSV Assay across operators, study sites, testing days, GeneXpert Instrument systems, runs, and reagent lots.

Two operators at three study sites tested a seven-member reproducibility panel in duplicate twice per day on six different days (2 operators x 3 sites x 7 panel members x 2 replicates x 2 runs x 6 days) for a total of 1008 runs. Each site used a different GeneXpert Instrument (Infinity-80, GeneXpert Dx, or Infinity-48) and three Xpert Xpress Flu/RSV reagent lots for the analysis. The reproducibility specimen panel consisted of one seasonal influenza A strain (Flu A/Victoria/361/2011), one influenza B strain (Flu B/Massachusetts/2/2012), and one RSV (A/Long/MD/56) strain. Each virus strain was prepared at two concentration levels: a “low positive” sample with an analyte concentration approximately 1X the limit of detection (LoD) and a “moderate positive” sample with an analyte concentration approximately 2X the LoD.

The reproducibility panel specimens were prepared using cultured influenza and RSV viruses spiked into negative simulated nasal matrix. The simulated matrix consisted of 2.5% (w/v) porcine mucin, 1% (v/v) human whole blood in 0.85% sodium chloride (NaCl) formulated in a 1X PBS solution with 15% glycerol, which was then diluted in UTM to a final concentration of 16.7%. A negative sample containing simulated matrix only was also included in the panel.

The expected results for each of the panel members are shown in Table 4 below. Overall percent agreement between the expected results and the reproducibility study results is presented in Table 5. Analysis of variance (CV) between sites, days, lots and operators for each reproducibility panel member is presented in Table 6.

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Table 4: Reproducibility Panel Members and Expected Results

| Panel sample (strain) | Expected concentration | Expected positivity rate |
| --- | --- | --- |
| Negative | 0 | 0% |
| Influenza A - low pos | 1X LoD | ~95% |
| Influenza A - mod pos | 2X LoD | 100% |
| Influenza B - low pos | 1X LoD | ~95% |
| Influenza B - mod pos | 2X LoD | 100% |
| RSV - low pos | 1X LoD | ~95% |
| RSV - mod pos | 2X LoD | 100% |

Table 5: Reproducibility Results - Percent Agreement

| Sample ID | Site 1/Infinity-80 | | | Site 2/DX | | | Site 3/Infinity-48 | | | % Total agreementa |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | 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% (144/44) |
| 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 | 95.8% (23/24) | 100% (24/24) | 97.9% (47/48) | 100% (24/24) | 100% (24/24) | 100% (48/48) | 100% (24/24) | 95.8% (23/24) | 97.9% (47/48) | 98.6% (142/144)c |
| RSV-low pos | 91.7% (22/24) | 87.5% (21/24) | 89.6% (43/48) | 100% (23/23) | 100% (24/24) | 100% (47/47) | 91.7% (22/24) | 95.8% (23/24) | 93.8% (45/48) | 94.4% (135/143)b |
| RSV-mod pos | 100% (24/24) | 100% (24/24) | 100% (48/48) | 100% (23/23) | 100% (24/24) | 100% (47/47) | 100% (24/24) | 100% (24/24) | 100% (48/48) | 100% (143/143)b |

a Agreement calculated based on expected result: Negative for Negative (targeted positivity: $0\%$ ); Positive for Low Pos (targeted positivity: $95\%$ ) and Mod Pos (targeted positivity: $100\%$ ) samples.
b Fives samples 2x indeterminate [Flu A Low Pos (1); Flu A Mod Pos (2); RSV Low Pos (1); RSV Mod Pos (1)]
c Two Flu B Mod Pos samples were positive for all three targets.

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Table 6: Reproducibility Results -Variance Analysis

| Sample | Assay channel (analyte) | Na | Mean Ct | 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 |
| RSV-low Pos | RSV | 135 | 36.5 | 0 | 0 | 0.6 | 1.7 | 0 | 0 | 0.5 | 1.3 | 0.9 | 2.6 | 1.2 | 3.3 |
| RSV-mod pos | RSV | 143 | 33.5 | 0.2 | 0.7 | 0.1 | 0.4 | 0 | 0 | 0 | 0 | 1.5 | 4.6 | 1.6 | 4.6 |

Results with non-zero Ct values of 144.

# b. Assay Cut-Off

The Xpert Xpress Flu/RSV Assay detects influenza A (Flu A), influenza B (Flu B) and RSV analytes. For each analyte, a manual threshold and a valid minimum and maximum cycle threshold (Ct) setting are defined. The manual threshold setting is the relative fluorescence value that, when exceeded, results in a reported Ct value. The manual threshold is chosen as low as possible to preserve assay sensitivity, but high enough to insure that noise values in the baseline do not trigger a threshold crossing erroneously.

The manual threshold setting for the Flu A 1 target was set at 40 based on a mean fluorescent background level of 232.4 in pre-clinical and analytical studies. The manual threshold settings for the Flu A 2, Flu B, and RSV targets were 50 (mean = 140.7), 40 (mean = 256.9) and 45 (mean = 288.0), respectively.

The valid Ct range for Flu A1, Flu A2, Flu B, and RSV targets is 10 to 40. These values were determined using pre-clinical and analytical data and were subsequently validated in the clinical study. The Ct cutoffs are included as automatic calculations in the assay definition file (ADF) provided with the Xpert Xpress Flu/RSV Assay.

# c. External Controls

Three Zeptrometrix NATtrol Molecular Controls, comprised of inactivated viruses in a purified protein matrix, were evaluated as external controls with the Xpert Xpress Flu/RSV Assay.

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Testing was performed with 20 replicates of three separate lots of the NATtrol Influenza A/B Positive Control (cat # NATFLUA/B-6C), NatTrol RSV Positive Control (Cat # NATRSV-6C), and NATtrol Influenza/RSV Negative Control (cat# NATCXVA9-C6C), totaling 180 runs. All 180 runs provided valid and accurate test results.

For all analytical studies reported in this submission, one replicate of the three external controls was run with the Xpert Xpress Flu/RSV Assay on each day of testing.

## d. Stability

### Kit Stability

Real-time kit stability data for the Xpert Xpress Flu/RSV Assay is currently available for six months at the extremes of the recommended storage temperature range of 2°C to 28°C. Results to date show that average Ct values from the stability testing meet the predefined performance criteria. Testing is ongoing for up to 37 months on three reagent lots.

### Specimen Stability

A stability study was conducted to establish transport and storage claims for eluted NP swab specimens to be analyzed with the Xpert Xpress Flu/RSV Assay. Specimens included one seasonal influenza A strain (A/Victoria/361/2011), one influenza B strain (B/Massachusetts/2/2012), and one RSV strain (A/Long/MD/56) prepared in simulated matrix to an analyte concentration of approximately 2X LoD.

Eight replicates of each of the positive specimens and a negative control (simulated matrix only) were stored at 2°C, 8°C, 15°C, and 30°C, and tested at multiple time points. Under the conditions of the study, all positive and negative specimens at all storage conditions and temperatures tested were correctly identified using the Xpert Xpress Flu/RSV Assay. The study data supports specimen stability claims under the recommended specimen storage conditions (15-30°C for up to 24 hours and 2-8°C for up to seven days).

### Cartridge Hold Time

Xpert Xpress Flu/RSV samples that are prepared for testing may wait up to four and a half hours on the GeneXpert Infinity Instruments before a module becomes available. During this wait time, targeted viral RNA may degrade or become unstable such that an originally low positive sample is rendered "NEGATIVE." Assay performance out to five and a half hours was evaluated under three storage conditions (ambient, 25°C/75% relative humidity (RH), and 35°C) with a single lot of the Xpert Xpress Flu/RSV Assay. Testing was performed on eight replicates each of one influenza A (A/Victoria/361/2011), one influenza B (B/Massachusetts/2/2012), and one RSV (A/Long/MD/56) strain spiked into negative simulated matrix at an analyte concentration between 1X and 3X the LoD, and negative specimens consisting of simulated matrix

15

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only. All positive and negative replicates were correctly reported using the Xpert Xpress Flu/RSV Assay, demonstrating acceptable performance following incubation for periods exceeding the maximum cartridge hold time of four and half hours.

# e. Limit of Detection

The LoD of the Xpert Xpress Flu/RSV Assay was established using two influenza A 2009 H1N1-like strains, two influenza A H3N2 strains, two influenza B strains, two RSV A strains, and two RSV B strains diluted into negative pooled NP clinical matrix. The LoD is defined as the lowest concentration (tissue culture infectious does, $\mathrm{TCID}_{50} / \mathrm{mL}$ ) 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 virus strain was initially tested in a range finding study at five different concentrations in replicates of 20 per concentration of virus. Testing was performed with two lots of reagents across three testing days. The higher LoD estimate observed per strain and per lot as determined by probit regression analysis was selected for verification. Verification of the estimated LoD claim was performed on one reagent lot across a minimum of three testing days using 20 replicates per strain.

The LoD study results are presented in Tables 7 to 11.

Table 7: Influenza A 2009 H1N1-like LoD (TCID ${}_{50}/\mathrm{{mL}}$ ) Results

| Virus Strain | Confirmed LoD |
| --- | --- |
| Influenza A/California/7/2009 | 0.02 |
| Influenza A/Florida/27/2011 | 0.04 |

Table 8: Influenza A H3N2 LoD (TCID ${}_{50}/\mathrm{{mL}}$ ) Results

| Virus Strain | Confirmed LoD |
| --- | --- |
| Influenza A/Perth/16/2009 | 0.01 |
| Influenza A/Victoria/361/2011 | 0.75 |

Table 9: Influenza B LoD (TCID ${}_{50}/\mathrm{{mL}}$ ) Results

| Virus Strain | Confirmed LoD |
| --- | --- |
| Influenza B/Massachusetts/2/2012 (Yamagata) | 0.40 |
| Influenza B/Wisconsin/01/2011 (Victoria) | 0.19 |

Table 10: RSV A LoD (TCID ${}_{50}/\mathrm{{mL}}$ ) Results

| Virus Strain | Confirmed LoD |
| --- | --- |
| RSV A/2/Australia/61 | 0.87 |
| RSV A/Long/MD/56 | 1.10 |

Table 11: RSV B LoD (TCID ${}_{50}/\mathrm{{mL}}$ ) Results

| Virus Strain | Confirmed LoD |
| --- | --- |
| RSV B/Wash/18537/62 | 0.79 |
| RSV B/9320/MA/77 | 2.30 |

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# f. Analytical Reactivity

An analytical reactivity study was conducted to evaluate the ability of the Xpert Xpress Flu/RSV Assay to detect multiple influenza and RSV strains that are temporally and geographically diverse. Testing was performed on 53 different strains (48 influenza and 5 RSV) in triplicate using cultured virus spiked into negative simulated matrix at analyte concentrations approximately $3\mathrm{X}$ the LoD. Influenza A strains included 14 A/H1N1 (pre-2009 seasonal and 2009 pandemic), 9 A/H3N2, and 12 avian strains (H5N1, H5N2, H6N2, H7N2, H2N2, H7N9, and H9N2 subtypes). The 13 Influenza B strains included both the Victoria and Yamagata lineage. RSV strains included RSV types A and B.

The Xpert Xpress Flu/RSV Assay exhibited a broad range of reactivity, as all influenza A, influenza B and RSV strains tested positive in all three replicates, except for one influenza A H1N1 strain (A/New Jersey/8/76) which tested positive in 2 of 3 replicates at 0.1 TCID50/mL. Results from the study are shown in Table 12.

Table 12: Reactivity Results

| Virus | Strain | Target concentration | Result | | |
| --- | --- | --- | --- | --- | --- |
| | | | Flu A | Flu B | RSV |
| No template Control | | NA | NEG | NEG | NEG |
| Influenza A H1N1 (pre-2009) | A/swine/Iowa/15/30 | 0.1 TCID50/mL | POS | NEG | NEG |
| | A/WS/33 | 0.1 TCID50/mL | POS | NEG | NEG |
| | A/PR/8/34 | 0.1 TCID50/mL | POS | NEG | NEG |
| | A/Mal/302/54 | 0.1 TCID50/mL | POS | NEG | NEG |
| | A/Denver/1/57 | 0.1 TCID50/mL | POS | NEG | NEG |
| | A/New Jersey/8/76 | 0.1 TCID50/mL | POS | NEG | NEG |
| | A/New Caledonia/20/1999 | 0.1 TCID50/mL | POS | NEG | NEG |
| | A/New York/55/2004 | 0.1 TCID50/mL | POS | NEG | NEG |
| | A/Soloman Island/3/2006 | 0.1 TCID50/mL | POS | NEG | NEG |
| | A/Taiwan/42/06 | 0.1 TCID50/mL | POS | NEG | NEG |
| | A/Brisbane/59/2007 | 0.1 TCID50/mL | POS | NEG | NEG |
| Influenza A H1N1 (pdm 2009) | A/swine/NY/02/2009 | 0.1 TCID50/mL | POS | NEG | NEG |
| | A/Colorado/14/2012 | 0.1 TCID50/mL | POS | NEG | NEG |
| | A/Washington/24/2012 | 0.1 TCID50/mL | POS | NEG | NEG |
| Influenza A H3N2 (seasonal) | A/Aichi/2/68 | 2.0 TCID50/mL | POS | NEG | NEG |
| | A/HongKong/8/68 | 2.0 TCID50/mL | POS | NEG | NEG |
| | A/Port Chalmers/1/73 | 2.0 TCID50/mL | POS | NEG | NEG |
| | A/Hawaii/15/2001 | 2.0 TCID50/mL | POS | NEG | NEG |
| | A/Wisconsin/67/05 | 2.0 TCID50/mL | POS | NEG | NEG |
| | A/Brisbane/10/2007 | 2.0 TCID50/mL | POS | NEG | NEG |
| | A/Minnesota/11/2010 (H3N2)v | 2.0 TCID50/mL | POS | NEG | NEG |
| | A/Indiana/08/2011 (H3N2)v | 2.0 TCID50/mL | POS | NEG | NEG |
| | A/Texas/50/2012 | 2.0 TCID50/mL | POS | NEG | NEG |

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| Virus | Strain | Target concentration | Result | | |
| --- | --- | --- | --- | --- | --- |
| | | | Flu A | Flu B | RSV |
| Avian Influenza A | A/duck/Hunan/795/2002 (H5N1) | ≤1ρg/μLa | POS | NEG | NEG |
| | A/chicken/Hubei/327/2004 (H5N1) | ≤1ρg/μLa | POS | NEG | NEG |
| | A/Anhui/01/2005 (H5N1) | ≤1ρg/μLa | POS | NEG | NEG |
| | A/Japanese white eye/HongKong/1038/2006 | ≤1ρg/μLa | POS | NEG | NEG |
| | A/mallard/WI/34/75 (H5N2) | ≤1ρg/μLa | POS | NEG | NEG |
| | A/chicken/CA431/00 (H6N2) | ≤1ρg/μLa | POS | NEG | NEG |
| | A/duck/LTC-10-82743/1943 (H7N2) | ≤1ρg/μLa | POS | NEG | NEG |
| | A/chicken/NJ/15086-3/94 (H7N3) | ≤1ρg/μLa | POS | NEG | NEG |
| | A/Anhui/1/2013 (H7N9) | N/Ab | POS | NEG | NEG |
| | A/Shanghai/1/2013 (H7N9) | N/Ab | POS | NEG | NEG |
| | A/chicken/Korea/38349-p96323/1996 (H9N2) | ≤1ρg/μLa | POS | NEG | NEG |
| | A/Mallard/NY/6750/78 (H2N2) | ≤1ρg/μLa | POS | NEG | NEG |
| Influenza B | B/Lee/40 | 1.0 TCID50/mL | NEG | POS | NEG |
| | B/Allen/45 | 1.0 TCID50/mL | NEG | POS | NEG |
| | B/GL/1739/54 | 1.0 TCID50/mL | NEG | POS | NEG |
| | B/Maryland/1/59 | 1.0 TCID50/mL | NEG | POS | NEG |
| | B/Panama/45/90c | 1.0 TCID50/mL | NEG | POS | NEG |
| | B/Florida/07/2004d | 1.0 TCID50/mL | NEG | POS | NEG |
| | B/Florida/02/06c | 1.0 TCID50/mL | NEG | POS | NEG |
| | B/Florida/04/06d | 1.0 TCID50/mL | NEG | POS | NEG |
| | B/Hong Kong/5/72 | 1.0 TCID50/mL | NEG | POS | NEG |
| | B/Wisconsin/01/2010d | 1.0 TCID50/mL | NEG | POS | NEG |
| | B/Malaysia/2506/04c | 1.0 TCID50/mL | NEG | POS | NEG |
| | B/Taiwan/2/62 | 1.0 TCID50/mL | NEG | POS | NEG |
| | B/Brisbane/60/2008c | 1.0 TCID50/mL | NEG | POS | NEG |
| RSV A | RSV-A/NY (clinical unknown) | 3.0 TCID50/mL | NEG | NEG | POS |
| | RSV-A/WI/629-8-2/2007 | 3.0 TCID50/mL | NEG | NEG | POS |
| | RSV-A/WI/629-11-1/2008 | 3.0 TCID50/mL | NEG | NEG | POS |
| RSV B | RSV-B/WV14617/85 | 7.0 TCID50/mL | NEG | NEG | POS |
| | RSV-B/CH93(18)-18 | 7.0 TCID50/mL | NEG | NEG | POS |

a Purified viral RNA in simulated matrix was used for avian influenza A viruses due to biosafety regulations.
b Inactivated avian influenza A (H7N9) viruses without viral titer was diluted 100,000 fold in simulated matrix and tested due to biosafety regulations.
c Known Victoria lineage.
d Known Yamagata lineage.

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Further in silico analysis was conducted to determine the predicted cross reactivity of additional influenza A 2009 H1N1-like strains (n= 4 strains/target). The results showed $100\%$ sequence homology for all primer target nucleotide sequences analyzed.

# g. Analytical Specificity

An analytical specificity study was carried out to determine the potential for cross-reactivity of the Xpert Xpress Flu/RSV Assay with common respiratory pathogens or those potentially encountered in the nasopharynx. The panel of microorganisms included 16 viral, 26 bacterial and 2 yeast strains. Three replicates of each viral strain were tested at concentrations of $\geq 1\mathrm{x}10^{5}$ TCID $_{50}$ /mL. Bacterial and yeast strains were also tested in triplicate at concentrations of $\geq 1\mathrm{x}10^{6}$ CFU/mL with the exception of Chlamydia pneumoniae, which was tested at $1\mathrm{x}10^{5}$ CFU/mL. Each microorganism was prepared in negative simulated matrix to the concentrations indicated in Table 13 below.

Based on the results of this study, as summarized in Table 13, the Xpert Xpress Flu/RSV Assay showed no cross-reactivity with any of the 44 common respiratory microorganisms tested.

Table 13: Analytical Specificity Study Results

| Organism | Concentration | Result | | |
| --- | --- | --- | --- | --- |
| | | Flu A | Flu B | RSV |
| No template control | N/A | NEG | NEG | NEG |
| Adenovirus Type 1 | 1.12E+06 TCID50/mL | NEG | NEG | NEG |
| Adenovirus Type 7 | 1.87E+05 TCID50/mL | NEG | NEG | NEG |
| Human coronavirus OC43 | 2.85E+05 TCID50/mL | NEG | NEG | NEG |
| Human coronavirus 229E | 1.00E+05 TCID50/mL | NEG | NEG | NEG |
| Cytomegalovirus | 1.00E+05 TCID50/mL | NEG | NEG | NEG |
| Echovirus | 3.31E+07 TCID50/mL | NEG | NEG | NEG |
| Enterovirus | 3.55E+05 TCID50/mL | NEG | NEG | NEG |
| Epstein Barr Virus | 7.16E+07 TCID50/mL | NEG | NEG | NEG |
| HSV | 8.90E+05 TCID50/mL | NEG | NEG | NEG |
| Measles | 6.31E+05 TCID50/mL | NEG | NEG | NEG |
| Human metapneumovirus | 1.00E+05 TCID50/mL | NEG | NEG | NEG |
| Mumps virus | 6.31E+06 TCID50/mL | NEG | NEG | NEG |
| Human parainfluenza Type 1 | 1.15E+06 TCID50/mL | NEG | NEG | NEG |
| Human parainfluenza Type 2 | 6.31E+05 TCID50/mL | NEG | NEG | NEG |
| Human parainfluenza Type 3 | 3.55E+06 TCID50/mL | NEG | NEG | NEG |
| Rhinovirus Type 1A | 1.26E+05 TCID50/mL | NEG | NEG | NEG |
| Acinetobacter baumannii | 1.00E+06 CFU/mL | NEG | NEG | NEG |
| Burkholderia cepacia | 3.30E+06 CFU/mL | NEG | NEG | NEG |
| Candida albicans | 3.20E+06 CFU/mL | NEG | NEG | NEG |
| Candida parapsilosis | 3.00E+06 CFU/mL | NEG | NEG | NEG |
| Bordetella pertussis | 3.30E+06 CFU/mL | NEG | NEG | NEG |
| Chlamydia pneumoniae | 1.00E+05 CFU/mL | NEG | NEG | NEG |

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| Organism | Concentration | Result | | |
| --- | --- | --- | --- | --- |
| | | Flu A | Flu B | RSV |
| Citrobacter freundii | 3.30E+06 CFU/mL | NEG | NEG | NEG |
| Corynebacterium sp. | 3.30E+06 CFU/mL | NEG | NEG | NEG |
| Escherichia coli | 1.00E+07 CFU/mL | NEG | NEG | NEG |
| Enterococcus faecalis | 1.30E+06 CFU/mL | NEG | NEG | NEG |
| Haemophilus influenzae | 1.00E+06 CFU/mL | NEG | NEG | NEG |
| Lactobacillus reuteri | 1.00E+06 CFU/mL | NEG | NEG | NEG |
| Legionella spp. | 1.00E+06 CFU/mL | NEG | NEG | NEG |
| Moraxella catarrhalis | 1.00E+07 CFU/mL | NEG | NEG | NEG |
| Mycobacterium tuberculosis (avirulent) | 1.00E+06 CFU/mL | NEG | NEG | NEG |
| Mycoplasma pneumoniae | 1.00E+06 CFU/mL | NEG | NEG | NEG |
| Neisseria meningitidis | 2.15E+06 CFU/mL | NEG | NEG | NEG |
| Neisseria mucosa | 1.00E+07 CFU/mL | NEG | NEG | NEG |
| Propionibacterium acnes | 2.40E+07 CFU/mL | NEG | NEG | NEG |
| Pseudomonas aeruginosa | 3.70E+06 CFU/mL | NEG | NEG | NEG |
| Staphylococcus aureus (protein A producer) | 2.20E+06 CFU/mL | NEG | NEG | NEG |
| Staphylococcus epidermidis | 3.40E+06 CFU/mL | NEG | NEG | NEG |
| Staphylococcus haemolyticus | 4.00E+06 CFU/mL | NEG | NEG | NEG |
| Streptococcus agalactiae | 3.50E+06 CFU/mL | NEG | NEG | NEG |
| Streptococcus pneumoniae | 1.00E+06 CFU/mL | NEG | NEG | NEG |
| Streptococcus pyogenes | 1.00E+07 CFU/mL | NEG | NEG | NEG |
| Streptococcus salivarius | 1.00E+07 CFU/mL | NEG | NEG | NEG |
| Streptococcus sanguinis | 3.10E+06 CFU/mL | NEG | NEG | NEG |

h. Assay Interference

# Potentially Interfering Substances

The performance of the Xpert Xpress Flu/RSV Assay was evaluated in the presence of medically and/or physiologically relevant concentrations of potentially interfering substances (listed in Table 14 below) that may be present in NP swab specimens.

Table 14: Interfering Substances Tested

| Substance/class | Description/active ingredient | Concentration tested |
| --- | --- | --- |
| Control | Simulated nasal matrix | 100% (v/v) |
| Beta-adrenergic bronchodilator | Albuterol sulfate | 0.83 mg/mL (equivalent to 1 dose per day) |
| Blood | Blood (Human) | 2% (v/v) |
| BD™ Universal Viral Transport System | Transport media | 100% (v/v) |
| Remel M4® | Transport media | 100% (v/v) |
| Remel M4RT® | Transport media | 100% (v/v) |

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| Substance/class | Description/active ingredient | Concentration tested |
| --- | --- | --- |
| Remel M5® | Transport media | 100% (v/v) |
| Remel M6® | Transport media | 100% (v/v) |
| Throat lozenges, oral anesthetic and analgesic | Benzocaine, Menthol | 1.7 mg/mL |
| Mucin | Purified mucin protein (bovine or porcine submaxillary 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, Histaminum hydrochloricum sulfur | 15% (w/v) |
| Nasal corticosteroid | Fluticasone propionate | 5 μg/mL |

* FluMist vaccine samples were unavailable for testing at the time this study was conducted. Samples containing FluMist may cause false positive results.

Eight negative replicates were tested per potentially interfering substance to determine the effects on the performance of the SPC. In addition, eight positive replicates were tested per substance for each of six influenza and four RSV strains included in this study. The panel of positive samples included two 2009 H1N1-like strains (A/California/7/2009 and A/Florida/27/2011), two influenza A H3N2 strains (A/Perth/16/2009 and A/Victoria/361/2011), two influenza B strains (B/Wisconsin/01/2011 and B/Massachusetts/02/2012), and four RSV strains (A/Long/MD/56, A/2/Australia/61, B/Wash/18537/62, and B/9320/MA/77) spiked into negative simulated matrix to an analyte concentration 3X the LoD.

No assay interference was observed in the presence of the substances at the concentrations tested in this study. All positive and negative replicates tested with the Xpert Xpress Flu/RSV Assay were correctly identified as summarized in Table 15 below.

Table 15: Potentially Interfering Substances Study Results

| Substance | Neg | Flu A 2009 H1N1 | | Flu A H3N2 | | Flu B | | RSV A | | RSV B | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | Strain 1 | Strain 2 | Strain 1 | Strain 2 | Strain 1 | Strain 2 | Strain 1 | Strain 2 | Strain 1 | Strain 2 |
| Albuterol | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 |
| Blood | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 |
| BD UVTM | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 |
| M4 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 |
| M4RT | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 |
| M5 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 |
| M6 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 |

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| Substance | Neg | Flu A 2009 H1N1 | | Flu A H3N2 | | Flu B | | RSV A | | RSV B | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | Strain 1 | Strain 2 | Strain 1 | Strain 2 | Strain 1 | Strain 2 | Strain 1 | Strain 2 | Strain 1 | Strain 2 |
| Menthol | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 |
| Mucin | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 |
| Mupirocin | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 |
| Saline spray | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 |
| Anefrin | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 |
| Phenylephrine | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 |
| Tamiflu | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 |
| Zicam | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 |
| Fluticasone propionate | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 | 8/8 |

# Potentially Interfering Microorganisms

An analytical interference study was conducted to assess the inhibitory effects of a select panel of commensal microorganisms common to the nasopharynx, on the performance of the Xpert Xpress Flu/RSV Assay. Seven viral strains (listed in Table 16) at $\geq 1\mathrm{x}10^{5}$ $\mathrm{TCID}_{50} / \mathrm{mL}$ were tested in the presence of one strain each of influenza A (A/Perth/16/2009), influenza B (B/Massachusetts/2/2012), RSV A (A/Long/MD/56) and RSV B (B/9320/MA/77) viruses spiked individually at $3\mathrm{X}$ the LoD into negative simulated matrix. Competitive interference of three commensal bacterial strains at $\geq$ $1\mathrm{x}10^{6}$ CFU/mL was also evaluated with each of the Xpert Xpress Flu/RSV Assay analytes. Testing was performed with replicates of eight for each of the target strain and each potentially interfering strain combination, and a negative simulated matrix control.

Under the conditions of the study, no inhibitory effects were observed for each of the strains tested in the presence of another analyte.

Table 16. Potentially Interfering Microorganisms Tested

| Microorganisms | Commensal Microorganism Concentration |
| --- | --- |
| Adenovirus Type 1 | ≥ 1x105TCID50/mL |
| Human Coronavirus OC43 | ≥ 1x105TCID50/mL |
| Human metapneumovirus | ≥ 1x105TCID50/mL |
| Human parainfluenza Type 1 | ≥ 1x105TCID50/mL |
| Human parainfluenza Type 2 | ≥ 1x105TCID50/mL |
| Human parainfluenza Type 3 | ≥ 1x105TCID50/mL |
| Rhinovirus Type 1A | ≥ 1x105TCID50/mL |
| Staphylococcus aureus | ≥ 1x106CFU/mL |
| Staphylococcus epidermidis | ≥ 1x106CFU/mL |
| Haemophilus influenzae | ≥ 1x106CFU/mL |

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# Potential Competitive Analyte Interference

Internal competitive interference caused by co-infections of the targets in the Xpert Xpress Flu/RSV Assay was evaluated by testing individual influenza and RSV strains near the LoD in the presence of different influenza or RSV strains at higher concentration. Analytical competitive interference was assessed using one seasonal influenza A H3N2 strain (A/Victoria/361/2011), one influenza B strain (B/Massachusetts/2/2012), one RSV A strain (A/2/Australia) and one RSV B strain (B/Wash/18537/62).

Replicates of 20 were tested for each target strain and each competitive strain combination. The normal binomial distribution with 20 replicate samples at the limit of detection 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.

The results for each analyte at the LoD in combination with another competitive anlayte are shown in Tables 17 to 20. The average Ct values for each strain tested at the LoD without a competitive analyte were taken directly from the Limit of Detection study.

Table 17: Competitive Interference Study Results for Influenza A/Victoria/361/2011

| Target strain titer (TCID50/mL) | Competitive strain | Competitive strain titer (TCID50/ml) | Positives/20 replicates | Average Ct | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | Flu A1 | Flu B | RSV | SPC |
| 0.8 | N/A | N/A | 20/20 | 33.9 | 0 | 0 | 32.4 |
| 0.8 | B/Mass/2/2012 | 1x103 | 20/20 | 34.6 | 22.6 | 0 | 32.2 |
| | RSV A/2/Australia/61 | 1x103 | 20/20 | 35.2 | 0 | 24.1 | 32.1 |
| | RSV B/Wash/18537/62 | 1x104 | 20/20 | 34.5 | 0 | 21.8 | 32.1 |

Table 18: Competitive Interference Study Results for Influenza B/Massachusetts/2/2012

| Target strain titer (TCID50/mL) | Competitive strain | Competitive strain titer (TCID50/ml) | Positives/20 replicates | Average Ct | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | Flu A1 | Flu B | RSV | SPC |
| 0.45 | N/A | N/A | 20/20 | 0 | 33.8 | 0 | 32.0 |
| 0.45 | A/Victoria/361/2011 | 1x103 | 3/20 | 23.5 | 36.9 | 0 | 32.8 |
| | | 1x102 | 19/20 | 27.0 | 35.3 | 0 | 31.7 |
| | RSV A/2/Australia/61 | 1x103 | 19/20 | 0 | 34.9 | 24.0 | 32.4 |
| | RSV B/Wash/18537/62 | 1x103 | 20/20 | 0 | 33.9 | 25.2 | 32.4 |

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Table 19: Competitive Interference Study Results for RSV A/2/Australia/61

| Target strain titer (TCID50/mL) | Competitive strain | Competitive strain titer (TCID50/ml) | Positives/20 replicates | Average Ct | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | Flu A1 | Flu B | RSV | SPC |
| 1.1 | N/A | N/A | 20/20 | 0 | 0 | 34.6 | 31.7 |
| 1.1 | A/Victoria/361/2011 | 1x103 | 4/20 | 23.6 | 0 | 38.5 | 32.6 |
| | | 1x102 | 20/20 | 27.1 | 0 | 36.2 | 31.9 |
| | B/Mass/2/2012 | 1x103 | 19/20 | 0 | 22.7 | 36.6 | 32.6 |

Table 20: Competitive Interference Study Results for RSV B/Wash/18537/62

| Target strain titer (TCID50/mL) | Competitive strain | Competitive strain titer (TCID50/ml) | Positives/20 replicates | Average Ct | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | Flu A1 | Flu B | RSV | SPC |
| 0.9 | N/A | N/A | 19/20 | 0 | 0 | 36.5 | 32.1 |
| 0.9 | A/Victoria/361/2011 | 1x102 | 11/20 | 26.9 | 0 | 37.3 | 31.8 |
| | | 1x101 | 19/20 | 30.1 | 0 | 36.6 | 31.8 |
| | B/Mass/2/2012 | 1x103 | 13/20 | 0 | 22.7 | 36.6 | 32.4 |
| | | 1x102 | 20/20 | 0 | 26.3 | 36.2 | 32.1 |
| 1.6 (2X LoD) | A/Victoria/361/2011 | 1x102 | 18/20 | 26.8 | 0 | 35.1 | 31.8 |
| | B/Mass/2/2012 | 1x103 | 19/20 | 0 | 22.7 | 35.0 | 32.5 |

The results from the study show potential for competitive inhibition in specimens with two different viruses in which one virus is present at the LoD and the other virus is present at $>2$ logs higher $\mathrm{TCID}_{50} / \mathrm{mL}$ . The observed inhibitory effects are listed below and addressed in the limitations section of the package insert.

- With influenza B/Massachusetts/2/2012 at a concentration near the LoD, competitive inhibitory effects were observed in the presence of $1 \times 10^{3} \mathrm{TCID}_{50} / \mathrm{mL}$ of influenza A/Victoria/361/2011.
- With RSV A/2/Australia/6 at a concentration near the LoD, competitive inhibitory effects were observed in the presence of $1 \times 10^{3} \mathrm{TCID}_{50} / \mathrm{mL}$ of influenza A/Victoria/361/2011.
- With RSV B/Wash/18537/62 at a concentration near the LoD, competitive inhibitory effects were observed in the presence of $1 \times 10^{2} \mathrm{TCID}_{50} / \mathrm{mL}$ of influenza A/Victoria/361/2011 and $1 \times 10^{3} \mathrm{TCID}_{50} / \mathrm{mL}$ of influenza B/Mass/2/2012.

i. Carry-Over/Cross-Contamination

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Single-use, self-contained Xpert Xpress Flu/RSV Assay cartridges were tested for their susceptibility to carry-over contamination when alternating high positive and negative samples are tested in the same GeneXpert module. In this study, negative simulated matrix samples were processed in the same module of a GeneXpert Dx System immediately following a high positive sample comprised of either an influenza A strain (A/Victoria/361/2011) at $2 \times 10^{7} \mathrm{TCID}_{50} / \mathrm{mL}$ or an RSV A strain (A/Long/MD/26) at $1 \times 10^{4} \mathrm{TCID}_{50} / \mathrm{mL}$ spiked into negative simulated matrix. This process was repeated 20 times for a total of 41 runs resulting in 20 positive and 21 negatives for each virus type. A different GeneXpert module was used for each virus type.

All of the 42 negative replicates were correctly reported as “Flu A NEGATIVE; Flu B NEGATIVE; RSV NEGATIVE,” with Ct values of zero.

The 20 high influenza A positive replicates were correctly reported as “Flu A POSITIVE; Flu B NEGATIVE; RSV NEGATIVE,” and the 20 high RSV positive replicates were correctly reported as “Flu A NEGATIVE; Flu B NEGATIVE; RSV POSITIVE.” No evidence of specimen or amplicon carry-over contamination was observed.

## 2. Comparison Studies

### a. Method Comparison with Predicate Device

Performance of the Xpert Xpress Flu/RSV Assay was evaluated against an FDA-cleared nucleic acid comparator method in a prospective clinical study (see below).

### b. Matrix Comparison

A matrix comparison study was conducted to determine whether the Xpert Xpress Flu/RSV Assay performs equivalently with NP swab clinical matrix and simulated nasal matrix. Leftover NP swab clinical matrix samples were supplied from commercial sources and pooled to create an NP swab clinical background matrix. The simulated nasal matrix was formulated with $2.5\%$ (w/v) porcine mucin, $1\%$ (v/v) human whole blood in $0.85\%$ sodium chloride (NaCl) in 1X PBS solution with $15\%$ glycerol, which was then diluted in Copan UTM to a final concentration of $16.7\%$. Both matrices were confirmed to be negative with the Xpert Xpress Flu/RSV Assay before being used to prepare positive samples. Positive samples included two influenza A strains, one influenza B strain, and two RSV strains prepared in both NP clinical swab matrix and simulated matrix at the four analyte concentrations listed in Table 21.

The percent agreement between samples prepared in NP clinical matrix and simulated matrix was $100\%$ (Lower $95\%$ CI = 94.42) using the Wilson-Score method. All 65 samples per strain and per matrix were correctly reported as positive.

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Table 21: Matrix Equivalency Study Samples

| Virus strain | # Replicates tested at each concentration for each matrix type | | | |
| --- | --- | --- | --- | --- |
| | 2X LoD | 5X LoD | 10X LoD | 100X LoD |
| A/Florida/27/2011 | N= 40 | N= 10 | N= 10 | N= 5 |
| A/Victoria/361/2011 | N= 40 | N= 10 | N= 10 | N= 5 |
| B/Mass/2/2012 | N= 40 | N= 10 | N= 10 | N= 5 |
| RSV A/Long/MD/56 | N= 40 | N= 10 | N= 10 | N= 5 |
| RSV B/9320/MA/77 | N= 40 | N= 10 | N= 10 | N= 5 |

c. Fresh versus Frozen Sample Comparison

The performance of the Xpert Xpress Flu/RSV assay with fresh and frozen specimens was evaluated for equivalency by testing individual influenza and RSV strains at three different concentrations representing low positives (2X LoD), moderate positives (5X LoD), and high positives (10X LoD) in pooled negative NP clinical swab matrix. The NP swab samples used in this study were prescreened with the Xpert Xpress Flu/RSV Assay. All the samples that resulted in a negative result ("Flu A NEGATIVE; Flu B NEGATIVE; and RSV NEGATIVE") were pooled for this study. Negative samples consisted of NP swab matrix only.

The influenza and RSV viruses selected for this study were one seasonal influenza A H3N2 strain (A/Victoria/361/2011), one influenza B strain (B/Massachusetts/2/2012), one RSV A strain (RSVA/2/Australia/61), and one RSV B strain (RSV B/Wash/18537/62). Replicates of at least 20 were tested for each specimen condition and virus concentration. All positive and negative samples were tested under three conditions: fresh, after one freeze-thaw cycle, and after two freeze-thaw cycles. For each freeze-thaw cycle, the samples were placed at $-80^{\circ}\mathrm{C}$ for 24 hours after which they were thawed on ice for one hour prior to analysis with the Xpert Xpress Flu/RSV Assay. There was no statistically significant effect in the performance of the Xpert Xpress Flu/RSV Assay between fresh virus dilution and two sequential freeze thaw cycles for positive and negative samples. All positive and negative replicates were correctly identified using the Xpert Xpress Flu/RSV Assay with the exception of one of 20 influenza A replicate tests at 2X the LoD which was reported as "Flu A POSITIVE; Flu B NEGATIVE; RSV POSITIVE" after two freeze thaw cycles by the Xpert Xpress Flu/RSV Assay.

3. Clinical studies

a. Clinical Sensitivity and Specificity

Clinical performance characteristics of the Xpert Xpress Flu/RSV Assay were evaluated at 11 investigational sites in the U.S. during the 2015-2016 influenza season. To be enrolled in the prospective study, patients had to be exhibiting signs and symptoms of respiratory infection at the participating sites, and had to provide informed consent for the collection of an NP swab specimen.

Site personnel collected one NP swab from each patient with the Cepheid Xpert

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Nasopharyngeal Sample Collection Kit. Prospectively collected NP swab specimens were tested with the Xpert Xpress Flu/RSV Assay according to the product insert instructions. Remaining sample specimen was stored at $2 - 8^{\circ}\mathrm{C}$ for no more than 24 hours and shipped to the reference laboratory for comparator testing with an FDA-cleared molecular assay.

Due to a low prevalence of influenza viruses and the difficulty in obtaining fresh influenza and RSV-positive specimens, the specimen population for this study was supplemented with consecutively collected, frozen specimens. To be eligible for the clinical study, specimens had to be collected from individuals with signs and symptoms of respiratory infection whose routine care called for collection of NP swab specimens for influenza and/or RSV testing. Additionally, the leftover frozen sample volume had to have a minimum volume of $1.4\mathrm{mL}$ Copan UTM.

A total of 2580 NP swab specimens were enrolled in the clinical study, of which 2503 were eligible for inclusion. From the 2503 eligible specimens, 2065 (1142 prospective fresh and 923 consecutive frozen specimens) swabs were tested by both the Xpert Xpress Flu/RSV Assay and comparator assay and were included in the final dataset used for the performance analysis. Patient age and gender distribution for each of the 2065 specimens are presented in Tables 23 and 24, respectively.

Table 23: Patient Age Demographics

| Age group (years) | Number | Percent of patients |
| --- | --- | --- |
| ≤5 | 360 | 17.4% |
| 6 to 21 | 226 | 10.9% |
| 22 to 59 | 737 | 35.7% |
| ≥ 60 | 741 | 35.9% |
| Unknown | 1 | < 0.1% |
| Total | 2065 | 100% |

Table 24: Patient Gender Demographics

| Sex | Number | Percent of Patients |
| --- | --- | --- |
| Male | 964 | 46.7% |
| Female | 1101 | 53.3% |
| Total | 2065 | 100% |

Of the Xpert Xpress Flu/RSV 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. 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%.

For the fresh, prospectively collected NP swab specimens, the Xpert Xpress Flu/RSV Assay demonstrated a PPA and NPA of $94.6\%$ and $99.3\%$ for influenza A; $100\%$ and

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99.1% for influenza B; and 100% and 99.6% for RSV, respectively.

For the consecutively collected, frozen NP swab specimens, the Xpert Xpress Flu/RSV Assay demonstrated a PPA and NPA of 100% and 97.2% for influenza A; 100% and 98.2% for influenza B; and 97.9% and 97.7% for RSV, respectively.

For the combined dataset, the Xpert Xpress Flu/RSV Assay demonstrated a PPA and NPA of 98.1% and 98.4% for influenza A; 100% and 98.7% for influenza B; and 98.5% and 98.8% for RSV, respectively.

The performance results of the Xpert Xpress Flu/RSV Assay relative to the comparative assay are summarized in Table 25. Data are stratified by analyte (influenza A, influenza B and RSV) and by sample type (prospective, fresh and consecutive, frozen). Data were shown to be poolable across specimen types, assay lots, study sites, gender, and patient age group.

Table 25. Xpert Xpress Flu/RSV Assay Performance on NP Swab Specimens

| Specimen Type | Target | N | TP | FP | TN | FN | PPA (95% CI) | NPA (95% CI) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Fresh, prospectively collected | Flu A | 1142 | 35 | 8^{a} | 1097 | 2^{b} | 94.6% (82.3-98.5) | 99.3% (98.6-99.6) |
| | Flu B | 1142 | 42 | 10^{c} | 1090 | 0 | 100.0% (91.6-100.0) | 99.1% (98.3-99.5) |
| | RSV | 1142 | 17 | 5^{d} | 1120 | 0 | 100.0% (81.6-100.0) | 99.6% (99.0-99.8) |
| Frozen, consecutively collected | Flu A | 923 | 69 | 24^{e} | 830 | 0 | 100.0% (94.7-100.0) | 97.2% (95.9-98.1) |
| | Flu B | 923 | 36 | 16^{f} | 871 | 0 | 100.0% (90.4-100.0) | 98.2% (97.1-98.9) |
| | RSV | 923 | 47 | 20^{g} | 855 | 1^{h} | 97.9% (89.1-99.6) | 97.7% (96.5-98.5) |
| Combined^{1} | Flu A | 2065 | 104 | 32^{i} | 1927 | 2^{b} | 98.1% (93.4-99.5) | 98.4% (97.7-98.8) |
| | Flu B | 2065 | 78 | 26^{j} | 1961 | 0 | 100.0% (95.3-100.0) | 98.7% (98.1-99.1) |
| | RSV | 2065 | 64 | 25^{k} | 1975 | 1^{h} | 98.5% (91.8-99.7) | 98.8% (98.2-99.2) |

a Testing results by sequencing: 3 of 8 were Flu A Positive; 4 of 8 were Flu A Negative; 1 of 8 insufficient specimen.
b Testing results by sequencing: 2 of 2 were Flu A Negative.
c Testing results by sequencing: 6 of 10 were Flu B Positive; 3 of 10 were Flu B Negative; 1 of 10 insufficient specimen.
d Testing results by sequencing: 0 of 5 were RSV Positive; 3 of 5 were RSV Negative; 2 of 5 insufficient specimen.
e Testing results by sequencing: 8 of 24 were Flu A Positive; 11 of 24 were Flu A Negative; 5 of 24 insufficient specimen.
f Testing results by sequencing: 7 of 16 were Flu B Positive; 3 of 16 were Flu B Negative; 6 of 16 insufficient specimen.
g Testing results by sequencing: 3 of 20 were RSV Positive; 8 of 20 were RSV Negative; 9 of 20 insufficient specimen.
h Testing results by sequencing: 1 of 1 was RSV Negative.
i Testing results by sequencing: 11 of 32 were Flu A Positive; 15 of 32 were Flu A Negative; 6 of 32 insufficient specimen.
j Testing results by sequencing: 13 of 26 were Flu B Positive; 6 of 26 were Flu B Negative; 7 of 26 insufficient specimen.
k Testing results by sequencing: 3 of 25 were RSV Positive; 11 of 25 were RSV Negative; 11 of 25 insufficient specimen.
1 Nine specimens (8 Flu A FP; 9 Flu B FP; 7 RSV FP) were positive for all three targets.

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In addition to the NP swab samples tested above, 102 pre-selected frozen NP swab specimens were also tested. The results of this testing were analyzed separately and are as follows: the Xpert Xpress Flu/RSV Assay demonstrated a PPA and NPA of $100\%$ and $95.8\%$ , for influenza A, respectively; $100\%$ and $94.5\%$ for influenza B, respectively; and $100\%$ and $95.7\%$ , for RSV, respectively, relative to the comparator assay.

# b. Clinical Cut-off

Please refer to the Assay Cut-Off section of this document.

# c. Expected Values/Reference Range

Expected prevalence values of influenza A, influenza B and RSV infections were calculated using the data acquired from the 2065 prospectively and consecutively collected NP swab specimens tested with the Xpert Xpress Flu/RSV Assay. Results are shown in Table 26 below.

Table 26: Expected Values for Influenza A, Influenza B and RSV by Xpert Xpress Flu/RSV Assay in NP swabsa

| Age group (years) | Number of patients | Flu A | | Flu B | | RSV | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | Number positive | Positivity rate | Number positive | Positivity rate | Number positive | Positivity rate |
| ≤5 | 360 | 25 | 6.9% | 18 | 5.0% | 28 | 7.8% |
| 6-21 | 226 | 19 | 8.4% | 31 | 13.7% | 8 | 3.5% |
| 22-59 | 737 | 57 | 7.7% | 30 | 4.1% | 23 | 3.1% |
| ≥60 | 741 | 35 | 4.7% | 25 | 3.4% | 30 | 4.0% |
| Unknown | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| Total | 2065 | 136 | 6.6% | 104 | 5.0% | 89 | 4.3% |

a Eleven subjects had multi-infections by Xpert Xpress Flu/RSV Assay and are therefore counted more than once in this table: Flu A, Flu B &amp; RSV POS [(7); 1 sample RSV POS by comparator assay; 1 sample Flu A POS by comparator assay; 5 samples NEG for all targets by comparator assay]; Flu A &amp; RSV POS [(3); 1 sample Flu A POS by comparator assay; 2 samples NEG for all targets by comparator assay], and Flu A &amp; Flu B POS [(1); Flu A POS by comparator assay].

# N. Instrumentation/System Description

# 1. Instrument Name:

GeneXpert Dx Systems (GX-I, GX-II, GX-IV, GX-XVI) with GeneXpert Dx software version 4.6a or higher

GeneXpert Infinity-48 System with Xpertise software version 4.6a

GeneXpert Infinity-80 and Infinity-48s Systems with Xpertise software version 6.2a or higher

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2. System Description:

The GeneXpert Instrument System family (GeneXpert Dx and Infinity Systems) automates and integrates sample purification, nucleic acid amplification and detection of target sequences within compatible, assay-specific, single-use cartridges. The instrument systems each contain a computer and preloaded software for running tests and viewing the results.

3. Software:

FDA has reviewed applicant’s Hazard Analysis and software development processes for this line of product types.

Yes ☐ X ☐ or No ☐

4. Level of Concern

Moderate

5. Software Description

The GeneXpert Instrument Systems are provided with a computer, preloaded with software for running tests and viewing results. Each instrument (Dx and Infinity) contains random access, closed-system, computer-based software and embedded firmware which run dedicated microprocessor-controlled modules to integrate sample preparation, amplification and real-time detection in a single system.

The GeneXpert Infinity modules contain extra robotic features for cartridge handling. The Xpertise software utilized by the Infinity Systems is the user interface and provides the ordering of tests as well as automates loading and unloading of cartridges into GeneXpert modules within the system. The Xpertise user interface builds upon the existing core software functionality for handling GeneXpert modules for cartridge fluidics control, temperature control, optics control, and data analysis by the addition of automation handling for the robotic arm.

6. Specimen Identification

Specimens are manually loaded into the Xpert Xpress Flu/RSV Assay cartridge by the user. The user can then either scan or type the sample and patient ID into the system. Prior to placing the cartridge into the GeneXpert Instrument System, the barcode on the Xpert Xpress Flu/RSV Assay cartridge is scanned. The information contained in the assay barcode is utilized by the software to run the appropriate assay definition file (ADF). If an assay is being run that does not already exist in the GeneXpert database, the user must import the ADF before starting the test.

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7. Calibration

Not required.

8. Quality Control

## Sample Processing Control

The sample processing control (SPC) is a non-infectious armored RNA pseudovirus that is included in each cartridge to verify that adequate processing of the sample has occurred. The SPC verifies that nucleic acids have been released from the target viruses if the organism is present and detects specimen-associated PCR and RT-PCR inhibitors. The SPC should be POSITIVE in a sample that is negative for all three influenza A, influenza B and RSV target analytes, and can be NEGATIVE or POSITIVE in a sample containing detectable levels of one or more of the target analytes.

## Probe Check Control

Before the start of the amplification process, the GeneXpert Instrument Systems measures the fluorescence signal from the probes to monitor bead rehydration, reaction tube filling, probe integrity, and dye stability. All assay reagents must be present and intact for the PCC to pass the validated acceptance criteria. If any of the PCC conditions fail, the result is reported as an ERROR and the test must be repeated using a new assay cartridge.

## O. Other supportive Instrument Characteristics Data Not Covered in the "Performance Characteristics" Section Above

N/A

## P. Proposed Labeling

The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

## Q. Conclusion

The submitted information in the premarket notification is complete and supports a substantial equivalence decision.

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