XPERT FLU

K123191 · Cepheid · OCC · Dec 21, 2012 · Microbiology

Device Facts

• Record ID: K123191
• Device Name: XPERT FLU
• Applicant: Cepheid
• Product Code: OCC · Microbiology
• Decision Date: Dec 21, 2012
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3980
• Device Class: Class 2

Intended Use

The Cepheid Xpert® Flu Assay, performed on the GeneXpert® Instrument Systems, is an automated, multiplex real-time RT-PCR assay intended for the in vitro qualitative detection and differentiation of influenza A, influenza B and 2009 H1N1 influenza viral RNA. The Xpert Flu Assay uses nasal aspirates/washes and nasopharyngeal swab specimens collected from patients with signs and symptoms of respiratory infection in conjunction with clinical and epidemiological risk factors. The Xpert Flu Assay is intended as an aid in the diagnosis of influenza. Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions. Performance characteristics for influenza A were established during the 2009-2010 influenza season when 2009 H1N1 influenza was the predominant influenza A virus in circulation. Performance characteristics for influenza A were confirmed when influenza A/H3 and influenza A/2009 H1N1 were the predominant influenza A viruses in circulation (2009-2010, 2010-2011 and 2011-2012). When other influenza A viruses are emerging, performance characteristics may vary. If infection with a novel influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to state or local health department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.

Device Story

The Xpert Flu Assay is an automated, in vitro diagnostic test for qualitative detection and differentiation of influenza A, influenza B, and 2009 H1N1 viral RNA. It utilizes single-use, multi-chambered fluidic cartridges containing PCR reagents. The user loads the specimen (nasal aspirate/wash or nasopharyngeal swab) into the cartridge and inserts it into the GeneXpert Instrument System. The system automates sample preparation, nucleic acid extraction, and real-time RT-PCR amplification and detection. Each module features a syringe drive for fluid movement, an ultrasonic horn for cell lysis, and an I-CORE thermocycler. The process is completed in 75 minutes. Results are provided via the system user interface to healthcare providers to aid in influenza diagnosis. The device is intended for use in clinical settings by laboratory personnel (CLIA moderate or high complexity).

Clinical Evidence

Retrospective clinical study of 166 archived clinical samples (NP swabs and NA/W) compared to the predicate device. Results demonstrated high positive and negative percent agreement for Influenza A, 2009 H1N1, and Influenza B. Contrived specimens (n=10) spiked at LoD were also tested and correctly identified. Analytical performance included LoD studies for 12 strains and analytical specificity testing against 40 common respiratory pathogens/flora, showing 100% specificity.

Technological Characteristics

Multiplex real-time RT-PCR assay; integrated sample preparation and detection in single-use fluidic cartridges; utilizes GeneXpert® Instrument Systems (1-80 modules); ICORE® thermocycler; ultrasonic horn for cell lysis; automated fluidic movement via syringe drive/rotary valve; software-controlled; qualitative output.

Indications for Use

Indicated for patients with signs and symptoms of respiratory infection; used as an aid in the diagnosis of influenza A, influenza B, and 2009 H1N1 influenza viral RNA; requires clinical and epidemiological risk factor assessment.

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

• Cepheid Xpert® Flu Assay (K120911)

Submission Summary (Full Text)

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K123191

#### 510(k) Summary

| As required by 21 CFR Section 807.92(c). | DEC 2 1 2012 |
|--------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Submitted by: | Cepheid<br>904 Caribbean Drive<br>Sunnyvale, CA 94089<br>Phone number: [phone]<br>Fax number: [phone] |
| Contact: | Kerry J. Flom, Ph.D. |
| Date of Preparation: | October 9, 2012 |
| Device: | |
| Trade name: | Xpert® Flu |
| Common name: | Xpert Flu Assay |
| Type of Test: | Automated, multiplex real-time reverse transcription-<br>polymerase chain reaction (rRT-PCR) assay intended for the<br><i>in vitro</i> qualitative detection and differentiation of influenza<br>A, influenza B and 2009 H1N1 influenza viral RNA. |
| Regulation number/<br>Classification name: | 866.3332/Reagents for detection of specific novel influenza A<br>viruses, and<br>866.3980/Respiratory viral panel multiplex nucleic acid assay |
| Product code: | OQW, OCC, OOI |
| Classification<br>Advisory Panel | Microbiology (83) |
| Predicate Device: | Cepheid Xpert Flu Assay [510(k) #K120911] |

### Device Description:

The Xpert Flu Assay is a rapid, automated in vitro diagnostic test for qualitative detection and differentiation of influenza A, influenza B and influenza A, subtype 2009 HIN1. The assay is performed on the Cepheid GeneXpert Instrument Systems. The GeneXpert Instrument Systems automate and integrate sample purification, nucleic acid amplification, and detection of the target sequence in simple or complex samples using real-time PCR and RT-PCR assays. The systems require the use of single-use disposable cartridges that hold the PCR reagents and host the PCR process. Because the cartridges are self-contained and specimens never come into contact with working parts of the instrument modules, cross-contamination between samples is minimized.

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The Xpert Flu Assay includes reagents for the detection and differentiation of influenza A, influenza B and influenza A, subtype 2009 HIN1 directly from nasal aspirates/washes (NA/W) and nasopharyngeal (NP) swab specimens from patients suspected of having influenza. A Sample Processing Control (SPC) and a Probe Check Control (PCC) are also included. The SPC is present to control for adequate processing of the target viruses and to monitor the presence of inhibitors in the PCR reaction. The Probe Check Control (PCC) verifies reagent rehydration, PCR tube filling in the cartridge, probe integrity and dye stability.

The liquid specimen (NA/W) or swab specimen (NP) is collected according to the institution's standard procedures and placed into Universal Transport Medium (3mL UTM tubes). Following a brief mixing by inverting the UTM tube five times, the eluted material and one single-use reagent (Reagent 1), that is provided with the assay, are transferred to different, uniquely-labeled chambers of the disposable fluidic cartridge (the Xpert Flu cartridge). The user initiates a test from the system user interface and places the cartridge into the GeneXpert instrument platform, which performs hands-off realtime, multiplex polymerase chain reaction (PCR) for detection of DNA. In this platform, additional sample preparation, amplification, and real-time detection are all fullyautomated and completely integrated.

The single-use, multi-chambered fluidic cartridges are designed to complete sample preparation and real-time PCR for detection and differentiation of influenza A. influenza B and influenza A, subtype 2009 H1N1 in 75 minutes. The GeneXpert Instrument Systems, which consist of the GeneXpert Dx System, the GeneXpert Infinity-48 System, and the GeneXpert Infinity-80 System, have 1 to 80 randomly accessible modules that are each capable of performing separate sample preparation and real-time PCR tests. Each module contains a syringe drive for dispensing fluids (i.e., the syringe drive activates the plunger that works in concert with the rotary valve in the cartridge to move fluids between chambers), an ultrasonic horn for lysing cells or spores, and a proprietary I-CORE® thermocycler for performing real-time PCR and detection.

#### Device Intended Use:

The Cepheid Xpert® Flu Assay, performed on the GeneXpert® Instrument Systems, is an automated, multiplex real-time RT-PCR assay intended for the in vitro qualitative detection and differentiation of influenza A, influenza B and 2009 H1N1 influenza viral RNA. The Xpert Flu Assay uses nasal aspirates/washes and nasopharyngeal swab specimens collected from patients with signs and symptoms of respiratory infection in conjunction with clinical and epidemiological risk factors. The Xpert Flu Assay is intended as an aid in the diagnosis of influenza.

Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions.

Performance characteristics for influenza A were established during the 2009-2010 influenza season when 2009 HIN1 influenza was the predominant influenza A virus in circulation. Performance characteristics for influenza A were confirmed when influenza

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A/H3 and influenza A/2009 H1N1 were the predominant influenza A viruses in circulation (2009-2010, 2010-2011 and 2011-2012). When other influenza A viruses are emerging, performance characteristics may vary.

If infection with a novel influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to state or local health department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.

#### Substantial Equivalence:

The Xpert Flu Assay is substantially equivalent to the current Xpert Flu Assay [510(k) #K120911].

Similarities and differences between the Cepheid Xpert Flu Assay and the predicate device are shown in Table 5.1.

A clinical comparison study was conducted using prospectively collected archived and/or pre-selected banked specimens to compare the modified Xpert Flu Assay performance to the previously cleared Xpert Flu Assay.

| | Device | Predicate |
|--------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Item | Modified Cepheid Xpert Flu | Cepheid Xpert Flu |
| 510(k) Number | #K123191 | #K120911 |
| Regulation | Same | 866.3332 and 866.3980 |
| Product Code | Same | OQW, OCC, OOI |
| Device Class | Same | II |
| Technology/<br>Detection | Same | Multiplex real time RT/PCR |
| Intended Use | The Cepheid Xpert® Flu Assay,<br>performed on the GeneXpert®<br>Instrument Systems, is an automated,<br>multiplex real-time RT-PCR assay<br>intended for the <i>in vitro</i> qualitative<br>detection and differentiation of<br>influenza A, influenza B and 2009<br>H1N1 influenza viral RNA. The Xpert<br>Flu Assay uses nasal aspirates/washes<br>and nasopharyngeal swab specimens<br>collected from patients with signs and<br>symptoms of respiratory infection in | The Cepheid Xpert® Flu Assay is an<br>automated, multiplex real-time RT-PCR<br>assay intended for the <i>in vitro</i> qualitative<br>detection and differentiation of influenza<br>A, influenza B and 2009 H1N1 influenza<br>viral RNA. The Xpert Flu Assay uses<br>nasal aspirates/washes and<br>nasopharyngeal swab specimens collected<br>from patients with signs and symptoms of<br>respiratory infection in conjunction with<br>clinical and epidemiological risk factors. |

| | Table 5.1: Comparison of Similarities and Differences of the modified Xpert Flu | | | |
|--------------------------------------------------|----------------------------------------------------------------------------------|--|--|--|
| Assay with the Predicate Device, Xpert Flu Assay | | | | |

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| | Device | Predicate |
|------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Item | Modified Cepheid Xpert Flu<br>conjunction with clinical and<br>epidemiological risk factors. The Xpert<br>Flu Assay is intended as an aid in the<br>diagnosis of influenza.<br>Negative results do not preclude<br>influenza virus infection and should not<br>be used as the sole basis for treatment or<br>other patient management decisions.<br>Performance characteristics for<br>influenza A were established during the<br>2009-2010 influenza season when 2009<br>H1N1 influenza was the predominant<br>influenza A virus in circulation.<br>Performance characteristics for<br>influenza A were confirmed when<br>influenza A/H3 and influenza A/2009<br>H1N1 were the predominant influenza<br>A viruses in circulation (2009-2010,<br>2010-2011 and 2011-2012). When<br>other influenza A viruses are emerging,<br>performance characteristics may vary.<br>If infection with a novel influenza A<br>virus is suspected based on current<br>clinical and epidemiological screening<br>criteria recommended by public health<br>authorities, specimens should be<br>collected with appropriate infection<br>control precautions for novel virulent<br>influenza viruses and sent to state or<br>local health department for testing. Viral<br>culture should not be attempted in these<br>cases unless a BSL 3+ facility is<br>available to receive and culture<br>specimens. | The Xpert Flu Assay is intended as an aid<br>in the diagnosis of influenza.<br>Negative results do not preclude influenza<br>virus infection and should not be used as<br>the sole basis for treatment or other<br>patient management decisions.<br>Performance characteristics for influenza<br>A were established during the 2009-2010<br>influenza season when 2009 H1N1<br>influenza was the predominant influenza<br>A virus in circulation. When other<br>influenza A viruses are emerging,<br>performance characteristics may vary.<br>If infection with a novel influenza A virus<br>is suspected based on current clinical and<br>epidemiological screening criteria<br>recommended by public health<br>authorities, specimens should be collected<br>with appropriate infection control<br>precautions for novel virulent influenza<br>viruses and sent to state or local health<br>department for testing. Viral culture<br>should not be attempted in these cases<br>unless a BSL 3+ facility is available to<br>receive and culture specimens. |
| Indication for Use | Same | Patients with signs and symptoms of<br>respiratory infection in conjunction with<br>clinical and epidemiological risk factors. |
| Assay Targets | Same | Influenza A, influenza B, and influenza<br>A, subtype 2009 H1N1 |
| Specimen Types | Same | Nasal aspirates/washes (NA/W) and<br>Nasopharyngeal (NP) swabs |
| Technological<br>Principles | Same | RT/PCR |
| Nucleic Acid<br>Extraction | Same | Yes |
| | Device | Predicate |
| Item | Modified Cepheid Xpert Flu | Cepheid Xpert Flu |
| Extraction<br>Methods | Same | Sample preparation integrated in<br>GeneXpert Cartridge and GeneXpert<br>Instrumentation System |
| Assay Results | Same | Qualitative |
| Instrument System | Same | Cepheid GeneXpert® Instrument Systems |
| Assay Controls | Same | Encapsulated (armored) RNA<br>pseudovirus as a sample processing<br>control.<br>Available but not provided are inactivated<br>virus controls for Flu A/B and Flu A 2009<br>H1N1 as external positive controls and<br>Coxsackie virus as an external negative<br>control. |
| Rapid test results | Same | Total 75 minutes for sample preparation<br>and rRT-PCR |
| Primers and<br>probes for<br>influenza A,<br>influenza B, and<br>influenza A<br>subtype 2009<br>H1N1 | Same plus an additional primer for<br>Influenza A | Original primers and probes to detect the<br>presence of nucleic acid sequences of<br>influenza A, influenza B, and influenza,<br>subtype 2009 H1N1 |
| Laboratory Users | Same | CLIA Moderate or High Complexity |

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#### Non-Clinical Studies:

#### Analytical Sensitivity

#### Analytical Reactivity (Inclusivity)

The analytical reactivity of the Xpert Flu Assay was evaluated against forty (40) strains of influenza A (HINI, H3N2, H5N2, H5N1 and H7N3 subtypes), influenza A 2009 HINI and influenza B. Of these, influenza A subtype HINI (11), influenza A subtype H3N2 (9), influenza A subtype 2009 H1N1 (6), influenza A subtype H5N1 (1), influenza A subtype H5N2 (1), influenza A subtype H7N3 (1), and influenza B (11) were included. Twelve of the 40 influenza strains evaluated in this study were tested at the LoD concentration while all remaining strains were tested using viral stocks at 5-250 TCID50 mL. Three (3) replicates were tested for each strain. Results are shown in Table 5.2.

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| Viral Strain | Concentration<br>(TCID50/mL) | Influenza<br>A | Influenza A<br>2009 H1N1 | Influenza<br>B |
|----------------------------------------------------|------------------------------|----------------|--------------------------|----------------|
| Influenza A/Denver/1/57 (H1N1)a | 250 | + | - | - |
| Influenza A/Hawaii/15/2001 (H1N1)a | 50 | + | - | - |
| Influenza A/Mal/302/54 (H1N1)a | 50 | + | - | - |
| Influenza A/New Jersey/8/76 (H1N1)a | 250 | + | - | - |
| Influenza A/NWS/33 (H1N1)a | 5 | + | - | - |
| Influenza A/PR/8/34 (H1N1)a | 50 | + | - | - |
| Influenza A/Taiwan/42/06 (H1N1)a | 50 | + | - | - |
| Influenza A/Swine/1976/31 (Swine<br>H1N1)a | 250 | + | - | - |
| Influenza A/Swine/Iowa/15/30 (Swine<br>H1N1)a | 50 | + | - | - |
| Influenza A/Brisbane/59/07 (H1N1)a, b | 1 | + | - | - |
| Influenza A/NewCaledonia/20/1999<br>(H1N1)a,b | 5 | + | - | - |
| Influenza A/Brisbane/10/07 (H3N2)b | 1.25 | + | - | - |
| Influenza A/Victoria/361/2011<br>(H3N2)b | 1.25 | + | - | - |
| Influenza A/Victoria/3/75 (H3N2) | 250 | + | - | - |
| Influenza A/Aichi2/68 (H3N2) | 50 | + | - | - |
| Influenza A/Hong Kong/8/68 (H3N2) | 50 | + | - | - |
| Influenza A/NewYork/55/2004 (H3N2) | 50 | + | - | - |
| Influenza A/Port Chalmers/1/73<br>(H3N2) | 50 | + | - | - |
| Influenza A/Wisconsin/67/05 (H3N2)b | 10 | + | - | - |
| Influenza A/Perth/16/2009 (H3N2) | 50 | + | - | - |
| Influenza A/SwineNY/01/2009<br>(09 H1N1)b | 25 | + | + | - |
| Influenza A/SwineNY/02/2009<br>(09 H1N1)b | 1.25 | + | + | - |
| Influenza A/SwineNY/03/2009<br>(09 H1N1)b | 1.25 | + | + | - |
| Viral Strain | Concentration<br>(TCID50/mL) | Influenza<br>A | Influenza A<br>2009 H1N1 | Influenza<br>B |
| Influenza A/California/7/2009<br>(09 H1N1) | 5 | + | + | - |
| Influenza A/Canada/6294<br>(09 H1N1) | 50 | + | + | - |
| Influenza A/Wisconsin/629-S1<br>(09 H1N1) | 1 | + | + | - |
| Influenza A/Mallard/WI/34/75 (H5N2) | 3 pg/µL | + | - | - |
| Influenza A/Anhui/02/2005/PR8-<br>IBCDC-RG5 (H5N1) | 0.122 pg/µL | + | - | - |
| Influenza A/chicken/NJ/15086-3/94<br>(H7N3) | 50 pg/µL | + | - | - |
| Influenza B/Allen/45 | 50 | - | - | + |
| Influenza B/Florida/02/06b | 5 | - | - | + |
| Influenza B/Florida/04/06 | 50 | - | - | + |
| Influenza B/Florida/07/04b | 5 | - | - | + |
| Influenza B/GL/1739/54 | 50 | - | - | + |
| Influenza B/Hong Kong/5/72 | 50 | - | - | + |
| Influenza B/Lee/40 | 50 | - | - | + |
| Influenza B/Malaysia/2506/04 | 50 | - | - | + |
| Influenza B/Maryland/1/59 | 5 | - | - | + |
| Influenza B/Panama/45/90 | 250 | - | - | + |
| Influenza B/Taiwan/2/62 | 50 | - | - | + |

# Table 5.2: Analytical Reactivity (Inclusivity) Results of the Xpert Flu Assay

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ªSeasonal influenza A H1N1 (not 2009 H1N1)

b Strains (12) used in analytical LOD study and tested at limit of detection

°Concentration expressed in picograms/UL

#### Analytical Sensitivity

#### Limit of Detection

Studies were performed to determine the analytical limit of detection (LoD) of 2 seasonal influenza A (H1N1), 3 seasonal influenza A (H3N2), 5 influenza A 2009 H1N1 and 2 influenza B strains diluted into a surrogate nasopharyngeal matrix. The surrogate matrix consisted of 1% human blood, 2.5% mucin and 0.85% sodium chloride. The LoD is defined as the lowest concentration (tissue culture infective dose [TCID]50/mL) per sample that can be reproducibly distinguished from negative samples with 95%

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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 was determined empirically as the first concentration that had 19/20 or 20/20 positive results. The LoD values for each strain tested are summarized in Tables 5.3 -5.6.

| Strain ID - Influenza A subtype<br>H1N1 | Confirmed LoD<br>(TCID50/mL)<br>(at least 19/20 positive) |
|-----------------------------------------|-----------------------------------------------------------|
| Influenza A/H1/Brisbane/59/07 | 1 (20/20) |
| Influenza A/H1/New<br>Caledonia/20/1999 | 5 (20/20) |

Table 5.3: Confirmed LoD (TCID50/mL) – Seasonal Influenza A H1N1

Table 5.4: Confirmed LoD (TCID50/mL) – Seasonal Influenza A H3N2

| Strain ID - Influenza A subtype<br>H3N2 | Confirmed LOD<br>(TCID50/mL) (at least 19/20 positive) |
|-----------------------------------------|--------------------------------------------------------|
| Influenza A/H3/Brisbane/10/07 | 1.25 (20/20) |
| Influenza A/Victoria/361/2011 | 1.25 (20/20) |
| Influenza A/H3/Wisconsin/67/05 | 10 (20/20) |

Table 5.5: Confirmed LoD (TCID50/mL) – Influenza A 2009 H1N1

| Strain ID - Influenza A<br>subtype 2009 H1N1 | Confirmed LOD<br>(TCID50/mL) (at least 19/20 positive) |
|----------------------------------------------|--------------------------------------------------------|
| Influenza A/SwineNY/01/2009<br>(Lot #1) | 25 (19/20) |
| Influenza A/SwineNY/01/2009<br>(Lot #2) | 0.125 (19/20) |
| Influenza A/SwineNY/02/2009 | 1.25 (20/20) |
| Influenza A/SwineNY/03/2009 | 1.25 (20/20) |
| Influenza A/Canada/6294/2009 | 35 (19/20) |
| Influenza A/WI/629-S1/2009<br>(D00015) | 1 (20/20) |

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| Strain ID - Influenza B | Confirmed LOD<br>(TCID50/mL) (at least 19/20 positive) |
|---------------------------|--------------------------------------------------------|
| Influenza B/Florida/02/06 | 2 (19/20) |
| Influenza B/Florida/07/04 | 5 (20/20) |

Table 5.6: Confirmed LoD (TCID50/mL) - Influenza B

### Analytical Specificity (Exclusivity)

The analytical specificity of the Xpert Flu Assay was evaluated by testing a panel of 40 cultures consisting of 18 viral, 21 bacterial, and one yeast representing common respiratory pathogens or those potentially encountered in the nasopharynx. Three replicates of all bacterial and yeast strains were tested at concentrations ≥10° CFU/mL. Three replicates of all viruses were tested at concentrations ≥10° TCID30/mL. Purified nucleic acids (copies/mL) were tested for one virus strain (Cytomegalovirus) and one bacterial strain (Bordetella pertussis). Positive and negative controls were included in the study. The analytical specificity was 100%. Results are shown in Table 5.7.

| | Table 5.7: Analytical Specificity for Xpert Flu Assaya | |
|--|--------------------------------------------------------|--|
| | | |

| Strain | Concentration<br>(per Cartridge) | Influenza<br>A | Influenza A<br>2009 H1N1 | Influenza<br>B |
|-------------------------------------------------|----------------------------------|----------------|--------------------------|----------------|
| Positive Control 1 – Influenza<br>A/Influenza B | N/Ab | + | - | + |
| Positive Control 2 - Influenza A<br>2009 H1N1 | N/Ab | + | + | - |
| Negative Control | N/Ab | - | - | - |
| Adenovirus Type 7A | $1.1 x10^5$ TCID50/mL | - | - | - |
| Adenovirus Type 1 | $1.0 x10^7$ TCID50/mL | - | - | - |
| Human Coronavirus 229E | $2.5x10^4$ TCID50/mL | - | - | - |
| Human Coronavirus OC43 | $5.6 x10^4$ TCID50/mL | - | - | - |
| Cytomegalovirusc | $4.7x10^7$ Copies /mL | - | - | - |
| Enterovirus Type 71 | $3.5 x10^5$ TCID50/mL | - | - | - |
| Epstein-Barr Virus | $7.1x10^8$ TCID50/mL | - | - | - |
| Parainfluenzavirus Type 1 | $1.1 x10^5$ TCID50/mL | - | - | - |
| Strain | Concentration<br>(per Cartridge) | Influenza<br>A | Influenza A<br>2009 H1N1 | Influenza<br>B |
| Parainfluenzavirus Type 2 | 3.1 x107 TCID50/mL | - | - | - |
| Parainfluenzavirus Type 3 | 1.9 x106 TCID50/mL | - | - | - |
| Measles Virus | 6.3 x104 TCID50/mL | - | - | - |
| Human Metapneumovirus | 3.8 x105 TCID50/mL | - | - | - |
| Mumps Virus | 6.3 x106 TCID50/mL | - | - | - |
| Respiratory Syncytial Virus A | 5.3 x107 TCID50/mL | - | - | - |
| Respiratory Syncytial Virus B | 1.2 x107 TCID50/mL | - | - | - |
| Human HSV Type 1 | 3.1 x106 TCID50/mL | - | - | - |
| Human Rhinovirus Type 4 | 1.2 x105 TCID50/mL | - | - | - |
| Echovirus 11 | 3.3 x108 TCID50/mL | - | - | - |
| Bordetella pertussisc | 5000 ng/mL | - | - | - |
| Chlamydia pneumoniae | 5 x106 CFU/mL | - | - | - |
| Corynebacterium xerosis | 1x106 CFU/mL | - | - | - |
| Escherichia coli | 1x106 CFU/mL | - | - | - |
| Proteus vulgaris | 1x106 CFU/mL | - | - | - |
| Proteus mirabilis | 1x106 CFU/mL | - | - | - |
| Klebsiella pneumoniae | 1x106 CFU/mL | - | - | - |
| Haemophilus influenzae | 1x106 CFU/mL | - | - | - |
| Lactobacillus crispatus | 1x106 CFU/mL | - | - | - |
| Legionella pneumophila | 1x106 CFU/mL | - | - | - |
| Moraxella catarrhalis | 1x106 CFU/mL | - | - | - |
| Mycobacterium tuberculosis<br>(BCG strain) | 1x106 CFU/mL | - | - | - |
| Mycoplasma pneumoniae | 1x106 CFU/mL | - | - | - |
| Neisseria meningitides | 1x106 CFU/mL | - | - | - |
| Neisseria cinneria | 1x106 CFU/mL | - | - | - |
| Pseudomonas aeruginosa | 1x106 CFU/mL | - | - | - |
| Strain | Concentration<br>(per Cartridge) | Influenza<br>A | Influenza A<br>2009 H1N1 | Influenza<br>B |
| Staphylococcus aureus | 1x106 CFU/mL | - | - | - |
| Staphylococcus epidermidis | 1x106 CFU/mL | - | - | - |
| Streptococcus pneumoniae | 1x106 CFU/mL | - | - | - |
| Streptococcus pyogenes | 1x106 CFU/mL | - | - | - |
| Streptococcus salivarius | 1x106 CFU/mL | - | - | - |
| Candida albicans | 1x106 CFU/mL | - | - | - |

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4Cross reactivity with other swine-origin strains has not been tested. Concentration not available because these are inactivated viruses.

"Nucleic acid was tested for Cytomegalovirus and Bordetella pertussis.

### Interfering Substances

Potentially interfering substances that may be present in the nasopharynx were evaluated on representative Flu strains directly relative to the performance of the Xpert 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. These substances are listed in Table 5.8 with active ingredients and concentrations tested shown. Highly viscous samples resulting from the addition of 1.5% (w/v) and 2.5% (w/v) mucin yielded false-negative test results from the Xpert Flu Assay. Inhibition of the Xpert Flu Assay was also observed fr…