← Product Code [QOF](/submissions/MI/subpart-d%E2%80%94serological-reagents/QOF) · K231481

# Xpert Xpress CoV-2/Flu/RSV plus (K231481)

_Cepheid® · QOF · Aug 17, 2023 · Microbiology · SESE_

**Canonical URL:** https://fda.innolitics.com/submissions/MI/subpart-d%E2%80%94serological-reagents/QOF/K231481

## Device Facts

- **Applicant:** Cepheid®
- **Product Code:** [QOF](/submissions/MI/subpart-d%E2%80%94serological-reagents/QOF.md)
- **Decision Date:** Aug 17, 2023
- **Decision:** SESE
- **Submission Type:** Traditional
- **Regulation:** 21 CFR 866.3981
- **Device Class:** Class 2
- **Review Panel:** Microbiology

## Indications for Use

The Xpert® Xpress CoV-2/Flu/RSV plus test, performed on the GeneXpert® Dx and GeneXpert® Infinity Systems, is an automated multiplexed real-time reverse transcriptase polymerase chain reaction (RT-PCR) test intended for use in the simultaneous in vitro qualitative detection and differentiation of severe acute respiratory syndrome coronavirus (SARS-CoV-2), influenza A, influenza B, and/or respiratory syncytial virus (RSV) viral RNA in nasopharyngeal swab and anterior nasal swab specimens collected from individuals with signs and symptoms of respiratory tract infection. Clinical signs and symptoms of respiratory tract infection due to SARS-CoV-2, influenza A, influenza B, and RSV can be similar. The Xpert Xpress CoV-2/Flu/RSV plus is intended for use in the differential detection of SARS-CoV-2, influenza A, influenza B and/or RSV RNA and aids in the diagnosis of COVID-19, influenza and/or RSV infections if used in conjunction with other clinical and epidemiological information, and laboratory findings. SARS-CoV-2, influenza B, and RSV viral RNA are generally detectable in nasopharyngeal swab and anterior nasal swab specimens during the acute phase of infection. Positive results are indicative of the presence of the identified virus, but do not rule out bacterial infection or co-infection with other pathogens not detected by the test. The agent(s) detected by the Xpert Xpress CoV-2/Flu/RSV plus test may not be the definite cause of disease. Negative results do not preclude SARS-CoV-2, influenza A, influenza B, and/or RSV infection. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.

## Device Story

Device is a multiplexed, real-time RT-PCR assay for qualitative detection/differentiation of SARS-CoV-2, influenza A, influenza B, and RSV viral RNA. Input: nasopharyngeal or anterior nasal swab specimens collected in VTM or eNAT. Process: automated sample preparation (lysis, RNA extraction) and real-time RT-PCR amplification/detection within a self-contained, single-use cartridge. Instrument: GeneXpert Dx or Infinity systems. Output: qualitative results displayed on instrument software. Used in clinical settings by laboratory personnel. Aids diagnosis of COVID-19, influenza, and RSV infections when used with other clinical/epidemiological data. Benefits patient by providing rapid (approx. 36 min) differential diagnosis of common respiratory pathogens.

## Clinical Evidence

Prospective multi-center clinical study (n=6,121 evaluable specimens) comparing subject device to FDA-cleared molecular respiratory panels. Performance evaluated for SARS-CoV-2 (n=5,051), Flu A, Flu B, and RSV (n=5,954). SARS-CoV-2 PPA/NPA: 97.1%/98.2% (NPS), 98.2%/98.8% (NS). Flu A PPA/NPA: 99.0%/99.1% (NPS), 98.0%/99.3% (NS). Flu B PPA/NPA: 96.6%/100% (NPS), 100%/99.9% (NS). RSV PPA/NPA: 98.6%/100% (NPS), 93.3%/100% (NS).

## Technological Characteristics

Multiplexed real-time RT-PCR assay. Materials: single-use plastic cartridge with integrated chambers for sample processing and amplification. Sensing: fluorescent signal detection via I-CORE thermocycler. Energy: electrical (GeneXpert instrument). Connectivity: standalone or networked instrument systems. Sterilization: not specified. Software: automated interpretation of fluorescent signals via embedded algorithms.

## Regulatory Identification

A device to detect and identify nucleic acid targets in respiratory specimens from microbial agents that cause the SARS-CoV-2 respiratory infection and other microbial agents when in a multi-target test is an in vitro diagnostic device intended for the detection and identification of SARS-CoV-2 and other microbial agents when in a multi-target test in human clinical respiratory specimens from patients suspected of respiratory infection who are at risk for exposure or who may have been exposed to these agents. The device is intended to aid in the diagnosis of respiratory infection in conjunction with other clinical, epidemiologic, and laboratory data or other risk factors.

## Special Controls

*Classification.* Class II (special controls). The special controls for this device are:(1) The intended use in the labeling required under § 809.10 of this chapter must include a description of the following: Analytes and targets the device detects and identifies, the specimen types tested, the results provided to the user, the clinical indications for which the test is to be used, the specific intended population(s), the intended use locations including testing location(s) where the device is to be used (if applicable), and other conditions of use as appropriate.
(2) Any sample collection device used must be FDA-cleared, -approved, or -classified as 510(k) exempt (standalone or as part of a test system) for the collection of specimen types claimed by this device; alternatively, the sample collection device must be cleared in a premarket submission as a part of this device.
(3) The labeling required under § 809.10(b) of this chapter must include:
(i) A detailed device description, including reagents, instruments, ancillary materials, all control elements, and a detailed explanation of the methodology, including all pre-analytical methods for processing of specimens;
(ii) Detailed descriptions of the performance characteristics of the device for each specimen type claimed in the intended use based on analytical studies including the following, as applicable: Limit of Detection, inclusivity, cross-reactivity, interfering substances, competitive inhibition, carryover/cross contamination, specimen stability, precision, reproducibility, and clinical studies;
(iii) Detailed descriptions of the test procedure(s), the interpretation of test results for clinical specimens, and acceptance criteria for any quality control testing;
(iv) A warning statement that viral culture should not be attempted in cases of positive results for SARS-CoV-2 and/or any similar microbial agents unless a facility with an appropriate level of laboratory biosafety (
*e.g.,* BSL 3 and BSL 3+, etc.) is available to receive and culture specimens; and(v) A prominent statement that device performance has not been established for specimens collected from individuals not identified in the intended use population (
*e.g.,* when applicable, that device performance has not been established in individuals without signs or symptoms of respiratory infection).(vi) Limiting statements that indicate that:
(A) A negative test result does not preclude the possibility of infection;
(B) The test results should be interpreted in conjunction with other clinical and laboratory data available to the clinician;
(C) There is a risk of incorrect results due to the presence of nucleic acid sequence variants in the targeted pathogens;
(D) That positive and negative predictive values are highly dependent on prevalence;
(E) Accurate results are dependent on adequate specimen collection, transport, storage, and processing. Failure to observe proper procedures in any one of these steps can lead to incorrect results; and
(F) When applicable (
*e.g.,* recommended by the Centers for Disease Control and Prevention, by current well-accepted clinical guidelines, or by published peer-reviewed literature), that the clinical performance may be affected by testing a specific clinical subpopulation or for a specific claimed specimen type.(4) Design verification and validation must include:
(i) Detailed documentation, including performance results, from a clinical study that includes prospective (sequential) samples for each claimed specimen type and, as appropriate, additional characterized clinical samples. The clinical study must be performed on a study population consistent with the intended use population and compare the device performance to results obtained using a comparator that FDA has determined is appropriate. Detailed documentation must include the clinical study protocol (including a predefined statistical analysis plan), study report, testing results, and results of all statistical analyses.
(ii) Risk analysis and documentation demonstrating how risk control measures are implemented to address device system hazards, such as Failure Modes Effects Analysis and/or Hazard Analysis. This documentation must include a detailed description of a protocol (including all procedures and methods) for the continuous monitoring, identification, and handling of genetic mutations and/or novel respiratory pathogen isolates or strains (
*e.g.,* regular review of published literature and periodic in silico analysis of target sequences to detect possible mismatches). All results of this protocol, including any findings, must be documented and must include any additional data analysis that is requested by FDA in response to any performance concerns identified under this section or identified by FDA during routine evaluation. Additionally, if requested by FDA, these evaluations must be submitted to FDA for FDA review within 48 hours of the request. Results that are reasonably interpreted to support the conclusion that novel respiratory pathogen strains or isolates impact the stated expected performance of the device must be sent to FDA immediately.(iii) A detailed description of the identity, phylogenetic relationship, and other recognized characterization of the respiratory pathogen(s) that the device is designed to detect. In addition, detailed documentation describing how to interpret the device results and other measures that might be needed for a laboratory diagnosis of respiratory infection.
(iv) A detailed device description, including device components, ancillary reagents required but not provided, and a detailed explanation of the methodology, including molecular target(s) for each analyte, design of target detection reagents, rationale for target selection, limiting factors of the device (
*e.g.,* saturation level of hybridization and maximum amplification and detection cycle number, etc.), internal and external controls, and computational path from collected raw data to reported result (*e.g.,* how collected raw signals are converted into a reported signal and result), as applicable.(v) A detailed description of device software, including software applications and hardware-based devices that incorporate software. The detailed description must include documentation of verification, validation, and hazard analysis and risk assessment activities, including an assessment of the impact of threats and vulnerabilities on device functionality and end users/patients as part of cybersecurity review.
(vi) For devices intended for the detection and identification of microbial agents for which an FDA recommended reference panel is available, design verification and validation must include the performance results of an analytical study testing the FDA recommended reference panel of characterized samples. Detailed documentation must be kept of that study and its results, including the study protocol, study report for the proposed intended use, testing results, and results of all statistical analyses.
(vii) For devices with an intended use that includes detection of Influenza A and Influenza B viruses and/or detection and differentiation between the Influenza A virus subtypes in human clinical specimens, the design verification and validation must include a detailed description of the identity, phylogenetic relationship, or other recognized characterization of the Influenza A and B viruses that the device is designed to detect, a description of how the device results might be used in a diagnostic algorithm and other measures that might be needed for a laboratory identification of Influenza A or B virus and of specific Influenza A virus subtypes, and a description of the clinical and epidemiological parameters that are relevant to a patient case diagnosis of Influenza A or B and of specific Influenza A virus subtypes. An evaluation of the device compared to a currently appropriate and FDA accepted comparator method. Detailed documentation must be kept of that study and its results, including the study protocol, study report for the proposed intended use, testing results, and results of all statistical analyses.
(5) When applicable, performance results of the analytical study testing the FDA recommended reference panel described in paragraph (b)(4)(vi) of this section must be included in the device's labeling under § 809.10(b) of this chapter.
(6) For devices with an intended use that includes detection of Influenza A and Influenza B viruses and/or detection and differentiation between the Influenza A virus subtypes in human clinical specimens in addition to detection of SARS-CoV-2 and similar microbial agents, the required labeling under § 809.10(b) of this chapter must include the following:
(i) Where applicable, a limiting statement that performance characteristics for Influenza A were established when Influenza A/H3 and A/H1-2009 (or other pertinent Influenza A subtypes) were the predominant Influenza A viruses in circulation.
(ii) Where applicable, a warning statement that reads 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.
(iii) Where the device results interpretation involves combining the outputs of several targets to get the final results, such as a device that both detects Influenza A and differentiates all known Influenza A subtypes that are currently circulating, the device's labeling must include a clear interpretation instruction for all valid and invalid output combinations, and recommendations for any required followup actions or retesting in the case of an unusual or unexpected device result.
(iv) A limiting statement that if a specimen yields a positive result for Influenza A, but produces negative test results for all specific influenza A subtypes intended to be differentiated (
*i.e.,* H1-2009 and H3), this result requires notification of appropriate local, State, or Federal public health authorities to determine necessary measures for verification and to further determine whether the specimen represents a novel strain of Influenza A.(7) If one of the actions listed at section 564(b)(1)(A) through (D) of the Federal Food, Drug, and Cosmetic Act occurs with respect to an influenza viral strain, or if the Secretary of Health and Human Services determines, under section 319(a) of the Public Health Service Act, that a disease or disorder presents a public health emergency, or that a public health emergency otherwise exists, with respect to an influenza viral strain:
(i) Within 30 days from the date that FDA notifies manufacturers that characterized viral samples are available for test evaluation, the manufacturer must have testing performed on the device with those influenza viral samples in accordance with a standardized protocol considered and determined by FDA to be acceptable and appropriate.
(ii) Within 60 days from the date that FDA notifies manufacturers that characterized influenza viral samples are available for test evaluation and continuing until 3 years from that date, the results of the influenza emergency analytical reactivity testing, including the detailed information for the virus tested as described in the certificate of authentication, must be included as part of the device's labeling in a tabular format, either by:
(A) Placing the results directly in the device's labeling required under § 809.10(b) of this chapter that accompanies the device in a separate section of the labeling where analytical reactivity testing data can be found, but separate from the annual analytical reactivity testing results; or
(B) In a section of the device's label or in other labeling that accompanies the device, prominently providing a hyperlink to the manufacturer's public website where the analytical reactivity testing data can be found. The manufacturer's website, as well as the primary part of the manufacturer's website that discusses the device, must provide a prominently placed hyperlink to the website containing this information and must allow unrestricted viewing access.

## Predicate Devices

- BioFire Respiratory Panel 2.1 (RP2.1) ([DEN200031](/device/DEN200031.md))

## Submission Summary (Full Text)

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FDA

U.S. FOOD & DRUG

ADMINISTRATION

# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

## I Background Information:

A 510(k) Number
K231481

B Applicant
Cepheid

C Proprietary and Established Names
Xpert Xpress CoV-2/Flu/RSV plus

D Regulatory Information

|  Product Code(s) | Classification | Regulation Section | Panel  |
| --- | --- | --- | --- |
|  QOF | Class II | 21 CFR 866.3981 - Device To Detect And Identify Nucleic Acid Targets In Respiratory Specimens From Microbial Agents That Cause The SARS-Cov-2 Respiratory Infection And Other Microbial Agents When In A Multi-Target Test | MI - Microbiology  |
|  OOI | Class II | 21 CFR 862.2570 - Instrumentation for clinical multiplex test systems | CH - Clinical Chemistry  |

## II Submission/Device Overview:

### A Purpose for Submission:
To obtain 510(k) clearance for the Xpert Xpress CoV-2/Flu/RSV plus test for use on the GeneXpert Dx and Infinity Instrument Systems.

### B Measurand:
Conserved RNA sequences within the genes encoding the nucleocapsid protein (N), envelope protein (E) and RNA-dependent RNA polymerase protein (RdRP) of SARS-CoV-2 viruses; 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 protein (NS) of influenza B viruses; and the nucleocapsid protein of respiratory syncytial virus (RSV) A and B viruses.

### C Type of Test:
A multiplexed, real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay for the qualitative detection and differentiation of SARS-CoV-2, influenza A, influenza B, and RSV viral RNA from nasopharyngeal swab (NPS) specimens and anterior nasal swab (NS) specimens using the GeneXpert Instrument Systems platform.

Food and Drug Administration

10903 New Hampshire Avenue

Silver Spring, MD 20993-0002

www.fda.gov

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III Intended Use/Indications for Use:

A Intended Use(s):
See Indications for Use below.

B Indication(s) for Use:
The Xpert Xpress CoV-2/Flu/RSV plus test, performed on the GeneXpert Dx and GeneXpert Infinity Systems, is an automated multiplexed real-time reverse transcriptase polymerase chain reaction (RT-PCR) test intended for use in the simultaneous in vitro qualitative detection and differentiation of severe acute respiratory syndrome coronavirus (SARS-CoV-2), influenza A, influenza B, and/or respiratory syncytial virus (RSV) viral RNA in nasopharyngeal swab and anterior nasal swab specimens collected from individuals with signs and symptoms of respiratory tract infection. Clinical signs and symptoms of respiratory tract infection due to SARS-CoV-2, influenza A, influenza B, and RSV can be similar.

The Xpert Xpress CoV-2/Flu/RSV plus is intended for use in the differential detection of SARS-CoV-2, influenza A, influenza B and/or RSV RNA and aids in the diagnosis of COVID-19, influenza and/or RSV infections if used in conjunction with other clinical and epidemiological information, and laboratory findings. SARS-CoV-2, influenza A, influenza B, and RSV viral RNA are generally detectable in nasopharyngeal swab and anterior nasal swab specimens during the acute phase of infection.

Positive results are indicative of the presence of the identified virus, but do not rule out bacterial infection or co-infection with other pathogens not detected by the test. The agent(s) detected by the Xpert Xpress CoV-2/Flu/RSV plus test may not be the definite cause of disease.

Negative results do not preclude SARS-CoV-2, influenza A, influenza B, and/or RSV infection. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.

C Special Conditions for Use Statement(s):
Rx - For Prescription Use Only
For in vitro diagnostic use only

D Special Instrument Requirements:
GeneXpert Instrument Systems:
- GeneXpert Dx Systems (GX-I, GX-II, GX-IV, GX-XVI)
- GeneXpert Infinity Systems (Infinity-48s and Infinity-80)

IV Device/System Characteristics:

A Device Description:
The Xpert Xpress CoV-2/Flu/RSV plus test, performed on the GeneXpert Instrument (GeneXpert Dx, GeneXpert Infinity-48s and GeneXpert Infinity-80 systems), is a multiplex nucleic acid amplification test for the qualitative detection and differentiation of SARS-CoV-2, influenza A, influenza B, and RSV viral RNA from NPS and NS swab specimens.

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To perform the Xpert Xpress CoV-2/Flu/RSV plus test, NPS and NS swab specimens from patients are collected with nylon flocked swabs and placed into either 3 mL viral transport medium (VTM) or 2 mL eNAT. With a supplied transfer pipette, eluted NPS and NS swab specimens are loaded into the sample chamber of a single-use, self-contained Xpert Xpress CoV-2/Flu/RSV plus 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 CoV-2/Flu/RSV plus test in approximately 36 minutes. The Xpert Xpress CoV-2/Flu/RSV plus test can be run in one of five different modes, varying in the number of target analyte(s) tested for and reported. The five modes are: SARS-CoV-2 only; SARS-CoV-2 and Flu; SARS-CoV-2/Flu/RSV; Flu only; or Flu and RSV. For the SARS-CoV-2 only and Flu only modes, there is an option to activate an Early Assay Termination (EAT) function, which will provide earlier time to results in high titer specimens if the signal from the target nucleic acid reaches a predetermined threshold before the assay has finished running. When EAT is activated, the earliest time to a positive result is ~ 25 minutes. The GeneXpert Dx and 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 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.

## B Principle of Operation:

The Xpert Xpress CoV-2/Flu/RSV plus test is a nucleic acid-based test using real-time RT-PCR. After addition of the specimen to the Sample Chamber of the Xpert Xpress CoV-2/Flu/RSV plus test cartridge, the cartridge is loaded onto the GeneXpert Instrument System platform. The instrument then performs automated sample processing including RNA extraction, followed by reverse transcription, amplification, detection, and reporting of results. The results are interpreted automatically by the GeneXpert System and are shown in the View Results window.

## C Instrument Description Information:

1. Instrument Name:
- GeneXpert Dx Systems: GeneXpert Dx software version 4.7b or higher
- GeneXpert Infinity Systems: Xpertise software version 6.4b or higher

2. Specimen Identification:
To perform a test, the user selects the "Create Test" (Dx Systems) or "Orders" (Infinity Systems) icon, scans the cartridge barcode, enters the sample ID, or scans the sample ID, and

K231481 - Page 3 of 33

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loads the cartridge into the instrument module that has the green blinking light (for the Dx Systems) or onto the conveyor belt (for the Infinity Systems) to start the test.

## 3. Specimen Sampling and Handling:

NPS and NS swab specimens are collected using nylon flocked swabs and eluted into 3 mL VTM or 2 mL eNAT. At the testing facility, the operator mixes the specimen by rapidly inverting the specimen transport tube 5 times. A transfer pipette provided with the Xpert Xpress CoV-2/Flu/RSV plus is used to transfer an aliquot of the specimen into the Sample Chamber of the open test cartridge. After closing the cartridge lid, the operator loads the cartridge onto the applicable GeneXpert instrument for testing.

## 4. Calibration:

GeneXpert instruments are calibrated at the factory. Routine calibration of the GeneXpert Instrument systems may be performed by Cepheid Field Service Engineers during annual maintenance.

## 5. Quality Control:

The Xpert Xpress CoV-2/Flu/RSV plus test includes two internal controls: a sample processing control (SPC), and a probe check control (PCC).

### Sample Processing Control (SPC)

The sample processing control is a non-infectious armored RNA pseudovirus that ensures adequate lysis of target virus, 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 four SARS-CoV-2, 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 (PCC)

The probe check control 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.

### External Controls

Optional external quality control materials are available from Zeptometrix. Specifically, the following external controls may be used with the Xpert Xpress CoV-2/Flu/RSV plus test:

- External Positive Control: NATtrol Flu/RSV/SARS-CoV-2; Cat # NATFRC-6C-IVD
- External Negative Control: Coxsackievirus A9; Cat # NATCV9-6C-IVD

All external controls must be used in accordance with local, state, and federal accrediting organizations, as applicable.

## V Substantial Equivalence Information:

### A Predicate Device Name(s):

BioFire Respiratory Panel 2.1 (RP2.1)

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B Predicate 510(k) Number(s):

DEN200031

# C Comparison with Predicate(s):

|  Device & Predicate Device(s): | K231481 | DEN200031  |
| --- | --- | --- |
|  Device Trade Name | Xpert Xpress CoV-2/Flu/RSV plus | BioFire Respiratory Panel 2.1  |
|  Regulation Number and Name | Same | 21 CFR 866.3981; Devices to detect and identify nucleic acid targets in respiratory samples from microbial agents that cause the SARS-CoV-2 respiratory infection and other microbial agents when in a multi-analyte test.  |
|  Product Code | Same | QOF  |
|  Technology/Detection | Real-time reverse transcription polymerase chain reaction (RT-qPCR) | Nested multiplex RT-PCR followed by high resolution melting analysis to confirm identity of amplified product.  |
|  Intended Use/Indications for Use | The Xpert Xpress CoV-2/Flu/RSV plus test, performed on the GeneXpert Dx and GeneXpert Infinity Systems, is an automated multiplexed real-time reverse transcriptase polymerase chain reaction (RT-PCR) test intended for use in the simultaneous in vitro qualitative detection and differentiation of severe acute respiratory syndrome coronavirus (SARS-CoV-2), influenza A, influenza B, and/or respiratory syncytial virus (RSV) viral RNA in nasopharyngeal swab and anterior nasal swab specimens collected from individuals with signs and symptoms of respiratory tract infection. Clinical signs and symptoms of respiratory tract infection due to SARS-CoV-2, influenza A, influenza B, and RSV can be similar.The Xpert Xpress CoV-2/Flu/RSV plus is intended for use in the differential | The BioFire Respiratory Panel 2.1 (RP2.1) is a PCR-based multiplexed nucleic acid test intended for use with the BioFire FilmArray 2.0 or BioFire FilmArray Torch Systems for the simultaneous qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals suspected of respiratory tract infections, including COVID-19.Nucleic acids from the respiratory viral and bacterial organisms identified by this test are generally detectable in NPS specimens during the acute phase of infection. The detection and identification of specific viral and bacterial nucleic acids from individuals exhibiting signs and/or symptoms of respiratory infection is indicative of the presence of the identified microorganism and aids in the diagnosis of respiratory infection if used in  |

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|   | detection of SARS-CoV-2, influenza A, influenza B and/or RSV RNA and aids in the diagnosis of COVID-19, influenza and/or RSV infections if used in conjunction with other clinical and epidemiological information, and laboratory findings. SARS-CoV-2, influenza A, influenza B, and RSV viral RNA are generally detectable in nasopharyngeal swab and anterior nasal swab specimens during the acute phase of infection.

Positive results are indicative of the presence of the identified virus, but do not rule out bacterial infection or co-infection with other pathogens not detected by the test. The agent(s) detected by the Xpert Xpress CoV-2/Flu/RSV plus test may not be the definite cause of disease.

Negative results do not preclude SARS-CoV-2, influenza A, influenza B, and/or RSV infection. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. | conjunction with other clinical and epidemiological information. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.

The following organism types and subtypes are identified using the BioFire RP2.1:
• Adenovirus
• Coronavirus 229E
• Coronavirus HKU1
• Coronavirus NL63
• Coronavirus OC43
• Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)
• Human Metapneumovirus
• Human Rhinovirus/Enterovirus
• Influenza A, including subtypes H1, H3 and H1-2009
• Influenza B
• Parainfluenza Virus 1
• Parainfluenza Virus 2
• Parainfluenza Virus 3
• Parainfluenza Virus 4
• Respiratory Syncytial Virus
• Bordetella parapertussis
• Bordetella pertussis
• Chlamydia pneumoniae
• Mycoplasma pneumoniae

Negative results in the setting of respiratory illness may be due to infection with pathogens that are not detected by this test, or lower respiratory tract infection that may not be detected by an NPS specimen. Positive results do not rule out co-infection with other organisms. The agent(s) detected by the BioFire RP2.1 may not be the definite cause of disease. Additional laboratory testing (e.g., bacterial and viral culture, immunofluorescence, and radiography) may be necessary when  |
| --- | --- | --- |

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|   |  | evaluating a patient with possible respiratory tract infection.  |
| --- | --- | --- |
|  Test Format | Same | Single Use  |
|  Automated Test Processes | Same | Nucleic Acid Extraction, Detection and Results Interpretation  |
|  Results Reported | Same | Qualitative  |
|  Specimen Type | Nasopharyngeal (NPS) swab and Anterior Nasal (NS) swab | Nasopharyngeal (NPS) swab  |
|  Transport Media | Universal Transport Medium (UTM)/Viral Transport Medium (VTM), eNAT | Universal Transport Medium (UTM)/ Viral Transport Medium (VTM), saline  |
|  Internal Control | Sample Processing Control (SPC), Probe Check Control (PCC) | Sample Processing Control, PCR and Melt Analysis Control  |
|  Instrument Systems | GeneXpert Dx and Infinity Instrument Systems | BioFire FilmArray 2.0 or BioFire FilmArray Torch Systems  |
|  Time to Result | ~36 minutes for sample preparation and RT-PCR | ~45 minutes  |

VI Standards/Guidance Documents Referenced:

Standards
- ISO 14971. Medical Devices – Application of Risk Management to Medical Devices (2019)
- CLSI EP12-A2. User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline – Second Edition
- CLSI EP17-A2. Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline – Second Edition
- CLSI EP25-A. Evaluation of Stability of In Vitro Diagnostic Reagents; Approved Guideline.
- CLSI EP05-A3. Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline – Third Edition.
- CLSI MM03-A3. Molecular Diagnostic Methods for Infectious Diseases; Approved Guidance – Third Edition.
- CLSI EP07. Testing in Clinical Chemistry. Third Edition.
- CLSI MM17. Verification and Validation of Multiplex Nucleic Acid Assays. Second Edition.
- CLSI MM13. Collection, Transport, Preparation, and Storage of Specimens for Molecular Methods. Second Edition.

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## Special Controls

- Class II Special Controls as per 21 CFR 866.3981.

## Guidance Documents

- Guidance for Industry and FDA Staff, Format for Traditional and Abbreviated 510(k)s (September 13, 2019).
- Guidance for Industry and FDA Staff, Appropriate Use of Voluntary Consensus Standards in Premarket Submissions for Medical Devices (September 14, 2018).
- Guidance for Sponsors, Institutional Review Boards, Clinical Investigators and FDA Staff, Guidance on Informed Consent for In Vitro Diagnostic Device Studies Using Leftover Human Specimens that are Not Individually Identifiable (April 25, 2006).
- Guidance for Industry and FDA Staff, Class II Special Controls Guidance Document: Instrumentation for Clinical Multiplex Test Systems (March 10, 2005).
- Guidance for Industry and FDA Staff, Guidance for the Content of Premarket Submissions for Software Contained in Medical Devices (May 11, 2005).
- Guidance for Industry and FDA Staff, General Principles of Software Validation (January 11, 2002).
- Guidance for Industry and FDA Staff, Content of Premarket Submissions for Management of Cybersecurity in Medical Devices (October 2, 2014).
- Guidance for Industry, Cybersecurity for Networked Medical Devices Containing Off-the-Shelf (OTS) Software (January 14, 2005).
- Guidance for Industry and FDA Staff, Off-the-Shelf Software Use in Medical Devices (September 27, 2019).
- Guidance for Industry and FDA Staff, eCopy Program for Medical Device Submissions (April 27, 2020).
- Guidance for Test Developers and FDA Staff, Policy for Evaluating Impact of Viral Mutations on COVID-19 Tests (Revised); (January 12, 2023).
- Guidance for Industry, Other Stakeholders, and FDA Staff, Transition Plan for Medical Devices Issued Emergency Use Authorizations (EUA) During the Coronavirus Disease 2019 (COVID-19) Public Health Emergency (March 27, 2023).

## VII Performance Characteristics (if/when applicable):

### A Analytical Performance:

1. Precision/Reproducibility:

a. Within-Laboratory Precision

Within-laboratory precision was evaluated at a single site using the Xpert Xpress CoV-2/Flu/RSV plus test run on the GeneXpert Infinity System. A total of nine contrived panels containing known quantities of the target analytes were prepared in simulated matrix, consisting 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 at a 1:40 ratio to a concentration of 2.5% (v/v) in UTM/VTM. Data supporting the use of simulated matrix can be found in section VII Performance Characteristics.B.2. Sample Matrix and Transport Media Equivalency Study, later in this document. The viral materials used to generate the positive panel members are denoted in Table 1. The contrived positive panels consisted of single target spiked

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samples at low concentrations (~1.5x LoD) and moderate concentrations (~3x LoD) (Table 2). The study was conducted with one operator and one cartridge lot over the course of 20 days. Each panel member was tested in duplicate twice per day on 20 different days generating a total of 80 replicates per panel member (1 Site x 1 Operator x 1 Lot x 20 Days x 2 Runs per Day x 2 Replicates per run).

Table 1. Viral Strains for Within-Laboratory Precision Study

|  Description | Vendor | Catalog Number  |
| --- | --- | --- |
|  SARS-CoV-2 (USA-WA1/2020), inactivated | Zeptometrix | NATSARS(COV2)-ST  |
|  Influenza A/Idaho/07/2018, cultured | Influenza Reagent Resource (IRR) | NA  |
|  Influenza B/Wisconsin/10/2016, cultured | Influenza Reagent Resource (IRR) | NA  |
|  RSV B/Wash/18537/62, cultured | Cepheid (internal strain) | NA  |

Table 2. Precision Study Sample Panel

|  Panel ID | Description  |
| --- | --- |
|  1 | Negative  |
|  2 | SARS-CoV-2 (~1.5x LoD)  |
|  3 | SARS-CoV-2 (~3x LoD)  |
|  4 | Flu A (~1.5x LoD)  |
|  5 | Flu A (~3x LoD)  |
|  6 | Flu B (~1.5x LoD)  |
|  7 | Flu B (~3x LoD)  |
|  8 | RSV B (~1.5x LoD)  |
|  9 | RSV B (~3x LoD)  |

The qualitative (i.e., % agreement with expected results) and Ct results from the study are illustrated in Table 3 and Table 4, respectively.

Table 3. Within-Laboratory Precision Study – Qualitative Results

|  Panel ID | Panel Member | % Positive (pos n/valid n) | % Agreement with Expected Results (95% CI)  |
| --- | --- | --- | --- |
|  1 | Negative | 0% (0/80) | 100% (95.4-100%)  |
|  2 | SARS-CoV-2 (~1.5x LoD) | 98.8% (79/80) | 98.8% (93.3-99.8%)  |
|  3 | SARS-CoV-2 (~3x LoD) | 100% (80/80) | 100% (95.4-100%)  |
|  4 | Flu A (~1.5x LoD) | 97.5% (78/80) | 97.5% (91.3-99.3%)  |
|  5 | Flu A (~3x LoD) | 100% (80/80) | 100% (95.4-100%)  |
|  6 | Flu B (~1.5x LoD) | 96.3% (77/80) | 96.3% (89.5-98.7%)  |
|  7 | Flu B (~3x LoD) | 100% (80/80) | 100% (95.4-100%)  |
|  8 | RSV B (~1.5x LoD) | 97.5% (78/80) | 97.5% (91.3-99.3%)  |
|  9 | RSV B (~3x LoD) | 100% (80/80) | 100% (95.4-100%)  |

All low positive (~1.5x) panel members were positive ≥96.3%. All moderate positive (~3x) panel members were 100% positive for the spiked target analytes. The negative panel member was 0.0% positive for SARS-CoV-2, Flu A, Flu B, and RSV, (Table 3, above).

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Table 4. Within-Laboratory Precision Study - Ct Signal Variability Analysis Results

|  Panel Member | Target | N | Mean Ct | Between Days |   | Between Runs |   | Within Test |   | Total  |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |   |   |  SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%)  |
|  Negative | SPC | 80 | 28.93 | 0.31 | 1.1 | 0.21 | 0.7 | 0.76 | 2.6 | 0.85 | 2.9  |
|  SARS-CoV-2 ~1.5x LoD | SARS-CoV-2 | 79 | 36.95 | 0.00 | 0.0 | 0.45 | 1.2 | 1.05 | 2.8 | 1.14 | 3.1  |
|  SARS-CoV-2 ~3x LoD |   | 80 | 35.69 | 0.21 | 0.6 | 0.00 | 0.0 | 0.78 | 2.2 | 0.81 | 2.3  |
|  Flu A ~1.5x LoD | Flu A1 | 80 | 35.49 | 0.00 | 0.0 | 0.33 | 0.9 | 1.27 | 3.6 | 1.31 | 3.7  |
|   |  Flu A2 | 55 | 38.29 | 0.58 | 1.5 | 0.00 | 0.0 | 1.50 | 3.9 | 1.61 | 4.2  |
|  Flu A ~3x LoD | Flu A1 | 80 | 34.30 | 0.00 | 0.0 | 0.23 | 0.7 | 0.59 | 1.7 | 0.63 | 1.8  |
|   |  Flu A2 | 75 | 36.56 | 0.00 | 0.0 | 0.53 | 1.5 | 0.67 | 1.8 | 0.86 | 2.3  |
|  Flu B ~1.5x LoD | Flu B | 79 | 35.57 | 0.27 | 0.8 | 0.00 | 0.0 | 1.35 | 3.8 | 1.37 | 3.9  |
|  Flu B ~3x LoD |   | 80 | 34.35 | 0.00 | 0.0 | 0.00 | 0.0 | 0.84 | 2.5 | 0.84 | 2.5  |
|  RSV ~1.5x LoD | RSV | 80 | 35.78 | 0.24 | 0.7 | 0.00 | 0.0 | 1.02 | 2.8 | 1.04 | 2.9  |
|  RSV ~3x LoD |   | 80 | 34.75 | 0.00 | 0.0 | 0.34 | 1.0 | 0.61 | 1.7 | 0.69 | 2.0  |

Ct = cycle threshold; CV = coefficient of variation; SD = standard deviation

The mean and variability analysis between days, between runs, within runs, and overall (total) for Ct values is shown in Table 4. Overall  $\% \mathrm{CV}$  was  $\leq 4.2\%$ . The greatest source of variability was from Flu A at  $\sim 1.5\mathrm{x}$  LoD (Flu A2 Ct), which had a within-run  $\% \mathrm{CV}$  of  $3.9\%$ . Overall variability was low, and the study demonstrates assay variability within an acceptable range.

# b. Reproducibility

A blinded, multi-site reproducibility study was conducted to assess the total variability of the Xpert Xpress CoV-2/Flu/RSV plus test across operators, study sites, testing days, runs, instruments (GeneXpert Dx and Infinity Systems), and reagent lots.

The same nine contrived panels evaluated in the Within-Laboratory Precision Study (see Table 2) were also tested in the Reproducibility Study. The study was performed by two operators at each of three testing sites. At each site three cartridge reagent lots were evaluated. One site performed testing using the GeneXpert Infinity System, while the remaining two sites conducted testing with the GeneXpert Dx System. Each panel member was tested in duplicate twice per day on 6 different days generating a total of 144 replicates per panel member (3 Sites x 2 Operators per site x 3 Lots x 2 Days/Lot x 2 Runs per Day x 2 Replicates per run).

The qualitative (i.e., % agreement with expected results) and quantitative results from the study are illustrated in Table 5 and Table 6, respectively.

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Table 5. Reproducibility Study – Qualitative Results

|  Panel Member | % Agreement with Expected Results |   |   |   |   |   |   |   |   | Overall % Agreement (95% CI)  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  Site 1 (Infinity) |   |   | Site 2 (Dx) |   |   | Site 3 (Dx)  |   |   |   |
|   |  Op1 | Op2 | Site | Op1 | Op2 | Site | Op1 | Op2 | Site  |   |
|  Negative | 100% (24/24) | 100% (24/24) | 100% (48/48) | 100% (24/24) | 100% (24/24) | 100% (48/48) | 100% (24/24) | 100% (24/24) | 100% (48/48) | 100% (144/144) (97.4-100)  |
|  SARS-CoV-2 (~1.5x LoD) | 100% (24/24) | 100% (24/24) | 100% (48/48) | 100% (24/24) | 100% (24/24) | 100% (48/48) | 100% (24/24) | 100% (24/24) | 100% (48/48) | 100% (144/144) (97.4-100)  |
|  SARS-CoV-2 (~3x LoD) | 100% (24/24) | 100% (24/24) | 100% (48/48) | 100% (24/24) | 100% (24/24) | 100% (48/48) | 100% (24/24) | 100% (24/24) | 100% (48/48) | 100% (144/144) (97.4-100)  |
|  Flu A (~1.5x LoD) | 100% (24/24) | 100% (24/24) | 100% (48/48) | 100% (24/24) | 100% (24/24) | 100% (48/48) | 100% (24/24) | 100% (24/24) | 100% (48/48) | 100% (144/144) (97.4-100)  |
|  Flu A (~3x LoD) | 100% (24/24) | 100% (24/24) | 100% (48/48) | 100% (24/24) | 100% (24/24) | 100% (48/48) | 100% (24/24) | 100% (24/24) | 100% (48/48) | 100% (144/144) (97.4-100)  |
|  Flu B (~1.5x LoD) | 100% (24/24) | 100% (24/24) | 100% (48/48) | 95.8% (23/24) | 95.8% (23/24) | 95.8% (46/48) | 100% (24/24) | 100% (24/24) | 100% (48/48) | 98.6% (142/144) (95.1-99.6)  |
|  Flu B (~3x LoD) | 100% (24/24) | 100% (24/24) | 100% (48/48) | 100% (24/24) | 100% (24/24) | 100% (48/48) | 100% (23/23) | 95.8% (23/24) | 97.9% (46/47) | 99.3% (142/143)a (96.1-99.9)  |
|  RSV B (~1.5x LoD) | 100% (24/24) | 100% (24/24) | 100% (48/48) | 95.8% (23/24) | 100% (24/24) | 97.9% (47/48) | 100% (24/24) | 100% (24/24) | 100% (48/48) | 99.3% (143/144) (96.2-99.9)  |
|  RSV B (~3x LoD) | 100% (24/24) | 100% (24/24) | 100% (48/48) | 100% (24/24) | 100% (24/24) | 100% (48/48) | 100% (24/24) | 100% (24/24) | 100% (48/48) | 100% (144/144) (97.4-100)  |

Op = Operator
${}^{a}$  One replicate was excluded because it was run on a day when an external positive control produced an incorrect result but was inadvertently not retested.

The Xpert Xpress CoV-2/Flu/RSV plus test demonstrated  $100\%$  agreement with expected results for all panel members except for the Flu B low positive  $(98.6\%)$ , Flu B moderate positive  $(99.3\%)$ , and the RSV B low positive  $(99.3\%)$ . A lower percent agreement for low positive panel members was expected, since the analyte concentration of the panel member analytes was close to the limit of detection (i.e.,  $\sim 1.5\mathrm{x}$  LoD), which is expected to yield  $\geq 95\%$  detection rate. The results of the study demonstrate acceptable assay reproducibility for the Xpert Xpress CoV-2/Flu/RSV plus test.

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Table 6. Reproducibility Study – Ct Signal Variability Analysis Results

|  Panel Member | Target | N | Mean Ct | Variance Source  |   |   |   |   |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |   |   |  Site |   | Operator |   | Lot |   | Day |   | Run |   | Within-Run |   | Total  |
|   |   |   |   |  SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD  |
|  Negative | SPC | 144 | 30.8 | 0.1 | 0.4 | 0.0 | 0.0 | 0.9 | 2.9 | 0.5 | 1.5 | 0.0 | 0.0 | 1.3 | 4.2 | 1.6  |
|  SARS-CoV-2 Low Pos | SARS-CoV-2 | 144 | 37.4 | 0.0 | 0.0 | 0.2 | 0.5 | 0.1 | 0.2 | 0.0 | 0.0 | 0.3 | 0.7 | 0.4 | 1.1 | 0.5  |
|  SARS-CoV-2 Mod Pos |   | 144 | 36.2 | 0.0 | 0.1 | 0.1 | 0.3 | 0.0 | 0.0 | 0.1 | 0.3 | 0.2 | 0.4 | 0.4 | 1.0 | 0.4  |
|  Flu A Low Pos | Flu A1 | 144 | 35.7 | 0.2 | 0.6 | 0.0 | 0.0 | 0.1 | 0.2 | 0.0 | 0.0 | 0.0 | 0.0 | 0.6 | 1.6 | 0.6  |
|   |  Flu A2 | 135^{b} | 37.9 | 0.3 | 0.8 | 0.0 | 0.0 | 0.2 | 0.5 | 0.0 | 0.0 | 0.4 | 1.1 | 0.9 | 2.5 | 1.1  |
|  Flu A Mod Posa | Flu A1 | 144 | 34.7 | 0.0 | 0.0 | 0.1 | 0.2 | 0.0 | 0.0 | 0.1 | 0.3 | 0.0 | 0.0 | 0.4 | 1.2 | 0.4  |
|   |  Flu A2 | 144 | 36.6 | 0.0 | 0.1 | 0.0 | 0.0 | 0.2 | 0.4 | 0.0 | 0.0 | 0.0 | 0.0 | 0.5 | 1.5 | 0.6  |
|  Flu B Low Pos | Flu B | 144 | 36.3 | 0.3 | 0.8 | 0.0 | 0.1 | 0.0 | 0.0 | 0.1 | 0.2 | 0.3 | 0.7 | 0.7 | 2.1 | 0.8  |
|  Flu B Mod Pos |   | 142^{c} | 35.1 | 0.0 | 0.0 | 0.1 | 0.4 | 0.1 | 0.3 | 0.3 | 0.8 | 0.0 | 0.0 | 0.7 | 2.0 | 0.8  |
|  RSV B Low Pos | RSV | 144 | 35.8 | 0.1 | 0.2 | 0.0 | 0.0 | 0.2 | 0.4 | 0.0 | 0.0 | 0.0 | 0.0 | 0.6 | 1.7 | 0.6  |
|  RSV B Mod Pos |   | 144 | 34.8 | 0.1 | 0.2 | 0.0 | 0.0 | 0.1 | 0.4 | 0.0 | 0.0 | 0.2 | 0.5 | 0.5 | 1.4 | 0.5  |

Ct = cycle threshold; CV = coefficient of variation; SD = standard deviation
aOne replicate from Site 2 used the reagent lot 102 instead of the intended reagent lot 401. In the analysis, this replicate was included in the calculation for reagent lot 401.
bNine replicates were excluded due to not having Flu A2 Ct values.
cOne replicate was excluded due to not having a Flu B Ct value; another replicate was excluded because it was run on a day when an external positive control produced an incorrect result, but the sample was inadvertently not retested.

The mean and variability analysis between sites, operators, lots, days, runs, within-runs, and overall (total) for Ct values is shown in Table 6. Overall %CV was ≤ 5.3%. The greatest source of variability was from the negative samples, which had a within-run %CV of 4.2%. Overall variability was low, and the study demonstrates assay variability within an acceptable range.

2. Linearity:
Not Applicable; this is a qualitative assay.

3. Analytical Specificity/Interference:
Analytical Reactivity (Inclusivity)

a. Wet-Testing
An analytical reactivity study was conducted to evaluate the ability of the Xpert Xpress CoV-2/Flu/RSV plus test to detect multiple SARS-CoV-2, influenza and RSV strains that are temporally and geographically diverse. Testing was performed on 102 different strains (18 SARS-CoV-2, 48 Flu A, 21 Flu B, and 15 RSV) in triplicate using inactivated strains for SARS-CoV-2 and cultured virus for influenza and RSV, unless noted. Each strain was spiked into negative simulated matrix at analyte concentrations of ~3x the LoD. Data supporting the use of simulated matrix can be found in section VII
Performance Characteristics.B.2.Sample Matrix and Transport Media Equivalency

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Study, later in this document. The influenza A testing included 20 A/H1N1 (pre-2009 seasonal and 2009 pandemic) strains, 16 A/H3N2 strains, and 12 avian strains (including the following 9 subtypes: H5N1, H5N2, H6N2, H7N2, H7N3, H2N2, H7N9, and H9N2). The 21 Influenza B strains included both the Victoria and Yamagata lineage. The 15 RSV strains included RSV types A and B.

The Xpert Xpress CoV-2/Flu/RSV plus test exhibited a broad range of reactivity, as all SARS-CoV-2, influenza A, influenza B and RSV strains tested positive in all three replicates. Results from the study are shown in Table 7.

Table 7. Inclusivity Wet-Testing Study Results

|  Virus | Strain | Target Conc. | Result  |   |   |   |
| --- | --- | --- | --- | --- | --- | --- |
|   |   |   |  SARS-CoV-2 | Flu A | Flu B | RSV  |
|  SARS-CoV-2 | NATtrol SARS-CoV-2 USA-WA1/2020 | 412 copies/mL | POS | NEG | NEG | NEG  |
|   |  SARS-CoV-2/HongKong/VM20001061/2020 | 0.03 TCID50/mL | POSa | NEG | NEG | NEG  |
|   |  SARS-CoV-2/Italy-INMI1 | 1 TCID50/mL | POS | NEG | NEG | NEG  |
|   |  SARS-CoV-2/Africa/KRISPK005325/2020 (Beta) | 0.025 TCID50/mL | POS | NEG | NEG | NEG  |
|   |  SARS-CoV-2/England/204820464/2020 | 0.05 TCID50/mL | POS | NEG | NEG | NEG  |
|   |  SARS-CoV-2 (NY-Wadsworth-21033899-01/2021) P1_2021 (Gamma) | 0.01 TCID50/mL | POS | NEG | NEG | NEG  |
|   |  SARS-CoV-2 (NY-Wadsworth-21006055-01/2021) P2_2021 (Zeta) | 0.03 TCID50/mL | POS | NEG | NEG | NEG  |
|   |  SARS-CoV-2 (NYWadsworth-21025952-01/2021) B.1.526_2021 (Iota) | 0.1 TCID50/mL | POS | NEG | NEG | NEG  |
|   |  SARS-CoV-2 (NYWadsworth-103677-01/2020) B.1_2020 | 0.003 TCID50/mL | POS | NEG | NEG | NEG  |
|   |  SARS-CoV-2 (NYWadsworth-33126-01/2020) B.1.595_2020 | 0.0015 TCID50/mL | POS | NEG | NEG | NEG  |
|   |  SARS-CoV-2 (USA/CA-Stanford-15_S02/2021) B.1.617.1 (Kappa) | 1.7 TCID50/mL | POS | NEG | NEG | NEG  |
|   |  SARS-CoV-2 (USA/PHC658/2021) B.1.617.2 (Delta) | 0.01 TCID50/mL | POS | NEG | NEG | NEG  |
|   |  SARS-CoV-2 (USA/MDHP01542/2021) B.1.351 (Beta) | 100 (genome equivalents/mL) | POS | NEG | NEG | NEG  |
|   |  SARS-CoV-2 (USA/GA-EHC-2811C/20221) B.1.1.529 (Omicron) | 100 (genome equivalents/mL) | POS | NEG | NEG | NEG  |
|   |  SARS-CoV-2 RNA, USA/WA2/2020 (C09)b | 100 copies/mL | POS | NEG | NEG | NEG  |
|   |  SARS-CoV-2 RNA, England/205041766/2020 (C14) (alpha)b | 100 copies/mL | POS | NEG | NEG | NEG  |

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| Virus | Strain | Target Conc. | Result |
| --- | --- | --- | --- |
| SARS-CoV-2 | Flu A | Flu B | RSV |
|  | SARS-CoV-2 RNA, England/MILK-9E05B3/2020 (C15) (alpha)b | 200 copies/mL | POS | NEG | NEG | NEG |
| SARS-CoV-2 RNA /Japan (Brazil)/IC-0564/2021 (C17) (gamma)b | 100 copies/mL | POS | NEG | NEG | NEG |
|  |
| Flu A H1N1 (pre-2009) | A/swine/Iowa/15/30 | 10 TCID50/mL | NEG | POS | NEG | NEG |
| A/WS/33 | 0.6 CEID50/mL | NEG | POS | NEG | NEG |
| A/PR/8/34 | 1.25 CEID50/mL | NEG | POS | NEG | NEG |
| A/Mal/302/54 | 0.156 CEID50/mL | NEG | POS | NEG | NEG |
| A/Denver/1/57 | 1.5 CEID50/mL | NEG | POS | NEG | NEG |
| A/New Jersey/8/76 | 5 CEID50/mL | NEG | POS | NEG | NEG |
| A/New Caledonia/20/1999 | 0.10 TCID50/mL | NEG | POS | NEG | NEG |
| A/New York/55/2004 | 9 TCID50/mL | NEG | POS | NEG | NEG |
| A/Solomon Island/3/2006 | 0.0159TCID50/mL | NEG | POS | NEG | NEG |
| A/Taiwan/42/06 | 0.002 TCID50/mL | NEG | POS | NEG | NEG |
| A/Brisbane/59/2007 | 0.008 TCID50/mL | NEG | POS | NEG | NEG |
| A/Swine/NY/02/2009 | 3.2 TCID50/mL | NEG | POS | NEG | NEG |
|  |
| Flu A H1N1 (pdm 2009) | A/Colorado/14/2012 | 0.04 TCID50/mL | NEG | POS | NEG | NEG |
| A/Michigan/45/2015 | 15 CEID50/mL | NEG | POS | NEG | NEG |
| A/Iowa/53/2015 | 6 CEID50/mL | NEG | POS | NEG | NEG |
| A/Michigan/272/2017 | 0.07 TCID50/mL | NEG | POS | NEG | NEG |
| A/Idaho/07/2018 | 0.0159TCID50/mL | NEG | POS | NEG | NEG |
| A/Wisconsin/505/2018 | 0.08 TCID50/mL | NEG | POS | NEG | NEG |
| A/Hawaii/66/2019 | 100 CEID50/mL | NEG | POS | NEG | NEG |
| A/Indiana/02/2020 | NAc | NEG | POS | NEG | NEG |
|  |
| Flu A H3N2 | A/Aichi/2/68 | 2 CEID50/mL | NEG | POS | NEG | NEG |
| A/Hong Kong/8/68 | 0.25 CEID50/mL | NEG | POS | NEG | NEG |
| A/Port Chalmers/1/73 | 8 CEID50/mL | NEG | POS | NEG | NEG |
| A/Hawaii/15/2001 | 33 CEID50/mL | NEG | POS | NEG | NEG |
| A/Wisconsin/67/05c | 0.22 TCID50/mL | NEG | POS | NEG | NEG |
| A/Brisbane/10/2007 | 0.003 TCID50/mL | NEG | POS | NEG | NEG |
| A/Minnesota/11/2010 | 2.4 CEID50/mL | NEG | POS | NEG | NEG |
| A/Indiana/08/2011 | 0.02 TCID50/mL | NEG | POS | NEG | NEG |
| A/Texas/50/2012 | 0.008 TCID50/mL | NEG | POS | NEG | NEG |
| A/Alaska/232/2015 | 2 CEID50/mL | NEG | POS | NEG | NEG |
| A/Singapore/INFIMH-16-0019/2016 | 2.5 CEID50/mL | NEG | POS | NEG | NEG |
| A/Texas/71/2017 | 1 FFU/mL | NEG | POS | NEG | NEG |
| A/Kansas/14/2017 | 0.15 FFU/mL | NEG | POS | NEG | NEG |
| A/Wisconsin/04/2018d | 0.15 FFU/mL | NEG | POS | NEG | NEG |
| A/Arizona/45/2018 | 2 FFU/mL | NEG | POS | NEG | NEG |
| A/Hong Kong/45/2019 | 0.8 FFU/mL | NEG | POS | NEG | NEG |
|  |
| Avian Flu Ae | A/Mallard/NY/6750/78 (H2N2) | < 1 pg/uL | NEG | POS | NEG | NEG |
| A/duck/Hunan/795/2002 (H5N1) | < 1 pg/uL | NEG | POS | NEG | NEG |
| A/Vietnam/1194/2004 (H5N1) | < 1 pg/uL | NEG | POS | NEG | NEG |
| A/Anhui/01/2005 (H5N1) | < 1 pg/uL | NEG | POS | NEG | NEG |

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|  Virus | Strain | Target Conc. | Result  |   |   |   |
| --- | --- | --- | --- | --- | --- | --- |
|   |   |   |  SARS-CoV-2 | Flu A | Flu B | RSV  |
|   | A/Japanese white eye/Hong Kong/1038/2006 (H5N1) | < 1 pg/uL | NEG | POS | NEG | NEG  |
|   |  A/mallard/WI/34/75 (H5N2) | < 1 pg/uL | NEG | POS | NEG | NEG  |
|   |  A/turkey/Massachusetts/3740/1965 (H6N2) | 0.1 fg/uL | NEG | POS | NEG | NEG  |
|   |  A/duck/LTC-10-82743 (H7N2) | 5 fg/uL | NEG | POS | NEG | NEG  |
|   |  A/chicken/New Jersey/15086/3 (H7N3) | 4 fg/uL | NEG | POS | NEG | NEG  |
|   |  A/Anhui/1/2013 (H7N9) | 0.612 ng/uL | NEG | POS | NEG | NEG  |
|   |  A/Shanghai/1/2013 (H7N9) | NA† | NEG | POS | NEG | NEG  |
|   |  A/chicken/New Jersey/12220/1997 (H9N2) | 0.05 pg/uL | NEG | POS | NEG | NEG  |
|  |   |   |   |   |   |   |
|  Flu B | B/Lee/40 | 0.08 PFU/mL | NEG | NEG | POS | NEG  |
|   |  B/Allen/45 | 0.25 CEID50/mL | NEG | NEG | POS | NEG  |
|   |  B/GL/1739/54 | 0.50 CEID50/mL | NEG | NEG | POS | NEG  |
|   |  B/Maryland/1/59 | 0.2 CEID50/mL | NEG | NEG | POS | NEG  |
|   |  B/Taiwan/2/62 | 0.7 CEID50/mL | NEG | NEG | POS | NEG  |
|   |  B/Hong Kong/5/72 | 1 CEID50/mL | NEG | NEG | POS | NEG  |
|  |   |   |   |   |   |   |
|  Flu B (Victoria Lineage) | B/Panama/45/90 | 0.125 TCID50/mL | NEG | NEG | POS | NEG  |
|   |  B/Malaysia/2506/04 | 0.001 TCID50/mL | NEG | NEG | POS | NEG  |
|   |  B/Florida/02/06 | 0.004 TCID50/mL | NEG | NEG | POS | NEG  |
|   |  B/Brisbane/60/2008 | 0.005 TCID50/mL | NEG | NEG | POS | NEG  |
|   |  B/Maryland/15/2016 | 0.06 TCID50/mL | NEG | NEG | POS | NEG  |
|   |  B/Colorado/6/2017 | 0.01 TCID50/mL | NEG | NEG | POS | NEG  |
|   |  B/Hawaii/01/2018 | 1 TCID50/mL | NEG | NEG | POS | NEG  |
|   |  B/Missouri/12/2018 (NA D197E) | 1.2 TCID50/mL | NEG | NEG | POS | NEG  |
|   |  B/Washington/02/2019 | 60 TCID50/mL | NEG | NEG | POS | NEG  |
|  |   |   |   |   |   |   |
|  Flu B (Yamagata Lineage) | B/Florida/07/2004 | 0.03 TCID50/mL | NEG | NEG | POS | NEG  |
|   |  B/Florida/04/06 | 0.03 TCID50/mL | NEG | NEG | POS | NEG  |
|   |  B/Wisconsin/01/2010 | 0.025 CEID50/mL | NEG | NEG | POS | NEG  |
|   |  B/Wisconsin/10/2016 | 2 TCID50/mL | NEG | NEG | POS | NEG  |
|   |  B/Indiana/17/2017 | 0.5 TCID50/mL | NEG | NEG | POS | NEG  |
|   |  B/Oklahoma/10/2018 | 1 TCID50/mL | NEG | NEG | POS | NEG  |
|  |   |   |   |   |   |   |
|  RSV A | RSV-A/NY | 0.386 TCID50/mL | NEG | NEG | NEG | POS  |
|   |  RSV-A/WI-629.8.2/2007 | 0.50 TCID50/mL | NEG | NEG | NEG | POS  |
|   |  RSV-A/WI/629-11-1_2008 | 0.50 TCID50/mL | NEG | NEG | NEG | POS  |
|   |  RSV-A, Strain: 4/2015 Isolate #1 | 0.03 TCID50/mL | NEG | NEG | NEG | POS  |
|   |  RSV-A (2014, Isolate 342) | 0.38 IU/mL | NEG | NEG | NEG | POS  |
|   |  RSV-A (A2 cpts-248 mutant) | 1600 copies/mL | NEG | NEG | NEG | POS  |
|   |  RSV-A (2000/3-4) | 0.0015TCID50/mL | NEG | NEG | NEG | POS  |
|   |  RSV-A (2001/3-12) | 0.28 TCID50/mL | NEG | NEG | NEG | POS  |
|   |  RSV-A (1997/12-35) | 0.5 TCID50/mL | NEG | NEG | NEG | POS  |
|   |  RSV-A (Homo sapiens/ARG/177/2006) | 0.089 TCID50/mL | NEG | NEG | NEG | POS  |
|   |  RSV-A (1998/3-2) | 0.0089TCID50/mL | NEG | NEG | NEG | POS  |
|  |   |   |   |   |   |   |
|  RSV B | RSV-B/WV14617/85 | 0.04 TCID50/mL | NEG | NEG | NEG | POS  |

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|  Virus | Strain | Target Conc. | Result  |   |   |   |
| --- | --- | --- | --- | --- | --- | --- |
|   |   |   |  SARS-CoV-2 | Flu A | Flu B | RSV  |
|   | RSV-B-CH93(18)-18-01 | 0.004 TCID50/mL | NEG | NEG | NEG | POS  |
|   |  RSV-B (12/2014, Isolate #1) | 0.008 TCID50/mL | NEG | NEG | NEG | POS  |
|   |  RSV-B (cp23 Clone 1A2) | 4200 copies/mL | NEG | NEG | NEG | POS  |

${}^{a}$  One of three replicates was Invalid. The run was successfully repeated to obtain three valid replicates.
${}^{b}$  In vitro transcripts from Twist Biosciences.
$^{c}$ Influenza A/Indiana/02/2020 virus was without titer and the stock was diluted 48,000-fold in simulated matrix for testing.
dOne of three replicates yielded an ERROR result. The run was successfully repeated to obtain three valid replicates.
ePurified viral RNA in TE and diluted in simulated matrix was tested due to biosafety regulations.
$^{f}$ Inactivated avian influenza A (H7N9) viral RNA without viral titer was diluted 100,000-fold in simulated matrix for testing due to biosafety regulations.

# b. SARS-CoV-2 In silico Analysis

The inclusivity of the Xpert Xpress CoV-2/Flu/RSV plus test was evaluated using in silico analyses of assay amplicons in relation to the SARS-CoV-2 sequences available in the GISAID EpiCoV database as of June 15, 2022. For this analysis, the sequences were separated into lineages of interest based on the Pango Lineage assigned to each genome by GISAID, and those with ambiguous nucleotides were removed. BLAST (Basic Local Alignment Search Tool) was used to compare similarity between the amplicon sequences and the sequences in GISAID. The inclusivity analyses focused on the combined, nonambiguous sequences from variants of concern (VOC) or variants of interest (VOI) that may have important epidemiological, immunological, or pathogenic properties from a public health perspective, including Delta, Alpha, Omicron BA.1, Omicron BA.2, Omicron BA.2.12.1, Omicron BA.3, Omicron BA.4, Omicron BA.5, Eta, Iota, Kappa, Mu, Lambda, Zeta, and Theta, amongst others.

The evaluation included the following number of sequences per SARS-CoV-2 target: 10,310,839 sequences for the E target, 10,428,014 sequences for the N2 target, and 10,178,602 sequences for the RdRp target. The results of this analysis are shown in Table 8, stratified by the number of nucleotide mismatches between the amplicon and evaluated sequences.

Table 8. Results of SARS-CoV-2 In Silico Analyses

|  Amplicon | Exact Match | 1 Mismatch | 2 or More Mismatches | % Total <2 Mismatches  |
| --- | --- | --- | --- | --- |
|  E gene target | 99.5% (10,262,080 of 10,310,839) | 0.5% (47,959 of 10,310,839) | 0.01% (800 of 10,310,839) | 100%  |
|  N2 gene target | 98.1% (10,228,739 of 10,428,014) | 1.9% (194,319 of 10,428,014) | 0.05% (4,956 of 10,428,014) | 99.9%  |
|  RdRp gene target | 99.2% (10,092,873 of 10,178,602) | 0.8% (84,595 of 10,178,602) | 0.01% (1,134 of 10,178,602) | 100%  |

Based on the built-in redundancy of the Xpert Xpress CoV-2/Flu/RSV plus test's SARS-CoV-2 amplification system (i.e., 3 independent targets, only 1 of 3 must be detected to

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assign a positive result), it is not anticipated that any of the evaluated SARS-CoV-2 sequences would be missed by the Xpert Xpress CoV-2/Flu/RSV plus test.

# Cross-Reactivity/Microbial Interference

# a. Wet-Testing

# i. Cross-Reactivity

The analytical specificity (cross-reactivity) of the Xpert Xpress CoV-2/Flu/RSV plus test was evaluated by testing a panel of non-targeted microorganisms that may be found in a respiratory tract clinical specimen. Forty-eight (48) non-target microorganisms (Table 9) were evaluated in the study. Panels were composed of 1 to 8 different non-target microorganisms spiked into simulated matrix at  $\geq 1\times 10^{5}$ $\mathrm{TCID}_{50} / \mathrm{mL}$  (for viruses) and  $\geq 1\times 10^{6}$  CFU/mL (for bacteria and fungi), with the following exceptions: A live strain of Lactobacillus reutri was unavailable and therefore genomic DNA was tested at  $\geq 1\times 10^{6}$  copies/mL; similarly, whole, enveloped human coronavirus HKU1 was unavailable and therefore synthetic RNA was tested at  $\geq 1\times 10^{6}$  copies/mL. To evaluate cross-reactivity, each panel was tested in triplicate in the absence of the target microorganisms. No cross-reactivity was observed at the concentrations tested.

Table 9. Non-Target Microorganisms Evaluated During Cross-Reactivity Testing

|  Microorganism | Concentration | Microorganism | Concentration  |
| --- | --- | --- | --- |
|  Viruses |   | Bacteria  |   |
|  Human coronavirus NL63 | 1.17x105TCID50/mL | Acinetobacter baumannii | 1.30x107CFU/mL  |
|  MERS-coronavirus | 1.17x105TCID50/mL | Bordetella pertussis | 6.40x107CFU/mL  |
|  Human coronavirus 229E | 1.21x105TCID50/mL | Burkholderia cepacia | 1.90x108CFU/mL  |
|  Human coronavirus OC43 | 1.02x105TCID50/mL | Citrobacter freundii | 1.73x108CFU/mL  |
|  Human coronavirus HKU1a | 1.23x106copies/mL | Corynebacterium sp. | 1.27x107CFU/mL  |
|  Adenovirus Type 1 | 4.07x105TCID50/mL | Enterococcus faecalis | 5.87x107CFU/mL  |
|  Adenovirus Type 7 | 1.15x105TCID50/mL | Escherichia coli | 1.55x108CFU/mL  |
|  Cytomegalovirus | 1.00x105TCID50/mL | Hemophilus influenzae | 6.62x106CFU/mL  |
|  Echovirus | 1.14x105TCID50/mL | Lactobacillus reuteri b | 5.0x107copies/mL  |
|  Enterovirus | 2.80x105TCID50/mL | Legionella pneumophila | 1.42x108CFU/mL  |
|  Epstein Barr Virus | 5.60x106TCID50/mL | Moraxella catarrhalis | 2.46x106CFU/mL  |
|  HSV | 1.97x105TCID50/mL | Mycoplasma pneumoniae | 2.70x106CFU/mL  |
|  Human metapneumovirus | 4.07x105TCID50/mL | Neisseria meningitides | 4.20x106CFU/mL  |
|  Human parainfluenza Type 1 | 1.0x105TCID50/mL | Neisseria mucosa | 1.00x108CFU/mL  |
|  Human parainfluenza Type 2 | 1.2x105TCID50/mL | Propionibacterium acnes | 8.25x107CFU/mL  |
|  Human parainfluenza Type 3 | 1.2x105TCID50/mL | Pseudomonas aeruginosa | 1.05x107CFU/mL  |
|  Human parainfluenza Type 4 | 1.19x106TCID50/mL | Staphylococcus haemolyticus | 2.66x106CFU/mL  |
|  Measles | 1.20x105TCID50/mL | Staphylococcus aureus | 5.87x107CFU/mL  |
|  Mumps virus | 1.20x105TCID50/mL | Staphylococcus epidermidis | 2.47x107CFU/mL  |
|  Rhinovirus Type 1A | 1.00x105TCID50/mL | Streptococcus agalactiae | 1.75x107CFU/mL  |
|  Fungi |   | Streptococcus pneumoniae | 2.26x107CFU/mL  |
|  Candida albicans | 6.30x106CFU/mL | Streptococcus pyogenes | 9.00x106CFU/mL  |
|  Candida parapsilosis | 1.45x106CFU/mL | Streptococcus salivarius | 4.19x106CFU/mL  |
|   | Streptococcus sanguinis | 8.67x106CFU/mL  |   |
|   |   |  Chlamydia pneumoniae | 1.20x106CFU/mL  |
|   |   |  Mycobacterium tuberculosis (avirulent) | 1.20x106CFU/mL  |

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aLive virus was not available. Synthetic RNA was used.
bLive organism was not available. Genomic DNA was used.

## ii. Microbial Interference

A microbial interference study was conducted to assess potential inhibitory effects of a select panel of 10 non-target microorganisms (7 viruses and 3 bacteria) that may be found in a human respiratory specimen (Table 10). Viral strains were tested at ≥ 1×10⁵ TCID₅₀/mL (unless otherwise noted) and bacteria were tested at 1×10⁶ CFU/mL in the presence of one strain each of SARS-CoV-2 (USA/WA/1/2020), influenza A (A/Idaho/07/2018), influenza B (B/Washington/2/2019), RSV A (A/Australia/2/61) and RSV B (B/9320/MA/77) spiked individually at 3× LoD in negative simulated matrix. Data supporting the use of simulated matrix can be found in section VII Performance Characteristics.B.2.Sample Matrix and Transport Media Equivalency Study, later in this document. Testing was performed with replicates of eight for each of the target strain/potentially interfering strain combinations, and a negative simulated matrix control. Under the conditions of the study, no inhibitory effects were observed for each of the non-target microorganisms tested in the presence of the target analytes.

Table 10. Non-Target Microorganisms Tested in the Microbial Interference Study

|  Non-Target Microorganism | Concentration  |
| --- | --- |
|  Adenovirus Type 1C | 1.0×10⁵ TCID₅₀/mL  |
|  Human coronavirus OC43 | 1.0×10⁵ TCID₅₀/mL  |
|  Human Metapneumovirus 5, Type B1 | 1.0×10⁵ TCID₅₀/mL  |
|  Parainfluenza Type 1 | 1.0×10⁵ TCID₅₀/mL  |
|  Parainfluenza Type 2 | 1.0×10⁵ TCID₅₀/mL  |
|  Parainfluenza Type 3 | 1.0×10⁵ TCID₅₀/mL  |
|  Rhinovirus Type 1A | 1.0×10⁵ PFU/mL  |
|  Haemophilus influenzae | 1.0×10⁶ CFU/mL  |
|  Staphylococcus aureus | 1.0×10⁷ CFU/mL  |
|  Staphylococcus epidermidis | 1.0×10⁷ CFU/mL  |

## b. In silico

For SARS-CoV-2 in silico analyses, sequences for 45 microorganisms that are closely related or commonly encountered in a respiratory clinical specimen, were downloaded from GenBank. These sequences were analyzed with BLAST for homology to the primer/probe sequences for the SARS-CoV-2 targets included in the assay. For Flu and RSV in silico analyses, all sequences in GenBank (which encompasses essentially all species) were downloaded and were evaluated with BLAST for homology to the primer/probe sequences for the Flu A, Flu B, and RSV targets included in the assay. In addition to performing homology analyses with primer/probe sequences, homology to the amplicon was also assessed for SARS-CoV-2, Flu A, Flu B, and RSV. All sequences were downloaded from GenBank during the week of July 15, 2022.

For the SARS-CoV-2, Flu, and RSV in silico analyses, a non-target microorganism was determined to have no cross-reactivity when its sequence had &lt;80% homology with all the assay's primers and probes. When ≥80% homology was identified for a single assay, target primer or probe, an analysis of the remaining target primer/probe sequences was performed to determine if they exhibited ≥80% homology and thus may generate an

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amplicon that could be detected. For the SARS-CoV-2, Flu A, Flu B, and RSV in silico amplicon analyses, a non-target microorganism was determined to have no cross-reactivity if the amplicon shared  $&lt; 80\%$  homology to the non-target sequences.

For SARS-CoV-2, the E-gene target forward primer, reverse primer, and probe exhibited  $&gt;80\%$  homology to SARS-coronavirus from bats and humans and therefore these microorganisms may be amplified and detected. None of the remaining evaluated microorganisms have  $&gt;80\%$  homology to a SARS-CoV-2 target forward primer, reverse primer and probe and therefore are not expected to be amplified or detected.

For Flu A, Flu B, and RSV B, there were no sequences from organisms expected to be found in a human respiratory tract sample for which the forward primer, reverse primer, and probe all had  $\geq 80\%$  homology. The RSV A primer and probe oligonucleotides exhibited  $\geq 80\%$  homology with two Pangolin RSV A isolates. Therefore, the RSV A primers and probe may cross-react with Pangolin RSV A if the strain is circulating in a human population and present in a sample tested with the Xpert Xpress CoV-2/Flu/RSV plus test. While there was homology  $&gt;80\%$  to human genomic DNA, the matches were to different chromosomal regions, and there were no cases where a forward and reverse primer for a specific target matched to the same human genomic DNA fragment. For Flu A, Flu B, and RSV B, in silico amplicon analyses produced no matches with  $&gt;80\%$  homology.

While no matches of the RSV A amplicon to genomic sequences from non-RSV species sequences of  $&gt;80\%$  were observed, the RSV A amplicon shared a  $95\%$  identify with two Pangolin RSV A isolates.

# Interfering Substances

The performance of the Xpert Xpress CoV-2/Flu/RSV plus test was evaluated in the presence of medically and/or physiologically relevant concentrations of potentially interfering substances (listed in Table 11 below) that may be present in NPS or NS specimens.

Table 11. Potentially Interfering Substances Tested

|  Substance/Class | Description/Active Ingredient | Concentration Tested  |
| --- | --- | --- |
|  Control | Simulated NPS/NS Matrix | 100% (v/v)  |
|  Beta-Adrenergic Bronchodilator | Albuterol Sulfate | 0.83 mg/mL (equivalent to 1 dose per day)  |
|  Afrin Nasal Spray | Oxymetazoline | 15% (v/v)  |
|  BD Universal Transport Medium | Transport Medium | 100% (v/v)  |
|  Remel M4RT | Transport Medium | 100% (v/v)  |
|  Remel M5 | Transport Medium | 100% (v/v)  |
|  Copan Swab M Transport Medium | Transport Medium | 100% (v/v)  |
|  Blood | Blood (Human) | 2% (v/v)  |
|  Vaccine | FluMist Quadrivalent Vaccine | 6.7% (v/v)  |
|   |   |  6.7x10-4% (v/v)  |
|   |   |  6.7x10-6% (v/v)  |
|   |   |  6.7x10-7% (v/v)  |
|  Nasal Corticosteroid | Fluticasone Propionate | 5 ug/mL  |
|  Human Cells | Human Peripheral Blood Mononuclear Cells (PBMC) | 1x106cells/mL  |
|   |   |  0.5x106cells/mL  |

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For this study, 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 one SARS-CoV-2 strain, six influenza strains and four RSV strains. The panel of positive samples included one SARS-CoV-2 strain (USA-WA1/2020), two 2009 H1N1 strains (A/California/7/2009 and A/Idaho/07/2018), two influenza A H3N2 strains (A/Hong Kong/45/2019 and A/Victoria/361/2011), two influenza B strains (B/Wisconsin/10/2016 and B/Washington/2/2019), and four RSV strains (A/2/Australia/61, A/Long/MD/56, B/9320/MA/77, and B/WA/18537/62) spiked into simulated matrix to an analyte concentration of  $3\mathrm{x}$  LoD. Data supporting the use of simulated matrix can be found in section VII Performance Characteristics.B.2.Sample Matrix and Transport Media Equivalency Study, later in this document. If interference was observed for a particular substance /organism combination, the substance was diluted and retested until interference was no longer observed.

Interference was not observed for any of the evaluated substances, except FluMist, Human Peripheral Blood Mononuclear Cells (PBMCs), Snuff, and Zicam. The test results for these substances are shown in Table 12.

Table 12. Substances that Interfered with Detection of at Least One Target Organism

|  Substance | Conc. Tested | # Of Correct Results/Number Tested  |   |   |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |  No virus control | SARS-CoV-2 | Flu A 2009 H1N1a |   | Flu A H3N2b |   | Flu Bc |   | RSV Ad |   | RSV Be  |
|   |   |   |   |  1 | 2 | 1 | 2 | 1 | 2 | 1 | 2 | 1  |
|  FluMist | 6.7% | 8/8 | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA  |
|   |  6.7x10-4% (v/v) | NA | 7/8 | NA | NA | NA | NA | NA | NA | 0/8 | 0/8 | 2/8  |
|   |  6.7x10-6% (v/v) | NA | 8/8 | NA | NA | NA | NA | NA | NA | 8/8 | 7/8 | 8/8g  |

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Table 13. Competitive Interference Study Sample Panel Composition &amp; Study Results

|  Target 1 (Low conc.) |   | Target 2 (High conc.) |   | % Detected (# Detected / # Tested)  |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  Virus | Conc. | Virus | Conc. (RNA copies/mL) | SARS-CoV-2 | Flu A | Flu B | RSV |  |   |
|  SARS-CoV-2 | 414 copies/mL (3x LoD) | Flu A | 1.7x108 | 100% (3/3) | 100% (3/3) | 0% (0/3) | 0% (0/3) |  |   |
|   |   |  Flu B | 1.4x105 | 100% (3/3) | 0% (0/3) | 100% (3/3) | 0% (0/3) |  |   |
|   |   |  RSV A | 4.6x106 | 100% (3/3) | 0% (0/3) | 0% (0/3) | 100% (3/3) |  |   |
|  Virus | Conc. | Virus | Conc. (RNA copies/mL) | SARS-CoV-2 | Flu A | Flu B | RSV |  |   |
|   |   |  Flu A | 1.7x108 | 100% (3/3) | 100% (3/3) | 0% (0/3) | 0% (0/3) |  |   |
|   |   |  RSV B | 4.6x106 | 100% (3/3) | 0% (0/3) | 0% (0/3) | 100% (3/3) |  |   |

${}^{a}$  Flu A 2009 H1: (1) A/California/7/2009; (2) A/Idaho/07/2018
bFlu A H3N2: (1) A/Hong Kong/45/2019; (2) A/Victoria/361/2011
${}^{c}$  Flu B: (1) B/Wisconsin/10/2016; (2) B/Washington/2/2019
$^{\mathrm{d}}$ RSV A: (1) A/2/Australia/61; (2) A/Long/MD/56
eRSV B: (1) B/9320/MA/77; (2) B/WA/18537/62
One replicate reported NO RESULT. The run was successfully repeated to obtain the required number of valid replicates.
One replicate reported ERROR. The run was successfully repeated to obtain the required number of valid replicates.
${}^{\mathrm{b}}$  Two replicates reported ERROR. The two runs were successfully repeated to obtain the required number of valid replicates.

# Competitive Interference

The impact of competitive interference, caused by co-infections with on-target analytes, was evaluated for the Xpert Xpress CoV-2/Flu/RSV plus test by testing contrived samples consisting of individual SARS-CoV-2, Flu A, Flu B or RSV strains at 3x LoD in the presence of different target strains at high concentrations ( $\geq 1 \times 10^{5}$  RNA copies/mL, as determined by droplet digital PCR (ddPCR)). For this study, competitive interference was assessed using one strain each of SARS-CoV-2 (USA/WA/1/2020), influenza A (A/Idaho/07/2018), influenza B (B/Washington/2/2019), RSV A (A/Australia/2/61) and RSV B (B/9320/MA/77). Testing for each target strain (at low concentration) and each potential competitive strain (at high concentration) was performed in triplicate. No competitive interference was observed if all replicates for the low concentration target yielded positive results. If competitive interference was observed, the concentration of the competing virus was reduced by 10-fold increments until interference was no longer observed. The on-target analyte combinations evaluated, and the results of this evaluation are shown in Table 13.

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|   |  | RSV B | 1.9x105 | 100% (3/3) | 0% (0/3) | 0% (0/3) | 100% (3/3)  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  Flu A | 0.021 TCID50/mL (3x LoD) | SARS-CoV-2 | 1.0x106 | 100% (3/3) | 100% (3/3) | 0% (0/3) | 0% (0/3)  |
|   |   |  Flu B | 1.4x105 | 0% (0/3) | 100% (3/3) | 100% (3/3) | 0% (0/3)  |
|   |   |  RSV A | 4.6x106 | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3)  |
|   |   |  RSV B | 1.9x105 | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3)  |
|  Flu B | 38.7 CEID50/mL (3x LoD) | SARS-CoV-2 | 1.0x106 | 100% (3/3) | 0% (0/3) | 33% (1/3) | 0% (0/3)  |
|   |   |   |  1.0x105 | 100% (3/3) | 0% (0/3) | 100% (3/3) | 0% (0/3)  |
|   |   |  Flu A | 1.7x108 | 0% (0/3) | 100% (3/3) | 0% (0/3) | 0% (0/3)  |
|   |   |   |  1.7x107 | 0% (0/3) | 100% (3/3) | 0% (0/3) | 0% (0/3)  |
|   |   |   |  1.7x106 | 0% (0/3) | 100% (3/3) | 66.6% (2/3) | 0% (0/3)  |
|   |   |   |  1.7x105 | 0% (0/3) | 100% (3/3) | 100% (3/3) | 0% (0/3)  |
|   |   |  RSV A | 4.6x106 | 0% (0/3) | 0% (0/3) | 100% (3/3) | 100% (3/3)  |
|   |   |  RSV B | 1.9x105 | 0% (0/3) | 0% (0/3) | 100% (3/3) | 100% (3/3)  |
|  RSV A | 0.99 TCID50/mL (3x LoD) | SARS-CoV-2 | 1.0x106 | 100% (3/3) | 0% (0/3) | 0% (0/3) | 100% (3/3)  |
|   |   |  Flu A | 1.7x108 | 0% (0/3) | 100% (3/3) | 0% (0/3) | 0% (0/3)  |
|   |   |   |  1.7x107 | 0% (0/3) | 100% (3/3) | 0% (0/3) | 0% (0/3)  |
|   |   |   |  1.7x106 | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3)  |
|   |   |  Flu B | 1.4x105 | 0% (0/3) | 0% (0/3) | 100% (3/3) | 100% (3/3)  |
|  RSV B | 1.11 TCID50/mL (3x LoD) | SARS-CoV-2 | 1.0x106 | 100% (3/3) | 0% (0/3) | 0% (0/3) | 100% (3/3)  |
|   |   |  Flu A | 1.7x108 | 0% (0/3) | 100% (3/3) | 0% (0/3) | 100% (3/3)  |
|   |   |  Flu B | 1.4x105 | 0% (0/3) | 0% (0/3) | 100% (3/3) | 100% (3/3)  |

The study showed that Flu A at concentrations  $&gt;1.7 \times 10^{5}$  RNA copies/mL inhibited detection of Flu B at 3x LoD, and at Flu A concentrations  $&gt;1.7 \times 10^{6}$  RNA copies/mL inhibited detection of RSV A at 3x LoD (Table 13). In addition, SARS-CoV-2 at concentrations above  $1 \times 10^{5}$  RNA copies/mL inhibited detection of Flu B at 3x LoD (Table 13). No other competitive interference was observed for the potential co-infections evaluated in the study, at the concentrations tested.

# 4. Assay Reportable Range:

Not Applicable; this is a qualitative assay.

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5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):

a. Controls

External Control Evaluation

Two Zeptometrix NATtrol Molecular Controls, comprised of inactivated viruses in a purified protein matrix, were evaluated as external controls with the Xpert Xpress CoV-2/Flu/RSV plus test. Testing was performed with 20 replicates of three separate lots of the NATtrol SARS-CoV-2/Flu/RSV Positive Control (cat# NATFRC-6C-IVD) and the NATtrol SARS-CoV-2/Flu/RSV Negative Control (cat# NATCV9-6C-IVD) totaling 120 runs. All 120 runs provided valid and accurate results except for the following: A single Positive Control replicate was negative for Flu B and RSV.

b. Sample Stability

Room Temperature (15 to 30°C) and Refrigerated (2 to 8°C) Stability Studies

A stability study was conducted to establish transport and storage claims for eluted NPS specimens to be analyzed with the Xpert Xpress CoV-2/Flu/RSV plus test. Specimens included one SARS-CoV-2 strain (USA-WA1/2020), one influenza A strain (A/Idaho/07/2018), one influenza B strain (B/Washington/2/2019), and one RSV strain (RSV A/2/Australia/61) prepared in clinical NPS-UTM matrix and clinical NPS-eNAT matrix at a concentration of 3x LoD. Eight replicates of each of the positive specimens were tested at T0 (fresh) and at multiple time points following storage at 2°C, 8°C, 15°C, and 30°C. Negative samples were also included and tested in four replicates for each storage condition and temperature. 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 CoV-2/Flu/RSV plus. The study data supports the following specimen stability claims:

- VTM and eNAT: room temp (15-30°C) for up to 48 hours
- For VTM: refrigerated (2-8°C) for up to seven days
- eNAT: refrigerated (2-8°C) for up to six days

Freeze/Thaw Testing for Samples Frozen at -20°C or -80°C in VTM or eNAT

The performance of the Xpert Xpress CoV-2/Flu/RSV plus test with fresh and frozen specimens was evaluated for equivalency by testing co-spiked samples containing one SARS-CoV-2 strain (USA-WA1/2020), one influenza A strain (A/Idaho/07/2018), one influenza B strain (B/Washington/2/2019), and one RSV strain (RSV B/9320/MA/77) prepared in pooled negative clinical NPS-VTM matrix and pooled negative clinical NPS-eNAT matrix at a concentration of &lt;1x LoD, 2x LoD, and 5x LoD. Negative samples were also included and consisted of negative clinical NPS-VTM and negative clinical NPS-eNAT matrix.

Positive samples spiked at &lt;1x LoD, 5x LoD, and negative samples were tested in replicates of 10 per condition, while positive samples prepared at 2x were tested in replicates of 30 per condition. 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 either -20°C or -80°C for 24 hours after

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which they were thawed on ice prior to analysis with the Xpert Xpress CoV-2/Flu/RSV plus test.

For storage at -20°C under all conditions, all replicates for samples spiked at 2x and 5x LoD were positive, except for the following: one fresh sample spiked at 2x LoD in VTM was negative for Flu A, while a different fresh sample spiked at 2x LoD in VTM was negative for RSV. All negative samples were negative for all conditions tested. As expected, detection varied for samples spiked at &lt;1x LoD.

For storage at -80°C under all conditions, all samples spiked at 2x and 5x LoD were positive, while all negative samples were negative. As expected, detection varied for samples spiked at &lt;1x LoD.

The data from this study supports that NPS and NS samples collected in VTM and eNAT can undergo up to 1 freeze/thaw cycle when stored at -20°C or -80°C.

c. Kit Stability

Real-time kit stability data for the Xpert Xpress CoV-2/Flu/RSV plus test is currently available for eight 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 pre-defined performance criteria. Testing is ongoing for up to 37 months on seven cartridge lots.

d. Cartridge Hold Time

Xpert Xpress CoV-2/Flu/RSV plus 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 CoV-2/Flu/RSV plus test. Testing was performed on eight replicates each of one SARS-CoV-2 strain (USA-WA1/2020), one influenza A strain (A/Idaho/07/2018), one influenza B strain (B/Washington/2/2019), and one RSV strain (RSV A/2/Australia/61) spiked into negative simulated NPS matrix at an analyte concentration of 3x LoD. Negative specimens consisting of simulated NPS matrix were also included in the study. For negative specimens, eight replicates were collected for each condition and time point. The results of this study support a cartridge hold time of 4.5 hours.

6. Detection Limit:

The LoD of the Xpert Xpress CoV-2/Flu/RSV plus test was established using the WHO International Standard for SARS-CoV-2 (NIBSC, 20/146), 1 inactivated SARS-CoV-2 strain (USA-WA1/2020), and viral cultures of two Flu A H1 strains, 2 Flu A H3 strains, 2 Flu B strains (One Yamagata and one Victoria lineage), 2 RSV A strains, and 2 RSV B strains diluted into pooled negative clinical NPS matrix and negative clinical NS matrix. The LoD is defined as the lowest concentration for each strain at which 95% (19/20) of replicates yield a positive result.

Each virus strain was initially tested in a range finding study at ≥5 concentrations in replicates of 20 per concentration of virus. Range finding was performed with two cartridge

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lots for each virus/strain. Probit analysis was performed on the range finding results to estimate the LoDs, and the estimated LoDs were subsequently confirmed using two lots of Xpert Xpress CoV-2/Flu/RSV plus cartridges. For the confirmatory study, testing was also performed in replicates of 20 per concentration of virus. The highest (least sensitive) LoD value for the two lots evaluated in the confirmatory story was reported as the final LoD for the Xpert Xpress CoV-2/Flu/RSV plus test (see Table 14). The LoD for co-analyte spiked samples was also evaluated and shown to be equivalent to single analyte spiked samples.

Table 14. Xpert Xpress CoV-2/Flu/RSV plus Confirmatory LoD Study Results

|  Virus/Strain | Confirmed LoD Concentration  |   |
| --- | --- | --- |
|   |  NPS matrix | NS matrix  |
|  SARS-CoV-2, WHO International Standard | 94 IU/mL | 143 IU/mL  |
|  SARS-CoV-2, USA-WA1/2020 | 138 copies/mL | 64 copies/mL  |
|  Flu A/Idaho/07/2018 | 0.007 TCID50/mL | 0.012 TCID50/mL  |
|  Flu A/California/07/2009 | 0.0022 TCID50/mL | 0.0028 TCID50/mL  |
|  Flu A/Hong Kong/45/2019 | 0.44 FFU/mL | 0.49 FFU/mL  |
|  Flu A/Victoria/361/2011 | 0.05 TCID50/mL | 0.065 TCID50/mL  |
|  Flu B/Washington/2/2019 | 12.9 CEID50/mL | 26.3 CEID50/mL  |
|  Flu B/Wisconsin/10/2016 | 2.4 TCID50/mL | 2.41 TCID50/mL  |
|  RSV A/2/Australia/61 | 0.33 TCID50/mL | 0.28 TCID50/mL  |
|  RSV A/Long/MD/56 | 0.17 TCID50/mL | 0.22 TCID50/mL  |
|  RSV B/9320/MA/77 | 0.37 TCID50/mL | 0.24 TCID50/mL  |
|  RSV B/Wash/18537/62 | 0.2 TCID50/mL | 0.4 TCID50/mL  |

# 7. Assay Cut-Off:

The Xpert Xpress CoV-2/Flu/RSV plus test includes defined Ct value cutoffs for each of the viral targets as well as the SPC. These values were determined during pre-clinical testing and were subsequently confirmed in the clinical study. The Ct cutoffs are included as automatic calculations in the assay definition file (ADF) of the Xpert Xpress CoV-2/Flu/RSV plus test.

# 8. Accuracy (Instrument):

Not Applicable.

# 9. Carry-Over:

A study was conducted to assess whether the single-use, self-contained Xpert Xpress CoV-2/Flu/RSV plus cartridge prevents specimen and amplicon carryover by testing a negative sample immediately after testing of a high positive sample in the same GeneXpert module. The negative sample used in this study consisted of simulated NPS/NS matrix and the positive sample consisted of Flu B and SARS-CoV-2 spiked at high concentrations (i.e., Flu B/Wisconsin/10/2016 was spiked at  $1.0 \times 10^{6} \mathrm{TCID}_{50} / \mathrm{mL}$  and SARS-CoV-2 USA-WA1/2020 at  $1 \times 10^{4}$  copies/mL) into simulated NPS/NS matrix. The negative sample was tested in a GeneXpert module at the start of the study. Following the initial testing of the negative sample, the high positive sample was processed in the same GeneXpert module followed by another negative sample. This was repeated 20 times in the same module, resulting in 20 positives and 21 negatives for the module. The study was repeated using a second GeneXpert module for a total of 40 positive and 42 negative samples. All positive samples were correctly reported as positive, and all negative samples were correctly reported as negative. No specimen or amplicon carry-over contamination was observed.

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# B Comparison Studies:

1. Method Comparison with Predicate Device:
Not Applicable.

2. Matrix Comparison:

Sample Matrix and Transport Media Equivalency Study

The objective of this study was to establish equivalent performance of the Xpert Xpress CoV-2/Flu/RSV plus test between the following conditions:

a. clinical NPS matrix (in UTM/VTM), clinical NS matrix (in UTM/VTM), and simulated NPS/NS matrix in UTM/VTM.
b. clinical NPS matrix (in UTM/VTM) and clinical NPS matrix in eNAT.

Simulated NPS/NS background matrix consisted of 2.5% (w/v) porcine mucin and 1% (v/v) human whole blood in 0.85% sodium chloride (NaCl) formulated in 1x PBS solution with 15% glycerol and diluted into UTM/VTM at a 1:40 ratio. For this study, one SARS-CoV-2 strain (USA-WA1/2020), one influenza A strain (A/Idaho/07/2018), one Flu B strain (B/Wisconsin/10/2016) and two RSV strains (RSV A/2/Australia/61 and RSV B/9320/MA/77) were seeded into pooled negative matrices or simulated matrices at &lt;1x LoD, 1.5x LoD, 5x LoD, and 10x LoD. Aliquots of negative matrices were also included in the evaluation. Positive samples spiked at &lt;1x LoD, 5x LoD, 10x LoD and negative samples were tested in replicates of 10 per condition, while positive samples prepared at 1.5x LoD were tested in replicates of 30 per condition

For the conditions outlined in (a), all replicates for all samples spiked at 1.5x, 5x, and 10x LoD were positive for all evaluated matrices. All negative samples were negative for all matrices. As expected, detection varied for samples spiked at &lt;1x LoD.

For the conditions outlined in (b), all replicates for all samples spiked at 1.5x, 5x, and 10x LoD were positive for all evaluated matrices. All negative samples were negative for all matrices. As expected, detection varied for samples spiked at &lt;1x LoD.

The results for the conditions evaluated in (a) indicate that performance of the Xpert Xpress CoV-2/Flu/RSV plus test is equivalent with viruses seeded into clinical NPS matrix (in UTM/VTM), clinical NS matrix (in UTM/VTM), and simulated NPS/NS matrix in UTM/VTM and thus support use of simulated matrix in select analytical validation studies. The results for the conditions evaluated in (b) reveal that performance of the Xpert Xpress CoV-2/Flu/RSV plus test is equivalent with viruses seeded into clinical NPS matrix (in UTM/VTM) and clinical NPS matrix in eNAT.

# C Clinical Studies:

1. Prospective Study

The clinical performance of the Xpert Xpress CoV-2/Flu/RSV plus was established in a multi-center study conducted with nasopharyngeal swab (NPS) and anterior nasal swab (NS) specimens in VTM/UTM collected from individuals with signs and symptoms of respiratory

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infection. Specimens were prospectively collected (i.e., all comers between two time points that met the clinical study inclusion criteria) during portions of the 2021-2022 viral respiratory illness season. Due to low prevalence of Flu and RSV during the 2021-2022 viral respiratory illness season, archived prospectively collected specimens collected during the 2016-2017 influenza season were also enrolled. Specimens prospectively collected in 2022 were tested fresh (Category I) or after freezing (Category II). All specimens enrolled in 2016-2017 were Category II. In total, thirty-three (33) diverse clinical sites in the U.S. were involved in the prospective studies. Of these 33 sites, 5 sites participated in specimen collection only, 27 performed Xpert Xpress CoV-2/Flu/RSV plus testing and 1 site performed Xpert Xpress CoV-2/Flu/RSV plus testing, in addition to comparator and discordant analysis testing.

A total of 6,987 NPS and NS specimens were enrolled in the study. Of these, 6,100 specimens were enrolled in 2022 while the remaining 897 were collected in 2016-2017. Of the 6987 specimens, 19 were found to be ineligible after enrollment, 841 were excluded due to protocol deviations (e.g., specimen mislabeled, specimen not stored appropriately, etc.), and 6 were excluded because Xpert Xpress CoV-2/Flu/RSV plus test and/or comparator results were not provided by the testing sites. This left 6121 specimens for evaluation, of which 5331 (2672 NPS and 2659 NS) were collected in 2022 and 790 (422 NPS and 368 NS) were collected in 2016-2017. Patient demographic information for all evaluable specimens collected in 2022 and 2016-2017 is presented in Table 15.

Table 15. Demographic Data for Prospectively Collected Specimens

|  Specimens Collected in 2021-2022 | NPS (N=2672) | NS (N=2659) | Overall (N=5331)  |
| --- | --- | --- | --- |
|  Gender |   |   |   |
|  Female | 1568 (58.7%) | 1634 (61.5%) | 3202 (60.1%)  |
|  Male | 1104 (41.3%) | 1025 (38.5%) | 2129 (39.9%)  |
|  Age Group (Years) |   |   |   |
|  ≤5 | 9 (0.3%) | 183 (6.9%) | 192 (3.6%)  |
|  6-21 | 623 (23.3%) | 562 (21.1%) | 1185 (22.2%)  |
|  22-59 | 1676 (62.7%) | 1553 (58.4%) | 3229 (60.6%)  |
|  ≥60 | 364 (13.6%) | 361 (13.6%) | 725 (13.6%)  |
|  |   |   |   |
|  Specimens Collected in 2016-2017 | NPS (N=422) | NS (N=368) | Overall (N=790)  |
|  Gender |   |   |   |
|  Female | 211 (50.0%) | 223 (60.6%) | 434 (54.9%)  |
|  Male | 211 (50.0%) | 145 (39.4%) | 356 (45.1%)  |
|  Age Group (Years) |  |  |   |
|  ≤5 | 164 | 144 | 308  |
|  6-21 | 85 | 72 | 157  |

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|  22-59 | 134 | 111 | 245  |
| --- | --- | --- | --- |
|  ≥60 | 39 | 41 | 80  |

The Xpert Xpress CoV-2/Flu/RSV plus test was evaluated for SARS-CoV-2 performance by comparing to a U.S. FDA-cleared molecular respiratory panel that includes SARS-CoV-2. For SARS-CoV-2 performance evaluations, only specimens collected in 2022 were evaluated, as specimens collected prior to the COVID-19 pandemic (i.e., 2016-2017) were assumed to be SARS-CoV-2 negative. Of the 5331 evaluable specimens collected in 2022 and evaluated for investigational device SARS-CoV-2 performance, 261 were excluded due to non-evaluable/invalid comparator tests results, while an additional 19 were excluded because they were non-determinate (ND) upon retest. This left 5051 specimens (2536 NPS and 2515 NS) with valid Xpert Xpress CoV-2/Flu/RSV plus test and comparator SARS-CoV-2 results. Of the 2536 NPS specimens, 98.8% (2505/2536) were tested fresh, while 1.2% (31/2536) were tested frozen with the Xpert Xpress CoV-2/Flu/RSV plus. Of the 2515 NS specimens, 99.0% (2489/2515) were tested fresh, while 1.0% (26/2515) were tested frozen with the Xpert Xpress CoV-2/Flu/RSV plus.

The Xpert Xpress CoV-2/Flu/RSV plus test was evaluated for Flu A, Flu B, and RSV performance by comparing to a U.S. FDA-cleared molecular Flu A/B/RSV assay. For Flu and RSV performance calculations, specimens collected in both 2022 and 2016-2017 were evaluated. Of the 6,121 evaluable specimens collected during these time periods and evaluated for investigational device Flu and RSV performance, 142 were excluded due to non-evaluable/invalid comparator results, while an additional 25 were excluded because they yielded ND results upon retest. This left 5954 specimens (3011 NPS and 2943 NS) with valid Xpert Xpress CoV-2/Flu/RSV plus test and comparator Flu A, Flu B, and RSV results. Of the 3011 NPS specimens, 85.1% (2562/3011) were tested fresh, while 14.9% (449/3011) were tested frozen with the Xpert Xpress CoV-2/Flu/RSV plus. Of the 2943 NS specimens, 86.7% (2553/2943) were tested fresh, while 13.3% (390/2943) were tested frozen with the Xpert Xpress CoV-2/Flu/RSV plus.

The Xpert Xpress CoV-2/Flu/RSV plus test ND rate for specimens collected in 2022 and 2016-2017, combined, was initially 2.4% (148/6121) and decreased to 0.4% (25/6121) upon retest. For specimens collected in 2022, the ND was rate initially 2.5% (132/5331) and decreased to 0.4% (19/5331) upon retest. For specimens collected in 2016-2017, the ND rate was initially 2.0% (16/790) and decreased to 0.8% (6/790) upon retest.

A summary of the Xpert Xpress CoV-2/Flu/RSV plus test prospective clinical study performance is provided in Table 16 for NPS specimens and Table 17 for NS specimens. Positive Percent Agreement (PPA) was calculated as 100% × (TP / (TP + FN)). True positive (TP) indicates that both the Xpert Xpress CoV-2/Flu/RSV plus and the comparator method had a positive result for the specific analyte, and false negative (FN) indicates that the Xpert Xpress CoV-2/Flu/RSV plus was negative while the comparator result was positive. Negative Percent Agreement (NPA) was calculated as 100% × (TN / (TN + FP)). True negative (TN) indicates that both the Xpert Xpress CoV-2/Flu/RSV plus and the comparator method had negative results, and false positive (FP) indicates that the Xpert Xpress CoV-2/Flu/RSV plus was positive while the comparator result was negative. Specimens that obtained discordant SARS-CoV-2 results underwent additional testing with a U.S. FDA EUA SARS-CoV-2 molecular test, while specimens that obtained discordant Flu A, Flu B, or RSV results underwent additional testing with a U.S. FDA-cleared molecular respiratory panel.

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Table 16. Xpert Xpress CoV-2/Flu/RSV plus Assay Clinical Performance with NPS Specimens

|  Analyte | Positive Percent Agreement |   |   | Negative Percent Agreement  |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |  TP/(TP+FN) | % | 95% CI | TN/(TN+FP) | % | 95% CI  |
|  SARS-CoV-2 | Fresh | 454/468 | 97.0 | 95.0-98.2 | 2000/2037 | 98.2 | 97.5-98.7  |
|   |  Frozen | 8/8 | 100 | 67.6-100 | 23/23 | 100 | 85.7-100  |
|   |  Overall | 462/476a | 97.1 | 95.0-98.2 | 2023/2060b | 98.2 | 97.5-98.7  |
|  Flu A | Fresh | 98/100 | 98.0 | 93.0-99.5 | 2451/2462 | 99.6 | 99.2-99.8  |
|   |  Frozen | 93/93 | 100 | 96.0-100 | 343/356 | 96.3 | 93.9-97.9  |
|   |  Overall | 191/193c | 99.0 | 96.3-99.7 | 2794/2818d | 99.1 | 98.7-99.4  |
|  Flu B | Fresh | NA | NA | NA | 2562/2562 | 100 | 99.9-100  |
|   |  Frozen | 57/59 | 96.6 | 88.5-99.1 | 390/390 | 100 | 99.0-100  |
|   |  Overall | 57/59e | 96.6 | 88.5-99.1 | 2952/2952 | 100 | 99.9-100  |
|  RSV | Fresh | 12/12 | 100 | 75.8-100 | 2550/2550 | 100 | 99.8-100…

---

**Source:** [https://fda.innolitics.com/submissions/MI/subpart-d%E2%80%94serological-reagents/QOF/K231481](https://fda.innolitics.com/submissions/MI/subpart-d%E2%80%94serological-reagents/QOF/K231481)

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