Simplexa Bordetella Direct, Simplexa Bordetella Positive Control Pack

K173498 · Diasorin Molecular, LLC · OZZ · Aug 13, 2018 · Microbiology

Device Facts

• Record ID: K173498
• Device Name: Simplexa Bordetella Direct, Simplexa Bordetella Positive Control Pack
• Applicant: Diasorin Molecular, LLC
• Product Code: OZZ · Microbiology
• Decision Date: Aug 13, 2018
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3980
• Device Class: Class 2

Indications for Use

The DiaSorin Simplexa Bordetella Direct MOL2750 assay is an in vitro diagnostic test intended for use on the LIAISON MDX instrument for the qualitative detection and differentiation of Bordetella pertussis and Bordetella parapertussis nucleic acids from frozen nasopharyngeal (NPS) specimens from patients with signs and symptoms of Bordetella infection of the respiratory tract. The Simplexa Bordetella Direct MOL2750 assay is performed on the LIAISON MDX instrument and utilizes real-time PCR amplification to detect B. pertussis by targeting the IS481 insertional element of the B. pertussis genome and to detect B. parapertussis by targeting the IS1001 insertional element of the B. parapertussis genome. The IS481 insertional element can also be present in B. holmesii and B. bronchiseptica. Specimens collected from patients with respiratory infection caused by B. pertussis, B. holmesii or B. bronchiseptica may yield positive test results in IS481 assays. B. holmesii infection may cause clinical illness similar to B. pertussis, and mixed outbreaks involving both B. pertussis and B. holmesii infection have been reported. Additional testing should be performed if necessary to differentiate B. holmesii and B. pertussis. B. bronchiseptica is a rare cause of infection in humans. When clinical factors suggest that B. pertussis may not be the cause of respiratory infection, other clinically appropriate investigation(s) should be carried out in accordance with published guidelines. Negative results for the Simplexa Bordetella Direct MOL2750 assay do not preclude Bordetella infection and positive results do not rule out co-infection with other respiratory pathogens. Results from the Simplexa Bordetella Direct assay MOL2750 should be used with other clinical findings and epidemiological information as an aid in diagnosis of Bordetella infection. Test results should not be used as the sole basis for treatment or other patient management decisions. The Simplexa Bordetella Positive Control Pack MOL2760 is intended to be used as a control with the Simplexa Bordetella Direct kit. This control is not intended for use with other assays or systems.

Device Story

Real-time PCR assay for direct amplification/detection of B. pertussis (IS481 target) and B. parapertussis (IS1001 target) DNA from unprocessed nasopharyngeal swabs; no nucleic acid extraction required. Used on LIAISON MDX instrument; utilizes Direct Amplification Disc. Internal control included to monitor PCR inhibition/failure. Results interpreted automatically by LIAISON MDX Studio Software. Intended for clinical laboratory use to aid diagnosis; results used alongside clinical/epidemiological findings. IS481 target cross-reacts with B. holmesii/B. bronchiseptica; requires clinical correlation.

Clinical Evidence

Prospective study of 1,113 frozen NPS specimens. Performance compared to composite reference method (two real-time PCR assays plus bi-directional sequencing). B. pertussis PPA 91.9% (68/74), NPA 98.7% (1026/1039). B. parapertussis PPA 100% (13/13), NPA 99.6% (1096/1100). Contrived samples used to supplement B. parapertussis data.

Technological Characteristics

Real-time PCR; targets IS481 (B. pertussis) and IS1001 (B. parapertussis) insertion sequences. Direct amplification from unprocessed NPS (no extraction). Fluorescence-based detection. LIAISON MDX instrument platform. Assay definition loaded via QR barcode. 40-cycle PCR protocol.

Indications for Use

Indicated for qualitative detection and differentiation of B. pertussis and B. parapertussis nucleic acids in frozen nasopharyngeal (NPS) specimens from patients with signs/symptoms of respiratory tract infection.

Regulatory Classification

Identification

A respiratory viral panel multiplex nucleic acid assay is a qualitative in vitro diagnostic device intended to simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or viral culture. The detection and identification of a specific viral nucleic acid from individuals exhibiting signs and symptoms of respiratory infection aids in the diagnosis of respiratory viral infection when used in conjunction with other clinical and laboratory findings. The device is intended for detection and identification of a combination of the following viruses:(1) Influenza A and Influenza B; (2) Influenza A subtype H1 and Influenza A subtype H3; (3) Respiratory Syncytial Virus subtype A and Respiratory Syncytial Virus subtype B; (4) Parainfluenza 1, Parainfluenza 2, and Parainfluenza 3 virus; (5) Human Metapneumovirus; (6) Rhinovirus; and (7) Adenovirus.

Special Controls

*Classification.* Class II (special controls). The special controls are:(1) FDA's guidance document entitled “Class II Special Controls Guidance Document: Respiratory Viral Panel Multiplex Nucleic Acid Assay;” (2) For a device that detects and identifies Human Metapneumovirus, FDA's guidance document entitled “Class II Special Controls Guidance Document: Testing for Human Metapneumovirus (hMPV) Using Nucleic Acid Assays;” and (3) For a device that detects and differentiates Influenza A subtype H1 and subtype H3, FDA's guidance document entitled “Class II Special Controls Guidance Document: Testing for Detection and Differentiation of Influenza A Virus Subtypes Using Multiplex Nucleic Acid Assays.” See § 866.1(e) for the availability of these guidance documents.

Predicate Devices

• ARIES® Bordetella Assay for use with the ARIES® Systems (K163626)

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

A. 510(k) Number:
K173498

B. Purpose for Submission:
To obtain a substantial equivalence determination for the Simplex Bordetella Direct assay system for the direct amplification, detection and differentiation of *Bordetella pertussis* and *Bordetella parapertussis* DNA from unprocessed nasopharyngeal swabs (NPS).

C. Measurand:
Target DNA sequences of *Bordetella pertussis* insertion sequence, IS481 and *Bordetella parapertussis* insertion sequence, IS1001.

D. Type of Test:
Qualitative real-time polymerase chain reaction assay

E. Applicant:
DiaSorin Molecular LLC

F. Proprietary and Established Names:
Simplexa Bordetella Direct MOL2750
Simplexa Bordetella Positive Control Pack MOL 2760

G. Regulatory Information:
1. Regulation section:
866.3980
Respiratory viral panel multiplex nucleic acid assay
2. Classification:
Class II

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3. Product code:
OZZ, OOI

4. Panel:
Microbiology (83)

H. Intended Use:

1. Intended use(s):

Simplexa Bordetella Direct MOL2750

The DiaSorin Simplexa Bordetella Direct MOL2750 assay is an in vitro diagnostic test intended for use on the LIAISON MDX instrument for the qualitative detection and differentiation of *Bordetella pertussis* and *Bordetella parapertussis* nucleic acids from frozen nasopharyngeal (NPS) specimens from patients with signs and symptoms of Bordetella infection of the respiratory tract.

The Simplexa Bordetella Direct MOL2750 assay is performed on the LIAISON MDX instrument and utilizes real-time PCR amplification to detect B. pertussis by targeting the IS481 insertional element of the *B. pertussis* genome and to detect *B. parapertussis* by targeting the IS1001 insertional element of the *B. parapertussis* genome. The IS481 insertional element can also be present in *B. holmesii* and *B. bronchiseptica*. Specimens collected from patients with respiratory infection caused by *B. pertussis*, *B. holmesii* or *B. bronchiseptica* may yield positive test results in IS481 assays. *B. holmesii* infection may cause clinical illness similar to B. pertussis, and mixed outbreaks involving both *B. pertussis* and *B. holmesii* infection have been reported. Additional testing should be performed if necessary to differentiate *B. holmesii* and *B. pertussis*. *B. bronchiseptica* is a rare cause of infection in humans. When clinical factors suggest that *B. pertussis* may not be the cause of respiratory infection, other clinically appropriate investigation(s) should be carried out in accordance with published guidelines.

Negative results for the Simplexa Bordetella Direct MOL2750 assay do not preclude Bordetella infection and positive results do not rule out co-infection with other respiratory pathogens. Results from the Simplexa Bordetella Direct assay MOL2750 should be used with other clinical findings and epidemiological information as an aid in diagnosis of Bordetella infection. Test results should not be used as the sole basis for treatment or other patient management decisions.

Simplexa Bordetella Positive Control Pack MOL2760

The Simplexa Bordetella Positive Control Pack MOL2760 is intended to be used as a control with the Simplexa Bordetella Direct kit. This control is not intended for use with other assays or systems.

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2. Indication(s) for use:

Same as intended use.

3. Special conditions for use statement(s):

For prescription use only.

For in vitro diagnostic use only.

4. Special instrument requirements:

For use with the LIAISON MDX instrument.

I. Device Description:

The device is a real time PCR assay that enables the direct amplification, detection and differentiation of Bordetella pertussis and Bordetella parapertussis DNA from unprocessed nasopharyngeal swabs without nucleic acid extraction. The system consists of the Simplexa Bordetella Direct assay, the Liaison MDX (with Liaison MDX Studio Software), the Direct Amplification Disc and associated accessories. Primers and fluorescent probes are used together to amplify and detect Bordetella pertussis, Bordetella parapertussis and internal control targets. Insertion sequences IS481 and IS1001 are targeted to identify Bordetella pertussis and Bordetella parapertussis DNA respectively in the specimen. An internal control is used to detect PCR failure and/or inhibition. IS481 is also found in Bordetella holmseii which can cause clinical symptoms similar to Bordetella pertussis. Additional testing should be performed if necessary to differentiate between Bordetella holmesii and Bordetella pertussis.

Materials required but not supplied:

1. LIAISON MDX with LIAISON MDX Studio Software version 1.0 or higher.
2. Simplexa Bordetella Positive Control Pack (REF MOL2760).
3. 50 µL fixed volume pipette
4. Sterile, nuclease-free disposable pipette tips with filters (Extra Long tips ≥ 91 mm are recommended for pipetting directly from primary collection tubes.
5. Freezer (manual defrost) at -10 to -30°C (for kit components).
6. Refrigerator at 2 to 8°C (for specimens).
7. Disposable, powder-free gloves.
8. Freezer capable of -70°C (for specimens).
9. Vortex for mixing patient samples.

J. Substantial Equivalence Information:

1. Predicate device name(s):

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ARIES Bordetella Assay

2. Predicate 510(k) number(s):

K163626

3. Comparison with predicate:

Table 1. Similarities with Predicate Device

| Name | Predicate device (K163626): Aries Bordetella Assay for Aries Systems | Simplexa Bordetella Direct MOL2750 and Simplexa Bordetella Positive Control Pack MOL2760 (K173498) |
| --- | --- | --- |
| Intended Use | The ARIES Bordetella Assay is a real-time polymerase chain reaction (PCR) based qualitative in vitro diagnostic test for the direct detection and identification of Bordetella pertussis (B. pertussis) and Bordetella parapertussis (B. parapertussis) nucleic acid in nasopharyngeal swab (NPS) specimens obtained from individuals suspected of having a respiratory tract infection attributable to B. pertussis or B. parapertussis. The ARIES Bordetella Assay targets the B. pertussis toxin promoter and the B. parapertussis IS1001 insertion element in the genomes. When clinical factors suggest that B. pertussis or B. parapertussis may not be the cause of respiratory infection, other clinically appropriate investigation(s) should be carried out in accordance with published guidelines. Negative results for the ARIES Bordetella Assay do not preclude B. pertussis or B. parapertussis infection and positive results do not rule out co-infections with other respiratory pathogens. The direct detection and identification of B. pertussis and B. parapertussis nucleic acids from symptomatic patients aids in the diagnosis of B. pertussis and B. parapertussis respiratory infection in conjunction with other clinical findings and epidemiological | Simplexa Bordetella Direct MOL2750: The DiaSorin Molecular Simplexa Bordetella Direct MOL2750 assay is an in vitro diagnostic test intended for use on the LIAISON MDX instrument for the qualitative detection and differentiation of Bordetella pertussis and Bordetella parapertussis nucleic acids from frozen nasopharyngeal (NPS) specimens from patients with signs and symptoms of Bordetella infection of the respiratory tract. The Simplexa Bordetella Direct MOL2750 assay is performed on the LIAISON MDX instrument and utilizes real-time PCR amplification to detect B. pertussis by targeting the IS481 insertional element of the B. pertussis genome and to detect B. parapertussis by targeting the IS1001 insertional element of the B. parapertussis genome. The IS481 insertional element can also be present in B. holmesii and B. bronchiseptica. Specimens collected from patients with respiratory infection caused by B. pertussis, B. holmesii or B. bronchiseptica may yield positive test results in IS481 assays. B. holmesii infection may cause clinical illness similar to B. pertussis, and mixed outbreaks involving both B. pertussis and B. holmesii infection have been reported. Additional testing should be performed if necessary to differentiate B. holmesii and B. pertussis. B. bronchiseptica is a rare cause of |

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| Name | Predicate device (K163626): Aries Bordetella Assay for Aries Systems | Simplexa Bordetella Direct MOL2750 and Simplexa Bordetella Positive Control Pack MOL2760 (K173498) |
| --- | --- | --- |
| | information. The ARIES Bordetella Assay is indicated for use with the ARIES Systems. | infection in humans. When clinical factors suggest that B. pertussis may not be the cause of respiratory infection, other clinically appropriate investigation(s) should be carried out in accordance with published guidelines.

Negative results for the Simplexa Bordetella Direct MOL2750 assay do not preclude Bordetella infection and positive results do not rule out co-infection with other respiratory pathogens. Results from the Simplexa Bordetella Direct MOL2750 assay should be used with other clinical findings and epidemiological information as an aid in diagnosis of Bordetella infection. Test results should not be used as the sole basis for treatment or other patient management decisions.

Simplexa Bordetella Positive Control Pack MOL 2760:
The Simplexa Bordetella Positive Control Pack MOL2760 is intended to be used as a control with the Simplexa Bordetella Direct kit. This control is not intended for use with other assays or systems. |
| Assay Targets | B. pertussis Target B. pertussis and B. parapertussis toxin promoter (ptxA-pr), B. parapertussis Target IS1001 insertion element | The Simplexa Bordetella Direct assay detects the following multi-copy insertion sequence targets: Bordetella pertussis target IS481 and Bordetella parapertussis target IS1001 |
| Sample Types | Nasopharyngeal swabs in UTM, M5, M6 and ESwab | Nasopharyngeal swabs in Remel M5, Remel M6, UTM, and Liquid Aimes (ESwab) |
| Extraction Methods | None | None |
| Assay Methodology | Real-time system for detecting the presence / absence of Bordetella pertussis and Bordetella parapertussis DNA in clinical specimens. | PCR-based system for detecting the presence / absence of Bordetella pertussis and Bordetella parapertussis DNA in clinical specimens. |
| Detection Techniques | Different fluorescent reporter dyes for each target and melt analysis. | Multiplex assay using different reporter dyes for each target. |

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| Name | Predicate device (K163626): Aries Bordetella Assay for Aries Systems | Simplexa Bordetella Direct MOL2750 and Simplexa Bordetella Positive Control Pack MOL2760 (K173498) |
| --- | --- | --- |
| Optical Detection | Fluorescence Emissions and Detection | Fluorescence |
| Test Interpretation | Automated test interpretation and report generation. | Automated test interpretation and report generation. |

Table 2. Differences between Predicate Device

| Name | Aries Bordetella Assay for Aries Systems | Simplexa Bordetella Direct and Positive Control Pack |
| --- | --- | --- |
| B. pertussis target | Toxin promoter | IS481 |
| Sample types | Not applicable | Remel M4, Remel M4RT, VTM |
| Instrument | ARIES System, ARIES M1 System | LIAISON MDX |
| Time to result | Less than 2 hours | Approximately 1 hour |

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

Not applicable.

## L. Test Principle:

The Simplexa Bordetella Direct assay system is a real-time PCR assay that enables the direct amplification, detection and differentiation of *Bordetella pertussis* and *Bordetella parapertussis* DNA from unprocessed nasopharyngeal swabs (NPS) without nucleic acid extraction. The system consists of the Simplexa Bordetella Direct assay, the LIAISON MDX (with LIAISON MDX Studio Software), the Direct Amplification Disc and associated accessories.

In the Simplexa Bordetella Direct assay, primers and fluorescent probes are used together to amplify and detect *Bordetella pertussis*, *Bordetella parapertussis* and internal control targets. Insertion sequence regions of *Bordetella pertussis* (IS481) and *Bordetella parapertussis* (IS1001) are targeted to identify *Bordetella pertussis* and *Bordetella parapertussis* DNA respectively in the specimen. An internal control is used to detect PCR failure and/or inhibition. IS481 is also found in *Bordetella holmesii* which can cause clinical symptoms similar to *Bordetella pertussis*. Additional testing should be performed if necessary to differentiate between *Bordetella holmesii* and *Bordetella pertussis*.

## M. Performance Characteristics (if/when applicable):

1. Analytical performance:

**Precision/Reproducibility: Reproducibility Study:**

The Simplex Bordetella Direct assay was performed at three sites on five non-consecutive days per site. Each day of testing involved two runs with different operators

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between the runs. A panel of six parameters was assembled to complete this testing which consisted of a positive control, a negative NPS in UTM, and four contrived samples of analyte spiked into a negative matrix. The spiked samples included a low and medium level of detection for both Bordetella species. Ninety replicates (3 replicates x 2 runs x 5 days x 3 sites) were tested for each sample panel member. The control sets, positive control and negative UTM, were tested twice a day per instrument. The results for the inter-operator, Intra-operator, Inter-site, and Intra-site reproducibility studies show that the total percent coefficient of variation of $\leq 6.4\%$ .

Below are the summary result data:

Table 3. Qualitative Summary of Reproducibility

| Qualitative Summary of Reproducibility | | |
| --- | --- | --- |
| Sample Panel Member | Expected Qualitative Result | Observed Qualitative Result |
| Bordetella pertussis A639 – LP | ~95% of the replicates result into Bordetella pertussis Positive | 100.0% (90/90) Positive 95% CI: 95.9 to 100.0% |
| Bordetella pertussis A639 – MP | 100% of the replicates result into Bordetella pertussis Positive | 100.0% (90/90)* Positive 95% CI: 94.0 to 99.8% |
| Bordetella parapertussis A747 – LP | ~95% of the replicates result into Bordetella parapertussis Positive | 100.0% (90/90) Positive 95% CI: 95.9 to 100.0% |
| Bordetella parapertussis A747 – MP | 100% of the replicates result into Bordetella parapertussis Positive | 100.0% (90/90) Positive 95% CI: 95.9 to 100.0% |
| Native negative nasopharyngeal swab in UTM | 100% of the replicates result into Bordetella Negative | 100.0% (90/90) Negative 95% CI: 95.9 to 100.0% |
| Positive Control | 100% of the replicates result into Bordetella pertussis & Bordetella parapertussis Positive | 100.0% (90/90) Positive 95% CI: 95.9 to 100.0% |

*A dual detection was observed 1/90. Bordetella parapertussis was observed in a sample spiked only with Bordetella pertussis.

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Table 4. Qualitative Summary of Reaction Mix Inter-lot Reproducibility

| Qualitative Summary of Reaction Mix Inter-lot Reproducibility | | |
| --- | --- | --- |
| Frozen Panel Member | Expected Qualitative Result | Observed Qualitative Result |
| Bordetella pertussis A639 – Low Positive | ~95% of the tested replicates result into Bordetella pertussis Positive | 100.0% (36/36) BP Positive 95% CI: 90.4 to 100.0% |
| Bordetella pertussis A639 – Medium Positive | 100% of the tested replicates result into Bordetella pertussis Positive | 100.0% (36/36) BP Positive 95% CI: 90.4 to 100.0% |
| Bordetella parapertussis A747 - Low Positive | ~95% of the tested replicates result into Bordetella parapertussis Negative | 100.0% (36/36) BPP Positive 95% CI: 90.4 to 100.0% |
| Bordetella parapertussis A747 - Medium Positive | 100% of the tested replicates result into Bordetella parapertussis Positive | 100.0% (36/36) BPP Positive 95% CI: 90.4 to 100.0% |
| Negative | 100% of the tested replicates result into Bordetella Pertussis and Bordetella parapertussis Negative | 97.2% (35/36) Negative 95% CI: 85.5 to 99.9% |
| Positive Control | 100% of the tested replicates result into Bordetella Pertussis and Bordetella parapertussis Positive | 100.0% (36/36) Positive 95% CI: 90.4 to 100.0% |

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# Precision Study

# Positive Control Inter-lot Precision Study:

Three positive controls are 10 aliquots per kit at $100~\mu \mathrm{L}$ of inactivated Bordetella bacteria. Multiple lots were used to evaluate inter-lot reproducibility by performing 6 runs across three consecutive days with two runs per day by a single operator (. Each run included at least two replicates per positive control lot. A total of 37 runs were performed on four instruments. The results of this study provided $100\%$ qualitative reproducibility. The interlot coefficient of variance is $\leq 4.2\%$ . The Simplexa Bordetella Positive Control Pack (MOL2760) may be used as an external control for QC testing, training or proficiency testing. Each laboratory should establish its own QC ranges and frequency of QC testing based on applicable local laws, regulations, and good laboratory practice. Typical Ct values for the positive control range between 21 to 26. Detection of the Simplexa DNA internal Control is not required for a valid result.

Table 5. Mean Ct, SD and % CV for PC

| Quantitative Summary of PC Inter-Lot Reproducibility (Variance Components) | | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Analyte | N | Mea n | Inter-Day | | Inter-Run | | Inter-Lot | | Intra- | | Total | |
| | | | SD | %C | SD | %CV | SD | %C | SD | %C | SD | %CV |
| Bordetella pertussis (FAM) | 36 | 22.5 | 0.0 | 0.0 | 0.0 | 0.0 | 0.88 | 3.9 | 0.16 | 0.7 | 0.89 | 4.0 |
| Bordetella para-pertussis (CFR610) | 36 | 22.7 | 0.0 | 0.0 | 0.0 | 0.0 | 0.96 | 4.2 | 0.12 | 0.5 | 0.96 | 4.3 |
| IC (Q670) | 36 | 29.6 | 0.12 | 0.4 | 0.0 | 0.0 | 0.29 | 1.0 | 0.18 | 0.6 | 0.36 | 1.2 |

# Inter-lot Precision Studies:

The Simplexa Bordetella Direct assays was evaluated for lot-to-lot variability of the Simplexa Bordetella Direct Reaction Mix. Three lots of Reaction Mix were used to test six sample panel members, specifically a positive control, a negative NPS pooled UTM, and four contrived positive samples in negative matrix. The contrived samples were low and medium positive $B$ pertussis (strain A639) and low and medium positive $B$ parapertussis (strain A747). Each sample panel member was tested in duplicate with each Reaction mix for two runs per day for three non-consecutive days. In addition, a total of twelve sets of daily external controls were tested across four instruments during the study. A summary table of results is presented below:

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Table 6. Inter-lot Statistics of PC

| Descriptive Statistics of Ct Values by PC Lot & Overall | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Positive Control Lot # | | Bordetella pertussis Ct | | | | | | |
| | N | N | Mean | SD | %CV | Min | Max | Range |
| R31259 | 12 | 12 | 21.3 | 0.2 | 1.0 | 21 | 21.6 | 0.8 |
| R31261 | 12 | 12 | 23.1 | 0.3 | 1.2 | 23 | 23.7 | 0.9 |
| R31371 | 12 | 12 | 23.0 | 0.3 | 1.5 | 23 | 23.7 | 1.2 |
| All | 36 | 36 | 22.5 | 0.9 | 3.9 | 21 | 23.7 | 2.9 |

# b. Linearity/assay reportable range:

Not applicable because the Simplexa Bordetella Direct assay is qualitative.

# c. Traceability, Stability, Expected values (controls, calibrators, or methods):

The negative control for this device/assay is a non-templated control NPS sample and is not included in the kit. The Positive Control (MOL2761), which is the only component of the Simplexa Bordetella Positive Control Pack (MOL2760), shelf life was determined to be 12 months. Testing was conducted every 3 months past date of manufacture up to 12 months and one-month past expiration dating (OMPE). The same testing strategy was employed for the Simplexa Bordetella Reaction Mix (MOL2750) and all data supported 12-month stability for these reagents.

# d. Limit of detection:

The LoD for the Simplexa Bordetella Direct assay was determined on two $B$ pertussis strains (A639 and BAA-589) and two $B$ parapertussis strains (A747 and E595). Samples were prepared using quantified stocks serially diluted into native negative nasopharyngeal swab matrix in Universal Transport Media (UTM). The LoD was defined as the lowest concentration at which $95\%$ of replicates were positive for the respective species of Bordetella. For each strain, eight different concentrations of bacteria were spiked into UTM. Each concentration was tested in at least 32 replicates in a total of 201 runs over 17 non-consecutive days. A summary of the results for each species evaluated is provided in Table 7 below:

Table 7. Limits of Detection in CFU/mL

| Bordetella species | Bordetella strain | LoD Concentration (CFU/mL) |
| --- | --- | --- |
| Bordetella pertussis | A639 | 14.7 |
| | BAA-589 | 20.9 |
| Bordetella parapertussis | A747 | 347.3 |
| | E595 | 239.0 |

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The results of the LoD experiments showed that the LoD of the Simplexa Bordetella Direct assay for detection of $B$ pertussis is 14.7 and 20.9 CFU/mL for strains A639 and BAA-589, respectively. The LoD for $B$ parapertussis was is 347.3 and 239.0 CFU/mL for strains A747 and E595, respectively.

# e. Analytical Reactivity/Inclusivity:

The Simplexa Bordetella Direct assay was evaluated for analytical reactivity to 18 Bordetella strains (12 B. pertussis and 6 B. parapertussis) not tested in the LoD study. Samples were prepared by spiking each strain into negative NPS matrix at $2\mathrm{x}$ LoD. All 18 strains were detected as positive for B. pertussis at or below $80~\mathrm{CFU / mL}$ and for B. parapertussis at or below $590~\mathrm{CFU / mL}$ . In silico BLAST analysis demonstrated that the assay should detect at least 294 additional B. pertussis strains and 5 additional B. parapertussis strains.

Table 8. Inclusivity Results for B. pertussis

| Bordetella Strain | Concentration | Bordetella pertussis (IS481) Result (# detected/# tested) |
| --- | --- | --- |
| Bordetella pertussis BAA-1335 | 35.6 CFU/mL | 3/3 |
| Bordetella pertussis ATCC 8467 | 35.6 CFU/mL | 3/3 |
| Bordetella pertussis ATCC 9306 | 79.7 CFU/mL | 3/3 |
| Bordetella pertussis ATCC 12742 | 35.6 CFU/mL | 3/3 |
| Bordetella pertussis ATCC 51445 | 35.6 CFU/mL | 3/3 |
| Bordetella pertussis ATCC 53894 | 79.7 CFU/mL | 3/3 |
| Bordetella pertussis ATCC 8478 | 35.6 CFU/mL | 3/3 |
| Bordetella pertussis ATCC 12743 | 35.6 CFU/mL | 3/3 |
| Bordetella pertussis ATCC 9340 | 35.6 CFU/mL | 3/3 |
| Bordetella pertussis ATCC 9797 | 35.6 CFU/mL | 3/3 |
| Bordetella pertussis ATCC 10380 | 35.6 CFU/mL | 3/3 |
| Bordetella pertussis E431 | 35.6 CFU/mL | 3/3 |

Table 9. Inclusivity Results for B. parapertussis

| Bordetella Strain | Concentration | Bordetella parapertussis (IS1001) Result (# detected/# tested) |
| --- | --- | --- |
| Bordetella parapertussis ATCC 15311 | 586.3 CFU/mL | 3/3 |
| Bordetella parapertussis ATCC 15237 | 586.3 CFU/mL | 3/3 |
| Bordetella parapertussis ATCC 15989 | 586.3 CFU/mL | 3/3 |
| Bordetella parapertussis BAA-587 | 586.3 CFU/mL | 3/3 |
| Bordetella parapertussis C510 | 586.3 CFU/mL | 3/3 |
| Bordetella parapertussis E838 | 586.3 CFU/mL | 3/3 |

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# f. Analytical Specificity:

# Cross-Reactivity:

The Simplex Bordetella Direct was evaluated for potential cross reactivity with organisms that are closely related, cause similar clinical symptoms, or are present as normal flora in the nasopharynx. Microorganisms listed in Table 9 below were spiked at high concentrations into negative samples. Ninety-seven samples were tested with no cross-reactivity except for Bordetella holmesii which is expected to cross react since this species of Bordetella has the IS481 element present in Bordetella pertussis.

Table 10. Cross-reactivity Results

| Organism | Concentration | Bordetella pertussis(IS481)% Detection(# Detected/# Tested) | Bordetella parapertussis(IS1001)% Detection(# Detected/# Tested) |
| --- | --- | --- | --- |
| Baseline | N/A | 100% (45/45) | 100% (45/45) |
| Acinetobacter baumannii | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Acinetobacter lwoffii | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Adenovirus 1 | 1 x 105TCID50/mL | 100% (3/3) | 100% (3/3) |
| Adenovirus 31 | 1 x 105TCID50/mL | 100% (3/3) | 100% (3/3) |
| Arcanobacterium haemolyticum | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Bacillus cereus | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Bacteroides fragilis | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Bordetella avium | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Bordetella bronchiseptica | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Bordetella hinzi | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Bordetella holmseii F061 | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Bordetella petri | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Bordetella trematum | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Burkholderia cenocepacia | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Burkholderia cepacia | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Burkholderia multivorans | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Burkholderia thailandensis | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Candida albicans | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Candida glabrata | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Chlamydia pneumoniae | 1 x 106IFU/mL | 100% (3/3) | 100% (3/3) |
| Chlamydia trachmomatis | 1 x 106IFU/mL | 100% (3/3) | 100% (3/3) |
| Citrobacter freundii | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Clostridium difficile | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Coronavirus 229E | 1 x 105TCID50/mL | 100% (3/3) | 100% (3/3) |

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| Organism | Concentration | Bordetella pertussis (IS481) % Detection (# Detected/# Tested) | Bordetella parapertussis (IS1001) % Detection (# Detected/# Tested) |
| --- | --- | --- | --- |
| Coronavirus NL63* | 1 x 10^{4} TCID50/mL | 100% (3/3) | 100% (3/3) |
| Coronavirus OC43 | 1 x 10^{5} TCID50/mL | 100% (3/3) | 100% (3/3) |
| Corynebacterium diphtheriae | 1 x 10^{6} CFU/mL | 100% (3/3) | 100% (3/3) |
| Coxsackievirus A16 | 1 x 10^{5} TCID50/mL | 100% (3/3) | 100% (3/3) |
| Coxsackievirus B4 | 1 x 10^{5} TCID50/mL | 100% (3/3) | 100% (3/3) |
| Cytomegalovirus | 1 x 10^{5} TCID50/mL | 100% (3/3) | 100% (3/3) |
| Echovirus 6 | 1 x 10^{5} TCID50/mL | 100% (3/3) | 100% (3/3) |
| Echovirus 7 | 1 x 10^{5} TCID50/mL | 100% (3/3) | 100% (3/3) |
| Echovirus 9 | 1 x 10^{5} TCID50/mL | 100% (3/3) | 100% (3/3) |
| Echovirus 11 | 1 x 10^{5} TCID50/mL | 100% (3/3) | 100% (3/3) |
| Enterobacter aerogenes Z052 | 1 x 10^{6} CFU/mL | 100% (3/3) | 100% (3/3) |
| Enterobacter cloacae | 1 x 10^{6} CFU/mL | 100% (3/3) | 100% (3/3) |
| Enterococcus faecalis vanB | 1 x 10^{6} CFU/mL | 100% (3/3) | 100% (3/3) |
| Enterovirus 70 | 1 x 10^{5} TCID50/mL | 100% (3/3) | 100% (3/3) |
| Enterovirus 71 | 1 x 10^{5} TCID50/mL | 100% (3/3) | 100% (3/3) |
| Epstein-Barr Virus | 1 x 10^{5} copies/mL | 100% (3/3) | 100% (3/3) |
| Escherichia coli | 1 x 10^{6} CFU/mL | 100% (3/3) | 100% (3/3) |
| Fusobacterium necrophorum | 1 x 10^{6} CFU/mL | 100% (3/3) | 100% (3/3) |
| Haemophilus influenzae | 1 x 10^{6} CFU/mL | 100% (3/3) | 100% (3/3) |
| Haemophilus parainfluenzae | 1 x 10^{6} CFU/mL | 100% (3/3) | 100% (3/3) |
| HSV-1 (MacIntyre) | 1 x 10^{5} TCID50/mL | 100% (3/3) | 100% (3/3) |
| HSV-2 (G) | 1 x 10^{5} TCID50/mL | 100% (3/3) | 100% (3/3) |
| Influenza A/Swine/Iowa/15/30 H1N1* | 1 x 10^{4} TCID50/mL | 100% (3/3) | 100% (3/3) |
| Influenza B/Malaysia/2506/04* | 1 x 10^{4} TCID50/mL | 100% (3/3) | 100% (3/3) |
| Klebsiella oxytoca | 1 x 10^{6} CFU/mL | 100% (3/3) | 100% (3/3) |
| Klebsiella pneumoniae | 1 x 10^{6} CFU/mL | 100% (3/3) | 100% (3/3) |
| Lactobacillus acidophilus | 1 x 10^{6} CFU/mL | 100% (3/3) | 100% (3/3) |
| Lactobacillus plantarum 17-5 | 1 x 10^{6} CFU/mL | 100% (3/3) | 100% (3/3) |
| Legionella longbeachae | 1 x 10^{6} CFU/mL | 100% (3/3) | 100% (3/3) |
| Legionella pneumophila (Philadelphia) | 1 x 10^{6} CFU/mL | 100% (3/3) | 100% (3/3) |
| Listeria monocytogenes | 1 x 10^{6} CFU/mL | 100% (3/3) | 100% (3/3) |
| Measles | 1 x 10^{5} TCID50/mL | 100% (3/3) | 100% (3/3) |
| Metapneumovirus-9 | 1 x 10^{5} TCID50/mL | 100% (3/3) | 100% (3/3) |
| Moraxella catarrhalis Ne 11 | 1 x 10^{6} CFU/mL | 100% (3/3) | 100% (3/3) |

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| Organism | Concentration | Bordetella pertussis(IS481)% Detection(# Detected/# Tested) | Bordetella parapertussis(IS1001)% Detection(# Detected/# Tested) |
| --- | --- | --- | --- |
| Morganella morganii | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Mumps | 1 x 105TCID50/mL | 100% (3/3) | 100% (3/3) |
| Mycobacterium avium | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Mycobacterium tuberculosis(genomic DNA) | 1 x 106genome copies/mL | 100% (3/3) | 100% (3/3) |
| Mycoplasma hominis | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Mycoplasma pneumoniaeStrain M129 | 1 x 106CCU/mL | 100% (3/3) | 100% (3/3) |
| Neisseria elongata | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Neisseria gonorrhoeae | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Neisseria meningitidis | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Neisseria mucosa | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Parainfluenza 1 | 1 x 105TCID50/mL | 100% (3/3) | 100% (3/3) |
| Parainfluenza 2 | 1 x 105TCID50/mL | 100% (3/3) | 100% (3/3) |
| Parainfluenza 3* | 1 x 104TCID50/mL | 100% (3/3) | 100% (3/3) |
| Parainfluenza 4* | 1 x 104TCID50/mL | 100% (3/3) | 100% (3/3) |
| Parvimonas micra | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Peptostreptococcus anaerobius | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Proteus mirabilisZ050 | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Proteus vulgaris | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Pseudomonas aeruginosa | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Pseudomonas fluorescens | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Rhinovirus 1A* | 1 x 104TCID50/mL | 100% (3/3) | 100% (3/3) |
| RSV A | 1 x 105TCID50/mL | 100% (3/3) | 100% (3/3) |
| RSV B WV/14617/85 | 1 x 105TCID50/mL | 100% (3/3) | 100% (3/3) |
| Serratia liquefaciens | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Serratia marcescens | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Staphylococcus aureus(MRSA) | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Staphylococcus epidermidis(MRSE) | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Stenotrophomonas maltophilia | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Streptococcus anginosus | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Streptococcus canis | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Streptococcus dysgalactiae | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Streptococcus intermedius | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Streptococcus mitis | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Streptococcus mutans | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Streptococcus oryzae | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Streptococcus thermophilus | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Streptococcus thermophilus | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Streptococcus thermophilus | 1 x 106CFU/mL | 100% | 100% |
| Streptococcus thermophilus | 1 x 106CFU/mL | 100% | 100% |
| Streptococcus thermophilus | 1 x 106CFU/mL | 100% | 100% |
| Streptococcus thermophilus | 1 x 106CFU/mL | 100% | 100% |
| Streptococcus thermophilus | 1 x 106CFU/mL | 100% | 100% |

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| Organism | Concentration | Bordetella pertussis(IS481)% Detection(# Detected/# Tested) | Bordetella parapertussis(IS1001)% Detection(# Detected/# Tested) |
| --- | --- | --- | --- |
| Streptococcus pneumoniae | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Streptococcus pyogenes M1 | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Streptococcus salivarius | 1 x 106CFU/mL | 100% (3/3) | 100% (3/3) |
| Ureaplasma urealyticum | 1 x 106CCU/mL | 100% (3/3) | 100% (3/3) |
| Varicella Zoster Virus* | 1 x 104TCID50/mL | 100% (3/3) | 100% (3/3) |

# Interfering Substances:

Sixteen potentially interfering endogenous and exogenous substances were evaluated for potential interference with the Simplexa Bordetella Direct assay. The substances tested were Albuterol sulfate, Ampicillin powder, Azithromycin powder, Beclomethasone dipropionate, Blood, Chloraseptic sore throat spray, Ciprofloxacin, Erythromycin, Flonase Nasal Spray, Mucin, Mupirocin, Rifampicin, Robitussin DM, Saline Nasal spray, Sudafed PE, and Zicam spray. Each substance was evaluated in contrived samples that contained Bordetella pertussis and Bordetella parapertussis at low positive concentrations (approximately $1 - 2 \times \mathrm{LoD}$ ) and moderate positive concentrations (approximately $2 - 4 \times \mathrm{LoD}$ ). Results of the study demonstrated that there was no evidence of interference caused by the substances at the concentrations listed, both high and low concentrations.

# Competitive Interference:

The Simplexa Bordetella Direct assay was evaluated for competitive interference by testing whether the presence of a clinically relevant high concentration of either $B$ pertussis or $B$ parapertussis could affect detection of the other targeted Bordetella species when present at a low level. A low positive sample was contrived for each target by spiking B. pertussis or $B$ parapertussis separately (at approximately 2x LoD) into nasopharyngeal swab matrix in universal transport media (UTM) and a baseline Ct value was determined for each sample. High concentrations of the other Bordetella strain were then spiked into the low-level sample and tested. No competitive interference was observed. The results as shown in Table 10 below showed that a low level of $B$ pertussis was detected in the presence of a high level of $B$ parapertussis; similarly a low level of $B$ parapertussis was detected in the presence of a high level of $B$ pertussis.

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Table 11. Competitive Interference Results

| Baseline
(Low Concentration) | | Competitive Interferent
(High Concentration) | | Bordetella pertussis (IS481)
(#Detected/#Total) | Bordetella parapertussis
(IS1001)
(#Detected/#Total) |
| --- | --- | --- | --- | --- | --- |
| Species | 2 X LoD
CFU/mL | Species | CFU/mL | | |
| Bordetella pertussis | 29.4
CFU/mL | Bordetella parapertussis | 2x10^{7} CFU/mL | 3/3 | 3/3 |
| Bordetella parapertussis | 694.6
CFU/mL | Bordetella pertussis | 1X10^{7} CFU/mL | 3/3 | 3/3 |

g. Carry-Over/Cross-contamination Study:

The Simplexa Bordetella Direct assay was evaluated for the presence of contamination due to carry over in known negative specimens by alternating high positive samples with negative samples. No evidence of carry-over contamination was observed.

h. Assay cut-off:

The Simplex Bordetella Direct Ct cut-off for Bordetella pertussis and Bordetella parapertussis detection was determined during feasibility and verification studies. The preliminary Ct cut-off for IS481 target (Bordetella pertussis) and IS1001 target (Bordetella parapertussis) for analysis was set at 40 cycles. However, during verification, all studies were performed to 45 cycles for informational use. When verification data were evaluated, limit of detection (LoD) and clinical agreement results confirmed the setting of the Ct cut-off for both IS481 and IS1001 at 40 cycles. The verification LoD study detected all Bordetella pertussis and Bordetella parapertussis LoD concentration replicates prior to cycle 40. In addition, the verification clinical agreement study detected all Bordetella pertussis and Bordetella parapertussis positive clinical samples prior to cycle 40.

Based on the results of verification studies, the final Ct cut-off was established at 40 cycles for both IS481 (Bordetella pertussis) and IS1001 (Bordetella parapertussis) targets. Furthermore, the PCR cycling protocol was finalized at 40 cycles. The target Ct cut-offs and cycling protocol are stored in the Simplexa Bordetella Direct assay definition.

2. Comparison studies:

a. Method comparison with predicate device:

Not applicable

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a. Matrix comparison:

The Simplexa Bordetella Direct assay was evaluated for performance with five different collection and transport media types. The study also evaluated sample stability following multiple freeze-thaw cycles as well as extended storage in refrigerated and frozen states. The study was performed using eight LIAISON MDX instruments, and a single lot of Positive Control across 18 non-consecutive days. A total of 336 experimental runs were performed by four operators. Of these 336 runs, 194 were with contrived samples and 142 were for daily controls.

For this study, all samples were contrived in one of the following types of transport media: Copan Universal Transport Media (UTM; also known as Universal Viral Transport (BD UVT)), Remel M4, Remel M4RT, Remel M5 and Remel M6. For each media type, 42 samples were contrived at various concentrations from 2x LoD up to 50x LoD, for Bordetella pertussis and/or Bordetella parapertussis.

Results from the study demonstrated that Bordetella pertussis and/or Bordetella parapertussis were detected appropriately in each sample, in each collection and transport media type at each time point. There was no difference in the detectability of the samples between the media types. Detection of Bordetella pertussis and Bordetella parapertussis in samples prepared at 2x LoD was confirmed in all five collection and transport media types and across all storage conditions tested.

Fresh vs. Frozen

The Simplexa Bordetella Direct assay was evaluated for performance with five different collection and transport media types. Multiple freeze thaw cycles, as well as extended storage in refrigerated and frozen states, were evaluated for their performance on the assay. The types of media examined include: Copan Universal Transport Media, Remel M4, Remel M4RT, Remel M5, and Remel M6. For each media type 42 samples were contrived at various concentrations from 2x LoD up to 50x LoD for both B. pertussis and B. parapertussis. Samples were tested immediately following preparation, fresh, subjected to refrigeration for up to seven days followed by storage at -70°C for at least 30 days and then freeze-thawed four times. The results of this study showed no statistically significant effect due to the storage conditions and/or sample collection and transport media based on the two sided 95% confidence interval of the percent detection observed.

Liquid Amies media was also evaluated on the Simplexa Bordetella Direct assay. This study examined multiple freeze-thaw cycles as well as extended storage in refrigerated and frozen states. Samples were contrived with Copan Universal Transport Media or liquid Amies medium spiked with various concentrations from 2x LoD up to 50x LoD for both B. pertussis and B. parapertussis. A total of 136 experimental runs were performed by four operators over seven non-consecutive days. Samples were tested immediately following preparation, fresh, subjected to refrigeration for up to 7 days followed by storage at -70°C for at least 30 days and then freeze-thawed four times. The overall results of this study indicate a slightly increased average Ct value for samples in liquid Amies medium compared to samples in UTM; however, among the samples in Amies medium there was no effect due to the storage conditions.

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3. Clinical studies:

Prospective Study:

A total of one thousand one hundred thirteen (1,113) unique prospectively collected frozen nasopharyngeal swab specimens were obtained from female and male patients having signs and symptoms of Bordetella pertussis or Bordetella parapertussis from six (6) clinical sites. The Simplexa test was performed at each of the clinical sites while the comparator was performed at one central laboratory. All samples were tested within 72 hours of collection. The performance of the assay was compared to a composite reference method consisting of two well-characterized real-time PCR assays (for each bacterial pathogen) followed by confirmation of positive PCR amplification products with bi-directional sequencing. Samples were characterized as negative if both comparator PCR assays were negative.

B. pertussis Sequencing Assay 1

The assay targets the IS481 repeat element, nucleotides 740 to 1,050 of GeneBank sequence ID AB473880. AB473880 is 1,055 base pairs. The Bordetella pertussis Sequencing Assay 1 region is upstream from the IS481 region targeted by the Simplexa Bordetella Direct assay. There is no detection region overlap between Bordetella pertussis Sequence Assay 1 and the Simplexa Bordetella Direct assay.

B. parapertussis Sequencing Assay 1

The assay targets the IS1001 repeat element, nucleotides 834 to 1,145 of GeneBank sequence ID JX013521. JX013521 is 1,190 base pairs. The probe and primers utilized by the comparator method assay for IS1001 do not overlap with the Simplexa Bordetella Direct assay.

B. pertussis Sequencing Assay 2

The assay targets the IS481 repeat element, nucleotides 224 to 542 of GeneBank sequence ID AB473880. AB473880 is 1,055 base pairs. Bordetella pertussis Sequencing Assay 2 region is upstream from the region targeted by the Simplexa Bordetella Direct assay. There is no detection region overlap between Bordetella pertussis Sequencing Assay 2 and the Simplexa Bordetella Direct assay.

B. parapertussis Sequencing Assay 2

The assay targets the IS1001 repeat element, nucleotides 377 to 679 of GeneBank sequence ID JX013521. JX013521 is 1,190 base pairs. Bordetella parapertussis Sequencing Assay 2 region is downstream from the region targeted by the Simplexa Bordetella Direct assay. There is no detection region overlap between BPP Sequence Assay 2 and Simplexa Bordetella Direct assay.

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Discrepant analysis was performed for B. pertussis samples using a FDA cleared NAAT. The invalid rate for the prospective clinical study using Simplexa Bordetella Direct was 1.73% (30/1740 runs).

Contrived Specimen Study:

For B. parapertussis, 56 contrived specimens were evaluated at clinical study sites. Specimens were prepared at various concentrations across the clinical range of the assay (2-50 x LoD). Results are shown in Table 13.

The tables below show the agreement of Simplexa Bordetella Direct versus Bi-directional Sequencing.

Clinical Performance for Bordetella pertussis:

Table 12. Simplexa Bordetella Direct Assay performance for B. pertussis

| Specimen Description | PPA | | 95%CI | NPA | | 95% CI |
| --- | --- | --- | --- | --- | --- | --- |
| Prospective Banked (Frozen) | 68/74 | 91.9% | 83.4% - 96.2% | 1026/1039 | 98.7% | 97.9.% - 99.3% |
| Contrived (containing only Bordetella parapertussis) | 0/0 | N/A | N/A | 112/112 | 100.0% | 96.7% - 100.0% |

Clinical Performance for Bordetella parapertussis:

Table 13. Simplexa Bordetella Direct Assay performance for Bordetella parapertussis

| Specimen Description | PPA | | 95%CI | NPA | | 95% CI |
| --- | --- | --- | --- | --- | --- | --- |
| Prospective Banked (Frozen) | 13/13 | 100.0% | 77.2% - 100.0% | 1096/1100 | 99.6% | 99.1% - 99.9% |
| Contrived | 56/56 | 100.0% | 93.6% - 100.0% | 0/56 | 0.0% | N/A |

The overall positivity observed in the prospective clinical study with the Simplex Bordetella Direct Assay was 6.1% and 1.2% for Bordetella pertussis and Bordetella parapertussis, respectively. The range for positivity from site to site was 0.0% to 33.3% for Bordetella pertussis and 0.0% to 2.1% for Bordetella parapertussis (reference lab had contrived samples equivalent to 50.0%).

4. Clinical Cut-off

Not applicable

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# 5. Expected Values/Reference Ranges

The observed positivity for Bordetella in the prospective population during the Simplexa Bordetella Direct clinical studies varied between institutions. Results are shown in the following tables. The percent positivity with the Simplexa Bordetella Direct at each site varied from $3.3\%$ and $9.4\%$ for Bordetella pertussis to $0.0\%$ and $2.4\%$ for Bordetella parapertussis.

Table 14. Total Enrolled Specimens

| Prevalence | Bordetella pertussis | Bordetella parapertussis |
| --- | --- | --- |
| Overall | 7.2%(82/1134) | 1.5%(19/1310) |

Table 15. Enrolled Specimens by Site

| Site | Bordetella pertussis | Bordetella parapertussis |
| --- | --- | --- |
| 1 | 9.4% (20/213) | 2.4% (7/288) |
| 2 | 5.9% (3/51) | 1.7% (1/59) |
| 3 | 7.9% (29/369) | 1.2% (5/406) |
| 4 | N/A | N/A |
| 5 | 3.3% (4/122) | 0.7% (1/140) |
| 6 | 6.9% (26/379) | 1.2% (5/405) |
| 7 | N/A | 0.0% (0/12) |

# N. Instrument Name:

LIAISON MDX

# O. System Descriptions:

# 1. Modes of Operation:

Does the applicant's device contain the ability to transmit data to a computer, webserver, or mobile device?

Yes

Does the applicant's device transmit data to a computer, webserver, or mobile device using wireless transmission?

No

# 2. Software:

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

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Yes

3. Specimen Identification:

The QR barcode card contains a digital representation of all the assay parameters required to load the assay definition onto the system. It also contains lot specific information. The Assay Definition information is scanned into the software by using the card or by importing the Assay Definition file into the database. Valid assay definition files may be pre-loaded into the database or loaded at the time of installation by DiaSorin Molecular Customer Service personnel.

4. Specimen Sampling and Handling:

Nasopharyngeal swab specimens are used to collect a nasopharyngeal sample which is then placed in transport media (list the claimed transport media here). Samples are loaded on the DAD consumable and run on the LIAISON MDX machine.

5. Calibration:

The assay does not require calibration. The assay contains an internal control for PCR function. DiaSorin Molecular offers optional external QC materials that are intended for use with the assay. Controls are packaged in single use aliquots and stored frozen, once thawed the controls are stable for 30 minutes at laboratory temperature.

6. Quality Control:

The Simplexa Bordetella Direct assay, primers and fluorescent probes are used together to amplify and detect B. pertussis and B. parapertussis and internal control targets. Insertion sequences IS481 and IS1001 are targeted to identify B. pertussis and B. parapertussis, respectively, in the specimen. An internal control is used to detect PCR failure and/or inhibition.

Describe the external controls here or refer to the above section where the external control kit is described.

P. Other Supportive Instrument Performance Characteristics Data Not Covered In The "Performance Characteristics" Section above: N/A

Q. Proposed Labeling:

The labeling supports the finding of substantial equivalence for this device.

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R. Conclusion:

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

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