LYRA PARAINFLUENZA VIRUS ASSAY

K141927 · Quidel Corporation · OOU · Oct 9, 2014 · Microbiology

Device Facts

• Record ID: K141927
• Device Name: LYRA PARAINFLUENZA VIRUS ASSAY
• Applicant: Quidel Corporation
• Product Code: OOU · Microbiology
• Decision Date: Oct 9, 2014
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3980
• Device Class: Class 2
• Attributes: Pediatric

Indications for Use

The Lyra™ Parainfluenza Virus Assay is a Real-Time PCR assay for the qualitative detection and identification of human parainfluenza virus types 1, 2 and 3 viral RNA from nasal and nasopharyngeal swab specimens from symptomatic patients. It is intended for use as an aid in the differential diagnosis of parainfluenza virus types 1, 2 and 3. This test is not intended to detect Parainfluenza 4a or Parainfluenza 4b viruses. Negative results do not preclude parainfluenza virus infection and should not be used as the sole basis for treatment or other patient management decisions.

Device Story

The Lyra™ Parainfluenza Virus Assay is an in vitro diagnostic test for qualitative detection of HPIV-1, HPIV-2, and HPIV-3 viral RNA. Input: nasal or nasopharyngeal swab specimens. Process: nucleic acid extraction using bioMérieux NucliSENS® easyMAG®, followed by multiplex Real-Time RT-PCR amplification and detection on the Applied Biosystems® 7500 Fast Dx instrument. The assay uses target-specific primers and fluorescent-labeled probes (FAM, JOE, Tex Red, CY5) to identify viral targets and an internal process control. Output: qualitative identification of PIV-1, PIV-2, and PIV-3. Used in clinical laboratory settings by trained personnel. Results aid clinicians in differential diagnosis of respiratory infections; negative results are presumptive and do not exclude infection. Benefits include rapid, sensitive, and specific molecular identification of parainfluenza viruses compared to traditional culture or DFA methods.

Clinical Evidence

Prospective multi-center study (n=1241) and retrospective study (n=105). Prospective study compared Lyra assay to DSFA and cell culture with DFA. PIV-1 sensitivity 100% (10/10), specificity 99.8%; PIV-2 sensitivity 100% (5/5), specificity 100%; PIV-3 sensitivity 100% (17/17), specificity 99.6%. Retrospective study compared Lyra to Prodesse ProParaFlu+ assay; PPA/NPA were high across all types. Discrepant analysis using additional RT-PCR confirmed several Lyra-positive/comparator-negative samples as true positives.

Technological Characteristics

Real-Time RT-PCR assay. Targets: PIV-1 (nuclear protein gene), PIV-2 (phosphate protein gene), PIV-3 (phosphate protein gene). Detection via fluorescent probes (FAM, JOE, Tex Red, CY5). Instrumentation: Applied Biosystems 7500 Fast Dx. Extraction: bioMérieux NucliSENS® easyMAG®. Form factor: single-tube multiplex reaction. Connectivity: standalone instrument.

Indications for Use

Indicated for symptomatic patients requiring differential diagnosis of human parainfluenza virus types 1, 2, and 3 via nasal or nasopharyngeal swab specimens. Not indicated for detection of Parainfluenza 4a or 4b.

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

• Prodesse ProParaflu™+ Assay (K091053)

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY AND INSTRUMENT COMBINATION TEMPLATE

A. 510(k) Number:
K141927

B. Purpose for Submission:
To obtain a Substantial Equivalence Determination for a new 510(k) application for Lyra™ Parainfluenza Virus Assay

C. Measurand:
The Lyra™ Parainfluenza Virus Assay detects viral RNA from Parainfluenza 1, 2, and 3 viruses. The primer and probe sets amplify and detect the Parainfluenza type 1 nuclear protein gene, Parainfluenza type 2 phosphate protein gene, and Parainfluenza type 3 phosphate protein gene, using real time reverse transcriptase polymerase chain reaction.

D. Type of Test:
The Lyra™ Parainfluenza Virus Assay is a multiplex nucleic acid detection assay that uses real time polymerase chain reaction.

E. Applicant:
Quidel Corporation
12544 High Bluff Drive, Suite 200
San Diego, California 92130
Telephone: [phone]
Fax: [phone]

F. Proprietary and Established Names:
Lyra™ Parainfluenza Virus Assay

G. Regulatory Information:
1. Regulation section:
21CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay
2. Classification:
Class II
3. Product code:
OOU

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4. Panel:

Microbiology (83)

H. Intended Use:

1. Intended use(s):

The Lyra™ Parainfluenza Virus Assay is a Real-Time PCR assay for the qualitative detection and identification of human parainfluenza virus types 1, 2 and 3 viral RNA from nasal and nasopharyngeal swab specimens from symptomatic patients. It is intended for use as an aid in the differential diagnosis of parainfluenza virus types 1, 2 and 3. This test is not intended to detect Parainfluenza 4a or Parainfluenza 4b viruses.

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

2. Indication(s) for use:

Same as intended use.

3. Special conditions for use statement(s):

This is a prescription only test. The assay is intended for use in hospital, reference, or state laboratory settings. The device is not intended for point-of-care use.

4. Special instrument requirements:

The assay can be performed using the Applied Biosystems® 7500 Fast Dx.

I. Device Description:

The Lyra™ Parainfluenza Virus Assay detects viral RNA of Parainfluenza 1, 2 and 3 viruses in nasal and nasopharyngeal swab specimens. First the nucleic acid is extracted from the patient sample using the NucliSENS® easyMAG® System. The extracted nucleic acid is then introduced into the master mix which contains the primers, probes, revise transcriptase, and TaqMan® chemistry and the reaction is loaded into the Applied Biosystems® (ABI) 7500 Fast Dx instrument. First, the reverse transcriptase generates a complimentary DNA strand from the viral RNA. The primer sets hybridize to the Parainfluenza type 1 nuclear protein gene, Parainfluenza type 2 phosphate protein gene, and Parainfluenza type 3 phosphate protein gene, and transcription and DNA amplification of the target sequence occur using real time reverse transcriptase polymerase chain reaction. Fluorescent-labeled probes bind to the target sequences and the dye is released from the probe containing quencher via the TaqMan® polymerase 5'-3' exonuclease activity. The un-quenched dye is detected in the ABI 7500 Fast Dx and if sufficient fluorescence is detected the result is reported as positive.

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J. Substantial Equivalence Information:

1. Predicate device name(s):

Prodesse ProParaflu™+ Assay

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

K091053

3. Comparison with predicate:

| | Subject Device: Lyra Parainfluenza Virus Assay | Predicate Device: Prodesse ProParaflu™+ Assay K091053 |
| --- | --- | --- |
| Intended Use | The Lyra™ Parainfluenza Virus Assay is a Real-Time RT-PCR assay for the qualitative detection and identification of human parainfluenza virus types 1, 2 and 3 viral RNA from nasal and nasopharyngeal swab specimens from symptomatic patients. It is intended for use as an aid in the differential diagnosis of parainfluenza virus types 1, 2 and 3. This test is not intended to detect Parainfluenza 4a or Parainfluenza 4b viruses.
Negative results do not preclude parainfluenza virus infection and should not be used as the sole basis for treatment or other patient management decisions. | The ProParaflu+ Assay is a multiplex Real Time RT-PCR in vitro diagnostic test for the qualitative detection and discrimination of Parainfluenza I Virus, Parainfluenza 2 Virus and Parainfluenza 3 Virus (HPIV-1, HPIV-2 and HPIV-3) nucleic acids isolated and purified from nasopharyngeal (NP) swab specimens obtained from individuals exhibiting signs and symptoms of respiratory tract infections. This assay targets the conserved regions of the Hemagglutinin- Neuraminidase (HN) gene of HPIV-1, HPIV-2 and HPIV-3, respectively. The detection and discrimination of HPIV- I, HPIV-2 and HPIV-3 nucleic acids from symptomatic patients aid in the diagnosis of human respiratory tract parainfluenza infections if used in conjunction with other clinical and laboratory findings. This test is not intended to detect Parainfluenza 4a or Parainfluenza 4b Viruses.
Negative test results are presumptive and should be confirmed by cell culture. |

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| | | Negative results do not preclude Parainfluenza 1, 2 or 3 virus infections and should not be used as the sole basis for treatment or other management decisions. |
| --- | --- | --- |
| DNA Amplification Technology | Real time polymerase chain reaction | Same |
| Target Sequence Detected | Parainfluenza type 1 nuclear protein gene, Parainfluenza type 2 phosphate protein gene, Parainfluenza type 3 phosphate protein gene | Conserved regions of the Hemagglutinin-Neuraminidase (HN) gene of HPIV-1, HPIV-2 and HPIV-3 |
| Sample Types | Nasal and Nasopharyngeal swabs | Nasopharyngeal swabs |
| Extraction | NucliSENS® easyMAG™ (bioMérieux) | MagNA Pure LC System (Roche), NucliSENS® easyMAG™ (bioMérieux) |
| Detection Techniques | Applied Biosystems 7500 Fast Dx | Cepheid SmartCycler II |

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

Class II Special Controls Guidance Document: Respiratory Viral Panel Multiplex Nucleic Acid Assay

## L. Test Principle:

The assay detects viral nucleic acids that have been extracted from a patient respiratory sample. A multiplex Real-time RT-PCR reaction is carried out under optimized conditions in a single tube generating amplicons for PIV-1, PIV-2, PIV-3, and the Process Control (PRC). Identification of PIV-1, PIV-2, PIV-3, and the PRC occurs by the use of target-specific primers and fluorescent-labeled probes that hybridize to conserved regions in the genomes of PIV-1, PIV-2, PIV-3, and the PRC.

| Lyra™ Probe Targets | |
| --- | --- |
| Virus | Target |
| PIV-1 | Nuclear Protein gene |
| PIV-2 | Phosphate Protein gene |
| PIV-3 | Phosphate Protein gene |
| Lyra™ Probe Labels | |
| --- | --- |
| Target | Dye |
| PIV-1 | FAM |
| PIV-2 | JOE |
| PIV-3 | Tex Red |
| PRC | CY5 |

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M. Performance Characteristics (if/when applicable):

1. Analytical performance:

a. Precision/Reproducibility:

The reproducibility studies were conducted over 14 days and testing was performed on 5 non-consecutive days at two clinical sites and one in-house laboratory. Each site was provided with 5 complete panels, which were stored at -70°C until used. Operators were blinded to the sample identity, which was randomized each day. Testing was conducted following the package insert instructions for use. One kit lot was used to perform the study. One strain of each parainfluenza virus was used at 3 different concentrations, 5x LoD, 2x LoD and 0.5x LoD for the Moderate Positive, Low Positive and High Negative samples respectively. The following strains were used; Parainfluenza-1 strain C35, Parainfluenza-2 strain Greer, Parainfluenza-3 strain C243.

The following table shows the summary of results for the reproducibility study. The results show that the assay had appropriate reproducibility at both the external clinical sites and the in-house laboratory. The reproducibility for this device is acceptable. The instances where the expected result differed from the actual result are within what is reasonable for C₅ and C₉₅ samples.

| | Site 1 | | | Site 2 | | | Site 3 | | | Combined | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Rate of Detection | Ave. Ct | %CV | Rate of Detection | Ave. Ct | %CV | Rate of Detection | Ave. Ct | %CV | Rate of Detection | Ave. Ct | %CV |
| PIV-1 High Negative | 18/30 | 32.3 | 5.6 | 7/30 | 32.7 | 4.2 | 11/30 | 32.4 | 4.9 | 36/90 | 32.4 | 5 |
| PIV-1 Low Positive | 30/30 | 27.3 | 4.3 | 30/30 | 26.3 | 2.7 | 30/30 | 27.1 | 9.1 | 90/90 | 26.9 | 6.3 |
| PIV-1 Moderate Positive | 30/30 | 24.5 | 2.8 | 30/30 | 23.8 | 2.6 | 30/30 | 24.2 | 3.8 | 90/90 | 24.2 | 3.3 |
| PIV-1 Negative | 0/30 | N/A | N/A | 0/30 | N/A | N/A | 0/29 | N/A | N/A | 0/89 | N/A | N/A |
| | | | | | | | | | | | | |
| PIV-2 High Negative | 24/30 | 30.2 | 6.7 | 2/30 | 34.3 | 7 | 6/30 | 32.9 | 7.1 | 32/90 | 31 | 7.7 |
| PIV-2 Low Positive | 30/30 | 25.4 | 4.7 | 30/30 | 27 | 4.3 | 30/30 | 27.9 | 9.4 | 90/90 | 26.8 | 7.7 |
| PIV-2 Moderate Positive | 30/30 | 22.8 | 1.9 | 30/30 | 23.7 | 2.8 | 30/30 | 24.5 | 7.6 | 90/90 | 23.7 | 5.7 |
| PIV-2 Negative | 0/30 | N/A | N/A | 0/30 | N/A | N/A | 0/29 | N/A | N/A | 0/89 | N/A | N/A |
| | | | | | | | | | | | | |

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| PIV-3 High Negative | 30/30 | 29.1 | 5.8 | 29/30 | 32.2 | 4.4 | 14/30 | 32 | 3.6 | 73/90 | 30.9 | 6.7 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| PIV-3 Low Positive | 30/30 | 25.5 | 6 | 30/30 | 26.1 | 2.9 | 30/30 | 26.9 | 8.2 | 90/90 | 26.2 | 1.7 |
| PIV-3 Moderate Positive | 30/30 | 22.8 | 1.7 | 30/30 | 23.6 | 3 | 30/30 | 24.3 | 5.5 | 90/90 | 23.2 | 4.6 |
| PIV-3 Negative | 0/30 | N/A | N/A | 0/30 | N/A | N/A | 0/29 | N/A | N/A | 0/89 | N/A | N/A |
| | | | | | | | | | | | | |
| PIV-1 Positive Control | 30/30 | 20.6 | 9.1 | 30/30 | 22.6 | 8.7 | 30/30 | 19.3 | 3.4 | 90/90 | 21 | 11.2 |
| PIV-2 Positive Control | 30/30 | 17.2 | 1.8 | 30/30 | 19.4 | 4.3 | 30/30 | 20.6 | 2 | 90/90 | 19.1 | 8 |
| PIV-3 Positive Control | 30/30 | 16.7 | 7.3 | 30/30 | 19.2 | 8.2 | 30/30 | 18.5 | 2.3 | 90/90 | 18.3 | 11.5 |
| Negative Control | 0/30 | N/A | N/A | 0/30 | N/A | N/A | 0/30 | N/A | N/A | 0/90 | N/A | N/A |

* One (1) replicate had an invalid PRC value and was removed for analysis.

b. Baseline, Threshold and Cut-off:

Baseline:

The baseline was determined using 38 parainfluenza virus negative clinical specimens. Next, known positive samples were tested and analyzed using two different baseline settings, manual and auto baseline on the ABI 7500 Fast Dx. When using the manual baseline background noise was reduced for all channels but the Ct values for all samples were changed, in some cases more than 2Cts. This resulted in a false positive for PIV-1 in a PIV-2 sample. This did not occur with the auto baseline setting. Therefore the auto baseline setting was used for all future analysis and is the chosen baseline setting in the package insert.

Signal Threshold:

For the ABI 7500 Fast Dx Instrument, the threshold is defined as the level of fluorescence above which the ABI 7500 Fast Dx software will assign a "positive" call. The software uses the baseline and threshold values to calculate the threshold cycle (Ct).

On the ABI 7500 Fast Dx Platform, PIV-1 and PIV-3 targets required no threshold changes from the default threshold of $1.00\mathrm{E} + 05$ baseline-corrected normalized reporter units $(\Delta \mathrm{Rn})$. However, the initial PIV-2 target threshold of $1.00\mathrm{E} + 05\Delta \mathrm{Rn}$ resulted in a loss of one positive specimen previously determined positive by culture with DFA confirmation. After re-analyzing the PIV-2 target using a lower threshold $(8.00\mathrm{E} + 04\Delta \mathrm{Rn})$, this specimen had a detectable Ct value. Therefore, the data indicated that lowering the threshold for this target would allow for maximum sensitivity to detect late positive specimens, but did not decrease specificity (no false positives were detected with the lower threshold). The threshold for

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PIV-3 (1.60E+05 ΔRn) was raised to compensate for excess background fluorescence noise resulting from the auto baseline setting in this channel. These threshold levels are the levels used for all future analysis and are the chosen threshold settings in the package insert.

## Cut-Off:

The cut-off was determined using the threshold for PIV-1 (1.00E+05), PIV-2 (8.00E+04) and PIV-3 (1.60E+05). Auto baseline was used for all targets. These conditions follow the package insert directions. Most Ct values were recorded between 18-27 cycles; however, at least 15 percent of samples were recorded after 30 cycles. The figure below shows the Ct Distribution for all three PIV targets from the ABI 7500 Fast Dx. The highest Ct value was detected at 44 cycles. The Ct range on this platform was from 17 cycles to 44 cycles, indicating that the cutoff should be at 45 cycles. Forty five cycles is the cut-off indicated in the package insert.


## c. Stability:

## Kit Stability:

Three kit lots were used to determine the real-time stability of the kit reagents when being stored at 4°C. PIV-1, PIV-2 and PIV-3 samples were tested at 2x LoD for each time point. Testing was conducted on days 0, 5, 7, 10, 15, 21, 38, 56, 76 and 90 (3 months), testing was then conducted monthly from months 4-11 and will continue through month 25. The following strains were used; Parainfluenza-1 strain C35, Parainfluenza-2 strain Greer, Parainfluenza-3 strain C243. Testing was conducted according to the directions in the package insert. Testing showed there was no change in Ct value greater than 3 Ct, which would indicate a significant change in detection and loss of stability, during the first 11 months of storage. Stability of the kit will be listed as 12 months in the package insert, and will be modified as more real time stability data is available.

## Rehydrated Master Mix Stability:

The stability of rehydrated master mix, when stored at -20°C, 4°C, and at room temperature, was determined in this study. Testing was conducted on the ABI 7500 Dx according to the package instructions. Parainfluenza-1 strain C35, Parainfluenza-2 strain Greer, Parainfluenza-3 strain C243 were tested at 2x LoD. Testing for the 4°C and room

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temperature conditions was performed at 1, 2, 4, 8, 9, 24, 28, 48, 52, 72 and 76 hours. Testing for the $-20^{\circ}\mathrm{C}$ condition was performed at 24, 28, 48, 52, 72 and 76 hours. Viral RNA was extracted only once (a minimum of 24 extractions), the RNA was then pooled, aliquoted and stored at $-70^{\circ}\mathrm{C}$ until used. Each strain was tested in triplicate, with three separate vials of rehydrated master mix, for every time and temperature condition. At the end of each incubation period an aliquot of viral RNA was added to the rehydrated Master Mix and loaded into the ABI $7500\mathrm{Dx}$ . The time point selected for stability claims in the package insert is the time point one point earlier than where all replicates were not detected. The table below shows the stability of the rehydrated Master Mix.

| Storage Temperature | Stability Time Limit |
| --- | --- |
| Room Temperature | 2 hours |
| 2-8°C | 4 hours |
| -20°C | 52 hours |

# Extracted Specimen Stability:

The stability of extracted samples was tested at Room Temperature, $4^{\circ}\mathrm{C}$ , $-20^{\circ}\mathrm{C}$ , and $-70^{\circ}\mathrm{C}$ at various time points. Testing was conducted on the ABI 7500 Dx according to the package instructions. Parainfluenza-1 strain C35, Parainfluenza-2 strain Greer, and Parainfluenza-3 strain C243 were prepared at 3X LoD in negative nasal matrix and used in this study. Sufficient volume was prepared to extract RNA for testing all time points. Samples stored at $-20^{\circ}\mathrm{C}$ and $-70^{\circ}\mathrm{C}$ were also tested for stability after 1, 2, and 3 freeze/thaw cycles. The table below shows the different time points tested for each temperature. Testing showed that there was no change in Ct value greater than 3 Ct, which would indicate a significant change in detection and loss of stability, at any of the time points tested. The package insert will list the stability of extracted RNA as stable if stored at Room Temperature, $4^{\circ}\mathrm{C}$ , $-20^{\circ}\mathrm{C}$ and $-70^{\circ}\mathrm{C}$ for up to 30 days.

| Temperature | Hours post extraction | Days post extraction |
| --- | --- | --- |
| Room Temperature | 4, 8 and 9 | 1, 2, 3, 7, 14, 21, 28, 30, 31 |
| 4°C | 4, 8 and 9 | 1, 2, 3, 7, 14, 21, 28, 30, 31 |
| -20°C | N/A | 1, 2, 3, 7, 14, 21, 28, 30, 31 |
| -70°C | N/A | 1, 2, 3, 7, 14, 21, 28, 30, 31 |

# d. Expected values (controls, calibrators, or methods):

No external controls are provided with this assay.

There is an internal control called the 'process control' (PRC). It is the MS2 bacteriophage (Zeptometrix) which is a single stranded RNA virus that can be extracted, amplified, and detected by primers and probes included in the master mix. Detection of the PRC indicates that assay conditions allowed proper sample extraction, amplification, and detection.

# e. Limit of Detection:

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The limit of detection (LOD) (defined as the lowest $\mathrm{TCID}_{50} / \mathrm{mL}$ that is detected $\geq 95\%$ of the time) of the Lyra Parainfluenza Virus Assay was determined on the ABI 7500 Fast Dx platform using three separate validation lots of lyophilized master mix and limiting dilutions of viral stocks. Parainfluenza-1 strain C35, Parainfluenza-2 strain Greer, and Parainfluenza-3 strain C243 were used in this study. Twenty replicates for each dilution of each parainfluenza virus type were prepared and tested. Viral stocks were diluted in negative nasal matrix and then extracted and stored for up to one month at $-70^{\circ}\mathrm{C}$ . Testing was then conducted according to the package insert. Data showing the results for each parainfluenza virus strain using each validation lot was provided at one concentration above and below the determined LoD. The table below shows the LoD determined for each parainfluenza virus type.

| Virus | LoD on ABI 7500 Fast Dx | Average Ct Value at LoD |
| --- | --- | --- |
| Parainfluenza-1, strain C-35 | 2.50 TCID50/mL | 38.0 |
| Parainfluenza-2, strain Greer | 250 TCID50/mL | 37.0 |
| Parainfluenza-3, strain C243 | 80 TCID50/mL | 38.1 |

# f. Analytical Inclusivity:

In silico analysis to demonstrate inclusivity for all three parainfluenza virus types was performed. No laboratory studies were performed because there are a very limited number of characterized strains of any human parainfluenza virus type. In silico analysis was performed comparing the sequence of the chosen target region for each parainfluenza virus type with the NCBI database for human parainfluenza virus sequences. Three hundred and eight human parainfluenza virus type 1, 458 human parainfluenza virus type 2, and 509 parainfluenza virus type 3 full-genome sequences were analyzed. Primers and probes were designed against consensus sequences found for each virus type and have $100\%$ homology to all sequences analyzed but do not have homology with other parainfluenza virus types. Therefore, in silico analysis and primer and probe design demonstrates that the assay should detect all human parainfluenza viruses of types 1, 2, and 3.

# g. Analytical Specificity/Cross-Reactivity:

# Cross Reactivity:

This study was designed to determine potential cross-reactivity of the assay with non-parainfluenza respiratory viruses, yeast, and bacteria. Organisms were sourced from DHI, Zeptometrix, or ATCC. Bacteria were tested at $1.00\mathrm{E} + 6$ cfu/ml or higher and viruses were tested at $1.00\mathrm{E} + 05$ TCID50.mL or higher, stock concentrations permitting. Stocks were diluted in negative nasal matrix and the samples were extracted and amplified according to the package insert instructions. All samples were tested in triplicate starting from the extraction step. The results were analyzed for any cross-reactivity for all three parainfluenza virus types and are listed below. No Cross-reactivity was observed in this study.

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| Organism | Vendor | Final Concentration TCID50/mL or CFU/mL | Result (Avg. Ct) |
| --- | --- | --- | --- |
| Adenovirus type 1 | DHI | 1.00E+05 | Negative |
| Coronavirus 229E | DHI | 1.00E+05 | Negative |
| Coronavirus NL63 | Zeptometrix | 7.05E+04 | Negative |
| Coronavirus OC43 | DHI | 1.00E+05 | Negative |
| Coxsackievirus B4:ODH-42385 | DHI | 1.00E+05 | Negative |
| Coxsackievirus B5:ODH-594484 | DHI | 1.00E+05 | Negative |
| Cytomegalovirus | DHI | 1.00E+05 | Negative |
| Echovirus 6 | DHI | 7.60E+08 | Negative |
| Echovirus 7 | DHI | 1.00E+05 | Negative |
| Echovirus 9 | DHI | 1.00E+05 | Negative |
| Echovirus 11 | DHI | 1.00E+05 | Negative |
| Enterovirus 70 | DHI | 1.00E+05 | Negative |
| Enterovirus 71 | DHI | 1.00E+05 | Negative |
| Epstein Barr Virus | Zeptometrix | 9.27E+07 Copies/mL | Negative |
| HSV Type 1 MacIntyre Strain | Zeptometrix | 5.89E+06 | Negative |
| HSV Type 2 G strain | DHI | 1.00E+05 | Negative |
| Human Metapneumovirus (A1) | DHI | 1.00E+05 | Negative |
| Human Rhinovirus 45 | DHI | 2.94E+04 | Negative |
| Human Rhinovirus 52 | DHI | 2.63E+04 | Negative |
| Influenza A/Mexico/4108/2009 | DHI | 1.00E+05 | Negative |
| Influenza A/Port Chalmers | DHI | 1.00E+05 | Negative |
| Influenza B/Florida/04/2006 | DHI | 1.00E+05 | Negative |
| Measles | Zeptometrix | 1.95E+06 | Negative |
| Mumps Virus | Zeptometrix | 2.75E+08 | Negative |
| RSV A (Long) | DHI | 1.00E+05 | Negative |
| RSV B Strain (Wash/18537/62) | DHI | 1.00E+05 | Negative |
| Varicella Zoster Virus | DHI | 1.00E+05 | Negative |
| Bordetella pertussis | DHI | 1.00E+06 | Negative |
| Bordetella bronchiseptica | DHI | 1.00E+06 | Negative |
| Chlamydophilia pneumonia | ATC | 1.00E+06 copies/mL | Negative |
| Chlamydophilia trachomatis | DHI | 1.00E+06 | Negative |
| Legionella pneumophila | DHI | 1.00E+06 | Negative |
| Mycobacterium intracellulare | DHI | 1.00E+06 | Negative |
| Mycobacterium tuberculosis | DHI | 1.00E+06 | Negative |
| Mycobacterium avium | DHI | 1.00E+06 | Negative |
| Mycoplasma pneumoniae | Zeptometrix | 3.16E+06 Color Changing Unit/mL | Negative |
| Haemophilus influenzae | DHI | 1.00E+06 | Negative |
| Pseudomonas aeruginosa | DHI | 1.00E+06 | Negative |

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| Proteus vulgaris | DHI | 1.00E+06 | Negative |
| --- | --- | --- | --- |
| Proteus mirabilis | DHI | 1.00E+06 | Negative |
| Neisseria gonorrhoeae | DHI | 1.00E+06 | Negative |
| Neisseria meningitidis | DHI | 1.00E+06 | Negative |
| Neisseria mucosa | DHI | 1.00E+06 | Negative |
| Klebsiella pneumoniae | DHI | 1.00E+06 | Negative |
| Escherichia coli | DHI | 1.00E+06 | Negative |
| Moraxella catarrhalis | DHI | 1.00E+06 | Negative |
| Corynebacterium diptheriae | DHI | 1.00E+06 | Negative |
| Lactobacillus plantarum | DHI | 1.00E+06 | Negative |
| Streptococcus pneumoniae | DHI | 1.00E+06 | Negative |
| Streptococcus pyogenes | DHI | 1.00E+06 | Negative |
| Streptococcus salivarius | DHI | 1.00E+06 | Negative |
| Staphylococcus epidermidis | DHI | 1.00E+06 | Negative |
| Staphylococcus aureus | DHI | 1.00E+06 | Negative |
| Candida albicans | DHI | 1.00E+06 | Negative |
| Parainfluenza Type 4A | Zeptometrix | 1.04E+05 | Negative |
| Parainfluenza Type 1 | DHI | 1.00E+05 | 18.0 for PIV1 |
| Parainfluenza Type 2 | DHI | 1.00E+05 | 13.0 for PIV2 |
| Parainfluenza Type 3 | DHI | 1.00E+05 | 15.2 for PIV3 |
| Negative Matrix Control | DHI | N/A | Negative |

# Microbial Interference:

Parainfluenza virus types 1, 2 and 3 were spiked with clinically relevant concentrations of viruses, bacteria or yeast in order to evaluate possible interference from organisms not targeted by the assay. Parainfluenza virus types were used at a 2X LoD concentration; all dilutions were made in negative nasal matrix. Clinically relevant levels of viruses and bacteria were used when stock concentrations permitted. Samples were extracted and amplified according to the package insert instructions. All samples were tested in triplicate starting from the extraction step. The results were analyzed for any interference with any of the three parainfluenza virus types. The final concentration tested and results are listed in the table below. No interference was observed in the study.

| Organism Name | Concentration Tested (TCID50/ml or CFU/mL) | PIV-1 Avg. Ct | PIV-2 Avg. Ct | PIV-3 Avg. Ct | Interference (Yes/No) |
| --- | --- | --- | --- | --- | --- |
| Adenovirus 1 | 1.51E+05 | 33.9 | 34.5 | 34.6 | No |
| Coronavirus 229E | 2.46E+06 | 33.6 | 33.7 | 33.8 | No |
| Coronavirus NL63 | 1.41E+04 | 35.0 | 35.5 | 34.6 | No |
| Coronavirus OC43 | 2.42E+06 | 33.6 | 34.4 | 34.0 | No |
| Coxsackievirus B4 | 2.00E+06 | 34.3 | 34.7 | 33.9 | No |
| Coronavirus NL63 | 1.41E+04 | 35.0 | 35.5 | 34.6 | No |
| Coronavirus OC43 | 2.42E+06 | 33.6 | 34.4 | 34.0 | No |
| Coxsackievirus B4 | 2.00E+06 | 34.3 | 34.7 | 33.9 | No |

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| Coxsackievirus B5/10/2006 | 3.62E+05 | 33.8 | 34.5 | 34.4 | No |
| --- | --- | --- | --- | --- | --- |
| Cytomegalovirus | 2.14E+05 | 34.1 | 33.9 | 33.8 | No |
| Echovirus 6 | 1.52E+08 | 34.5 | 34.0 | 34.6 | No |
| Echovirus 7 | 4.58E+05 | 34.3 | 32.9 | 34.3 | No |
| Echovirus 9 | 2.17E+06 | 34.0 | 33.9 | 34.1 | No |
| Echovirus 11 | 2.17E+05 | 34.1 | 34.7 | 34.1 | No |
| Enterovirus 70 | 2.41E+05 | 34.4 | 34.4 | 33.9 | No |
| Enterovirus 71 | 2.03E+05 | 34.1 | 34.3 | 34.2 | No |
| Epstein Barr Virus | 9.27E+07 copies/mL | 35.8 | 34.9 | 35.3 | No |
| HSV Type 1 MacIntyre Strain | 5.89E+06 | 34.0 | 33.5 | 33.8 | No |
| HSV Type 2 Strain G | 1.96E+05 | 33.8 | 34.7 | 33.9 | No |
| Human Metapneumovirus (A1) | 3.66E+05 | 34.8 | 34.6 | 34.5 | No |
| Human Rhinovirus 45 | 1.47E+04 | 34.2 | 33.8 | 33.7 | No |
| Human Rhinovirus 52 | 1.31E+04 | 34.7 | 34.0 | 33.8 | No |
| Influenza A/Mexico/4108/2009 | 4.08E+05 | 34.5 | 35.3 | 34.7 | No |
| Influenza A/Port Chalmers | 3.55E+07 | 34.8 | 34.6 | 34.0 | No |
| Influenza B/Florida/04/2006 | 1.54E+05 | 34.2 | 34.5 | 33.8 | No |
| Measles/7/2000 | 1.95E+06 | 34.4 | 33.3 | 34.0 | No |
| Mumps Virus | 2.75E+08 | 34.4 | 34.6 | 34.9 | No |
| RSV A (Long) | 4.36E+04 | 34.5 | 34.5 | 34.5 | No |
| RSV B (Wash/18537/62) | 3.43E+05 | 33.9 | 34.2 | 34.1 | No |
| Varicella Zoster Virus | 1.11E+04 | 34.0 | 34.6 | 33.6 | No |
| Bordetella pertussis | 9.08E+07 | 33.7 | 35.4 | 34.2 | No |
| Bordetella bronchiseptica | 5.40E+07 | 33.3 | 33.8 | 34.0 | No |
| Chlamydophila pneumoniae | 22 ng/mL (DNA) | 33.6 | 33.8 | 34.1 | No |
| Chlamydia trachomatis | 2.10E+06 | 33.7 | 34.0 | 34.2 | No |
| Legionella pneumophila | 1.42E+08 | 34.8 | 34.1 | 34.6 | No |
| Mycobacterium intracellualre | 1.53E+08 | 33.9 | 33.9 | 35.6 | No |
| Mycobacterium tuberculosis | 9.30E+06 | 34.7 | 34.4 | 33.5 | No |
| Mycobacterium avium | 3.18E+08 | 33.5 | 34.0 | 34.9 | No |

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| Mycoplasma pneumoniae | 3.16E+06 color changing units | 34.3 | 34.0 | 34.3 | No |
| --- | --- | --- | --- | --- | --- |
| Haemophilus influenzae | 4.00E+07 | 34.6 | 34.7 | 33.4 | No |
| Pseudomonas aeruginosa | 1.32E+08 | 33.8 | 33.3 | 34.1 | No |
| Proteus vulgaris | 6.53E+07 | 33.7 | 33.3 | 34.0 | No |
| Proteus mirabilis | 1.19E+08 | 34.3 | 34.1 | 34.5 | No |
| Neisseria gonorrhoeae | 1.40E+08 | 34.4 | 34.2 | 35.0 | No |
| Neisseria meningitidis | 1.29E+07 | 33.9 | 34.6 | 34.4 | No |
| Neisseria mucosa | 1.61E+08 | 34.0 | 34.0 | 33.7 | No |
| Klebsiella pneumoniae | 9.75E+07 | 34.0 | 34.6 | 34.0 | No |
| Escherichia coli | 1.13E+08 | 34.8 | 34.7 | 34.7 | No |
| Moraxella catarrhalis | 1.26E+08 | 34.6 | 34.5 | 34.3 | No |
| Corynebacterium diphtheriae | 3.44E+07 | 34.2 | 34.4 | 34.2 | No |
| Lactobacillus plantarum | 3.18E+07 | 34.1 | 34.6 | 34.1 | No |
| Streptococcus pneumoniae | 1.43E+07 | 35.4 | 35.6 | 35.0 | No |
| Streptococcus pyogenes | 6.38E+07 | 34.6 | 34.8 | 34.2 | No |
| Streptococcus salivarius | 5.40E+07 | 35.1 | 34.2 | 34.4 | No |
| Staphylococcus epidermidis | 9.23E+07 | 34.6 | 34.6 | 34.0 | No |
| Staphylococcus aureus | 6.08E+07 | 35.1 | 36.1 | 34.4 | No |
| Candida albicans | 9.70E+07 | 34.9 | 34.2 | 34.2 | No |
| Parainfluenza Type 4A | 4.17E+04 | 34.6 | 34.8 | 34.2 | No |
| Parainfluenza Type 1 | | 35.0 | Neg | Neg | N/A |
| Parainfluenza Type 2 | | Neg | 34.2 | Neg | N/A |
| Parainfluenza Type 3 | | Neg | Neg | 34.7 | N/A |
| Negative Nasal Matrix | | Neg | Neg | Neg | N/A |

Competitive Interference:

The purpose of this study is to determine whether competitive interference exists between analytes when more than one parainfluenza type is present in the same reaction. All samples were tested in triplicate starting from the extraction step. The results were analyzed for any competitive interference with any of the three parainfluenza virus types. The final concentration tested and results are listed in the table below.

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| | Analyte Concentration TCID50/mL | | | | |
| --- | --- | --- | --- | --- | --- |
| Test Analyte (at 2xLoD) | PIV1 | PIV2 | PIV3 | Replicates of Test Analyte Detected | Competitive Interference (Yes/No) |
| PIV1 | 5.00E+00 | - | - | 3/3 | No |
| | 5.00E+00 | 2.50E+02 | - | 3/3 | No |
| | 5.00E+00 | 1.25E+03 | - | 3/3 | No |
| | 5.00E+00 | 1.25E+04 | - | 3/3 | No |
| | 5.00E+00 | 1.25E+05 | - | 3/3 | No |
| | 5.00E+00 | 1.25E+06 | - | 3/3 | No |
| | 5.00E+00 | 1.25E+07 | - | 0/3 | Yes |
| | 5.00E+00 | - | 8.00E+01 | 3/3 | No |
| | 5.00E+00 | - | 4.00E+02 | 3/3 | No |
| | 5.00E+00 | - | 4.00E+03 | 3/3 | No |
| | 5.00E+00 | - | 4.00E+04 | 3/3 | No |
| | 5.00E+00 | - | 4.00E+05 | 3/3 | No |
| | 5.00E+00 | - | 4.00E+06 | 0/3 | Yes |
| PIV2 | | 2.50E+02 | - | 3/3 | No |
| | 5.00E+00 | 2.50E+02 | - | 3/3 | No |
| | 2.50E+01 | 2.50E+02 | - | 3/3 | No |
| | 2.50E+02 | 2.50E+02 | - | 3/3 | No |
| | 2.50E+03 | 2.50E+02 | - | 3/3 | No |
| | 2.50E+04 | 2.50E+02 | - | 3/3 | No |
| | - | 2.50E+02 | 8.00E+01 | 3/3 | No |
| | - | 2.50E+02 | 4.00E+02 | 3/3 | No |
| | - | 2.50E+02 | 4.00E+03 | 3/3 | No |
| | - | 2.50E+02 | 4.00E+04 | 3/3 | No |
| | - | 2.50E+02 | 4.00E+05 | 3/3 | No |
| | - | 2.50E+02 | 4.00E+06 | 0/3 | Yes |
| PIV3 | - | - | 8.00E+01 | 3/3 | No |
| | 5.00E+00 | - | 8.00E+01 | 3/3 | No |
| | 2.50E+01 | - | 8.00E+01 | 3/3 | No |
| | 2.50E+02 | - | 8.00E+01 | 3/3 | No |
| | 2.50E+03 | - | 8.00E+01 | 3/3 | No |
| | 2.50E+04 | - | 8.00E+01 | 3/3 | No |
| | - | 2.50E+02 | 8.00E+01 | 3/3 | No |
| | - | 1.25E+03 | 8.00E+01 | 3/3 | No |
| | - | 1.25E+04 | 8.00E+01 | 3/3 | No |
| | - | 1.25E+05 | 8.00E+01 | 3/3 | No |
| | - | 1.25E+06 | 8.00E+01 | 3/3 | No |
| | - | 1.25E+07 | 8.00E+01 | 0/3 | Yes |

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The results indicate that competitive interference exists for PIV-1 when combined with PIV-2 at a concentration $1.25 \times 10^{7} \mathrm{TCID}_{50}$ or greater and at $4.0 \times 10^{6} \mathrm{TCID}_{50}$ or greater when combined with PIV-3. When evaluating PIV-2, it was determined that interference existed when combined with PIV-3 at a concentration $4.0 \times 10^{6} \mathrm{TCID}_{50}$ or greater. No interference was seen when PIV-2 was combined with PIV-1. For PIV-3, there was competitive interference when combined with PIV-2 at a concentration of $1.25 \times 10^{7} \mathrm{TCID}_{50}$ or greater. No interference was seen when PIV-3 was combined with PIV-1.

For all virus combinations, detectable interference was seen at concentrations equal to or greater than $4.0 \times 10^{6} \mathrm{TCID}_{50}$ . These titers are high and not likely to be found in clinical samples; it is therefore highly unlikely that competitive interference will be seen in the clinical settings. A limitation indicating competitive interference at high concentrations will be included in the package insert.

# h. Interfering Substances:

A study was conducted to demonstrate that the Lyra Parainfluenza Virus Assay specifically detects parainfluenza virus types 1, 2, and 3 in the presence of clinically relevant concentrations of potentially interfering substances. To prepare samples for testing, parainfluenza virus at $4\mathrm{x}$ LoD in negative nasal matrix was diluted 1:1 with each test substance. The final concentration after dilution for each substance is listed in the table below and the final concentration of parainfluenza virus was $2\mathrm{x}$ LoD. Each mixed sample was then extracted and tested according to the package insert.

Ct values for all testing were reported and there was no significant change from the control. No interference was detected in the presence of any of the substances tested.

| Name | Active Ingredients | Concentration to be Tested | Testing Level Rationale |
| --- | --- | --- | --- |
| Mucin (Bovine Submaxillary Gland, type I-S) | Purified mucin protein | 60μg/mL | 1000x maximum level present in serum |
| Blood (human), EDTA anticoagulated | N/A | 2% (vol/vol) | |
| Neo-Synephrine | Phenylephrine HCl | 15% (vol/vol) | 10% of total recommended dose |
| Afrin Nasal Spray | Oxymetazoline Hydrochloride | 15% (vol/vol) | 10% of total recommended dose |

{15}

| Zicam Homeopathic Non-Drowsy Allergy Relief No Drip Liquid Nasal Gel | Luffa Operculata, Galphimia Glauca, Histaminum Hydrochloricum, Sulphur | 5% (vol/vol) | 10% of total recommended dose |
| --- | --- | --- | --- |
| Saline Nasal Spray: | Sodium chloride with preservatives | 15% (vol/vol) of dose | 10% of total recommended dose |
| OTC Throat Lozenges: LNegens Wild Cherry | Menthol | 25% (mass/vol) | Same menthol level as other products |
| Zanamivir | Zanamivir | 3.3-5mg/mL (target 4mg/mL) | 10% of total spray dose |
| Tobramycin | Tobramycin | 4.0μg/mL | 10% of total recommended dose |
| Mupirocin | Mupirocin | 6.6-10mg/mL (target 8mg/mL) | 10% of total recommended dose in Mupirocin ointment |
| Oseltamivir phosphate (Tamiflu) | Oseltamivir phosphate | 7.5-25mg/mL (target 16mg/mL) | 10% of total recommended dose |

i. Carry-Over/Cross-Contamination:

Simulated parainfluenza high positive samples were analyzed in series alternating with negative samples (checkerboard pattern) in order to determine whether carry-over/cross-contamination occurs with the use of the Lyra Parainfluenza Virus Assay. The high positive samples in this study were at concentration of $1.0 \times 10^{7} \mathrm{TCID}_{50} / \mathrm{mL}$ in negative nasal matrix. Parainfluenza-1, Parainfluenza-2, and Parainfluenza-3 analytes were combined into one sample for this study. Samples were processed and tested according to the package insert. Testing was performed on 5 separate days where each 96-well plate contained 48 high positive samples alternating with 48 negative samples (negative nasal matrix only). No cross-contamination was observed during the study. All negative samples tested had negative results and all high positive samples had all three parainfluenza viruses detected.

2. Comparison studies:

a. Method comparison with predicate device: N/A

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# b. Matrix comparison:

This study was designed to verify that the assay does not have variable performance when samples are transported in various viral transport media. Parainfluenza-1 strain C35, Parainfluenza-2 strain Greer, Parainfluenza-3 strain C243 were used in this study. Viruses were diluted in the transport media to 2x LoD and testing was performed using six replicates per virus/media combination.

| Transport Media Name | Vendor | PIV-1 Avg Ct | PIV-2 Avg Ct | PIV-3 Avg Ct |
| --- | --- | --- | --- | --- |
| UTM | Copan | 34.8 | 33.6 | 34.4 |
| M4 | Remel | 35.2 | 34.0 | 33.5 |
| M4-RT | Remel | 35.2 | 33.8 | 34.8 |
| M5 | Remel | 34.5 | 34.1 | 33.9 |
| M6 | Remel | 33.8 | 34.0 | 34.3 |

Varying the transport media did not affect the performance of this assay, with no observed changes in the Ct value. The data demonstrates that all transport media included in the study are suitable for use with the Lyra Parainfluenza Virus assay.

# c. Fresh vs. Frozen sample comparison:

Parainfluenza virus stocks were diluted to 2X and 5X LoD using negative nasal matrix. The five panels created contained 2X LoD (combined PIV-1, PIV-2 and PIV-3), 5X LoD (combined PIV-1, PIV-2 and PIV-3) and negative samples. Each panel consisted of 30 aliquots of 5X LoD, 30 aliquots of 2X LoD and 10 aliquots of negative nasal matrix. Panel 1 was extracted on the day of preparation. Panels 2 and 3 were stored at $4^{\circ}\mathrm{C}$ and then extracted on day 7 and 8, respectively. Panels 4 and 5 were stored at $-20^{\circ}\mathrm{C}$ and then extracted on day 7 and 8, respectively. A single replicate of each panel member was extracted and tested according to the instructions in the package insert.

| | | 5X LoD | | | 2X LoD | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Panel # | Conditions | PIV1 | PIV2 | PIV3 | PIV1 | PIV2 | PIV3 | Neg. |
| 1 | Day 0 | 33.4 | 32.4 | 32.6 | 36.2 | 35.0 | 35.0 | NEG |
| 2 | Day 7, 4°C | 34.4 | 33.0 | 32.7 | 37.6 | 35.5 | 34.9 | NEG |
| 3 | Day 8, 4°C | 34.0 | 32.7 | 32.2 | 37.1 | 35.4 | 34.5 | NEG |
| 4 | Day 7, -20°C | 33.5 | 32.5 | 32.4 | 36.1 | 35.3 | 34.7 | NEG |
| 5 | Day 8, -20°C | 33.5 | 32.6 | 32.6 | 36.6 | 35.6 | 35.3 | NEG |

The average Ct values for all samples at 5x LoD and 2x LoD are substantially equivalent

{17}

among all conditions. The data presented demonstrates that the performance of this test with frozen samples is equal to the performance with fresh samples.

# 3. Clinical studies:

The evaluation of the Lyra™ Parainfluenza Virus Assay occurred in two separate studies: a prospective multi-center study using 1241 fresh specimens from the upper respiratory tract; and a retrospective study using 105 frozen specimens from the upper respiratory tract. In both studies the bioMérieux NucliSENS® easyMag® was used at all sites for the extraction of nucleic acids from the clinical specimens. The Applied Biosystems® 7500 Fast Dx Real-Time PCR Instrument was used with the Lyra™ Parainfluenza Virus Assay for the amplification and detection of the target nucleic acids.

The prospective specimens were also tested with direct specimen fluorescent antibody (DSFA) and cell culture with DFA (CCFA). Specimens for DSFA and CCFA were shipped to central location daily with cold packs, and were cultured within 72- hours of collection. The DSFA and CCFA of the specimens were performed at a central location, DHI reference laboratory (Diagnostic Hybrids, Athens, Ohio).

The study utilized fresh and frozen specimens from the 2013 and 2014 respiratory seasons. Clinical samples were collected at three geographically diverse locations, all clinical laboratories

# Prospective Study:

A prospective study was conducted with 1241 fresh specimens from male and female patients with age range of $\leq 2$ to 102 years. The table below shows the age and gender distribution of the patients in this study.

| Gender | Female | Male |
| --- | --- | --- |
| Total | 651 | 590 |
| Age | | |
| < 2 years | 111 | 158 |
| 2 to 5 years | 130 | 126 |
| 6 to 21 years | 150 | 129 |
| 22 to 59 years | 160 | 108 |
| ≥ 60 years | 100 | 69 |

Similar performance was observed at the 3 clinical sites participating in the study during the 2013 and 2014 respiratory seasons. Therefore, performance results from all sites were pooled and the summary data for PIV-1, PIV-2 and PIV-3 are presented in the tables below.

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19

| PIV-1 | | | |
| --- | --- | --- | --- |
| Lyra™ Parainfluenza Virus Assay Positive | Comparator: DSFA and Culture with DFA | | |
| | Positive | Negative | Total |
| | 10 | 3* | 13 |
| Negative | 0 | 1228 | 1228 |
| | 10 | 1231 | 1241 |
| 95% CI | | | |
| Sensitivity | 10/10 | 100% | 72.2% to 100% |
| Specificity | 1228/1231 | 99.8% | 99.3% to 99.9% |

* Two (2) of the three (3) samples positive by Lyra but negative by the comparator method were positive when tested by an additional RT-PCR assay.

| PIV-2 | | | |
| --- | --- | --- | --- |
| Lyra™ Parainfluenza Virus Assay Positive | Comparator: DSFA and Culture with DFA | | |
| | Positive | Negative | Total |
| | 5 | 0 | 5 |
| Negative | 0 | 1236 | 1241 |
| | 5 | 1236 | 1246 |
| 95% CI | | | |
| Sensitivity | 5/5 | 100% | 56.6% to 100% |
| Specificity | 1236/1236 | 100% | 99.7% to 100% |
| PIV-3 | | | |
| --- | --- | --- | --- |
| Lyra™ Parainfluenza Virus Assay Positive | Comparator: DSFA and Culture with DFA | | |
| | Positive | Negative | Total |
| | 17 | 5* | 22 |
| Negative | 0 | 1219 | 1219 |
| | 17 | 1224 | 1241 |
| 95% CI | | | |
| Sensitivity | 17/17 | 100% | 81.6% to 100% |
| Specificity | 1219/1224 | 99.6% | 99.0% to 99.8% |

* Five (5) of the five (5) positive by Lyra but negative by the comparator method, were positive when tested by an additional RT-PCR assay.

Retrospective Study:

A Retrospective study was also conducted at site 1 because of the low prevalence of parainfluenza virus seen at all clinical sites during the study period. One hundred five frozen specimens from the upper respiratory tract were tested concurrently with the Lyra™

{19}

Parainfluenza Virus Assay and the Prodesse ProParaFlu+ assay (K091053).

| PIV-1 | | | |
| --- | --- | --- | --- |
| Lyra™ Parainfluenza Virus Assay | Comparator: Prodesse ProParaFlu+ assay | | |
| | Positive | Negative | Total |
| | 24 | 1* | 25 |
| Negative | 0 | 80 | 80 |
| Total | 24 | 81 | 105 |
| 95% CI | | | |
| Positive Percent Agreement | 24/24 | 100% | 86.2% to 100% |
| Negative Percent Agreement | 80/81 | 98.8% | 93.3% to 99.8% |

* One (1) sample positive by Lyra but negative by the comparator method, was positive when tested by an additional RT-PCR assay.

| PIV-2 | | | |
| --- | --- | --- | --- |
| Lyra™ Parainfluenza Virus Assay | Comparator: Prodesse ProParaFlu+ assay | | |
| | Positive | Negative | Total |
| | 22 | 5* | 27 |
| Negative | 0 | 78 | 78 |
| Total | 22 | 83 | 105 |
| 95% CI | | | |
| Positive Percent Agreement | 22/22 | 100% | 85.1% to 100% |
| Negative Percent Agreement | 78/83 | 94.0% | 86.7% to 97.4% |

* Five (5) of five (5) positive samples were positive by Lyra but negative by the comparator method, were positive when tested by an additional RT-PCR assay.

| PIV-3 | | | |
| --- | --- | --- | --- |
| Lyra™ Parainfluenza Virus Assay | Comparator: Prodesse ProParaFlu+ assay | | |
| | Positive | Negative | Total |
| | 24 | 0 | 24 |
| Negative | 0 | 81 | 81 |
| Total | 24 | 81 | 105 |
| 95% CI | | | |
| Positive Percent Agreement | 24/24 | 100% | 86.2% to 100% |
| Negative Percent Agreement | 81/81 | 100% | 95.5% to 100% |

When performed on the Applied Biosystems® 7500 Fast Dx the Lyra™ Parainfluenza Virus Assay yielded good sensitivity and specificity when compared to direct specimen fluorescent antibody (DSFA) and cell culture with DFA (CCFA).

When performed on the Applied Biosystems® 7500 Fast Dx the Lyra™ Parainfluenza

{20}

Virus Assay yielded good Positive Percent Agreement and Negative Percent Agreement when compared to the Prodesse ProParaFlu+ assay.

Based on the performance data presented from the clinical study this device performs as well as the predicate device, Prodesse ProParaFlu+ (K0910533).

## 4. Clinical cut-off:

Please refer to the "Baseline, Threshold, Cut-off" sections of this document.

## 5. Expected values/Reference range:

The prevalence (positives as determined by the reference method) of PIV-1, PIV-2 and PIV-3 during the study (2013 and 2014 respiratory seasons) is presented in the table below. The highest prevalence of PIV1 was observed among adults 60 years or older (2.4%) and of PIV-3 was observed among children younger than 2 years of age (3.7%).

| Combined Study – Prevalence (N=1241) | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | PIV-1 | | | PIV-2 | | | PIV-3 | | |
| Age | Total # | Total Positive | Prevalence | Total # | Total Positive | Prevalence | Total # | Total Positive | Prevalence |
| < 2 years | 269 | 1 | 0.40% | 269 | 2 | 0.70% | 269 | 10 | 3.70% |
| 2 to 5 years | 256 | 2 | 0.80% | 256 | 2 | 0.80% | 256 | 8 | 3.10% |
| 6 to 21 years | 279 | 1 | 0.40% | 279 | 0 | N/A | 279 | 2 | 0.70% |
| 22 to 59 years | 268 | 5 | 1.90% | 268 | 1 | 0.40% | 268 | 2 | 0.80% |
| ≥ 60 years | 169 | 4 | 2.40% | 169 | 0 | N/A | 169 | 0 | N/A |

## N. Instrument Name:

Applied Biosystems® 7500 Fast Dx Real-Time PCR Instrument with the SDS Software version 1.4

## O. System Descriptions:

The Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument with the SDS Software version 1.4 is a real-time nucleic acid amplification and detection system that measures fluorescence and converts the signal to comparative quantitative readouts using fluorescent detection of dual-labeled hydrolysis probes. The 7500 Fast Dx is to be used only by technologists trained in laboratory techniques, procedures and on use of the analyzer.

## 1. Modes of Operation:

Instructions for programming the Lyra Direct Strep Assay, using the Version 1.4 software, are included in the package insert. The computer system is locked and controlled by ABI.

{21}

No additional software can be loaded by the end-user.

# 2. Software:

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

Yes __X__ or No __

# Level of Concern:

Moderate

# Software Description:

The Sequence Detection Software (SDS) version 1.4 for the 7500 Fast Dx Instrument is used for instrument control, data collection and data analysis. The software can measure cycle-by-cycle real-time signals from the sample. The software provides a variety of tools to help the user analyze the data extracted from the samples. The software also provides lamp-life monitoring and other instrument maintenance information. The software runs as an application on a Windows operating system.

# 3. Specimen Identification:

Specimen identification is described in the "Test Principle" and "Baseline, Threshold, Cutoff" sections of this document.

# 4. Specimen Sampling and Handling:

All specimen sampling and handling is manual.

# 5. Calibration:

N/A

# 6. Quality Control:

An internal control 'process control' (PRC) is included in the device. It is the MS2 bacteriophage (Zeptometrix) which is a single stranded RNA virus that can be extracted, amplified and detected by primers and probes included in the master mix. Detection of the PRC indicates that assay conditions allowed proper sample extraction, amplification and detection.

No external controls are provided with this assay, but they are available from Quidel. External controls for PIV-1, PIV-2 and PIV-3 from the Lyra Parainfluenza Control Set #M1XX are recommended to serve as external positive controls. Laboratories may use previously characterized specimens that are negative for PIV-1, PIV-2 and PIV-3 as negative controls.

{22}

P. Proposed Labeling:

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

Q. Conclusion:

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

23