ADENOVIRUS R-GENE US
Device Facts
| Record ID | K121942 |
|---|---|
| Device Name | ADENOVIRUS R-GENE US |
| Applicant | Argene SA |
| Product Code | OCC · Microbiology |
| Decision Date | Feb 8, 2013 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.3980 |
| Device Class | Class 2 |
| Attributes | Pediatric |
Indications for Use
Adenovirus R-gene® US assay is a Real Time PCR in vitro diagnostic test for the rapid and qualitative detection of Adenovirus viral DNA isolated and purified from nasopharyngeal swab or nasopharyngeal wash/aspirate specimens obtained from individuals exhibiting signs and symptoms of acute respiratory infection. The intended use for this test is to aid in the diagnosis of respiratory Adenovirus infections in humans in conjunction with other clinical and laboratory findings. The test detects, but does not differentiate, Adenovirus species (A, B, C, D, E, F and G). Negative results do not preclude Adenovirus infection and should not be used as the sole basis for treatment or other patient management decisions.
Device Story
Adenovirus R-gene® US is a qualitative real-time PCR assay for detecting Adenovirus DNA in nasopharyngeal swabs or wash/aspirates. Process: nucleic acid extraction via bioMérieux NucliSENS easyMAG system; amplification/detection on Cepheid SmartCycler II instrument using Dx software (v1.7b or 3.0). Input: purified viral DNA from patient specimens. Operation: manual sample preparation; automated extraction and PCR. Output: qualitative result (Positive/Negative/Unresolved/Invalid) displayed via software. Clinical utility: aids diagnosis of respiratory Adenovirus infections; results used alongside clinical/laboratory findings. Benefits: rapid detection of multiple Adenovirus species (A-G) to support patient management decisions.
Clinical Evidence
Prospective multi-center clinical study (n=1576) compared Adenovirus R-gene US to rapid culture/DFA. Swab specimens (n=1183): 91.7% sensitivity, 96.2% specificity. Wash/aspirate specimens (n=393): 100% sensitivity, 94.4% specificity. Analytical studies included LoD determination across 22 Adenovirus types, cross-reactivity testing against 61 microorganisms, and interference testing with common nasal substances.
Technological Characteristics
Taqman-based real-time PCR assay. Targets Hexon gene region. Uses magnetic silica-based nucleic acid extraction (NucliSENS easyMAG). Amplification/detection via Cepheid SmartCycler 2.0 (I-CORE module). Dual-labeled probes (FAM/HEX). Ready-to-use liquid reagents. Requires external instrumentation for extraction and amplification.
Indications for Use
Indicated for symptomatic patients of all ages (1 month to 101 years) suspected of acute respiratory infection. Specimens: nasopharyngeal swabs and nasopharyngeal wash/aspirates. Prescription use only.
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® ProAdeno™+ Assay (K102952)
Related Devices
- K132159 — PROADENO+ ASSAY · Gen-Probe Prodesse, Inc. · Aug 14, 2013
- K102952 — PRODESSE PROADENO · Gen-Probe Prodesse, Inc. · Dec 3, 2010
- K141931 — LYRA ADENOVIRUS ASSAY · Quidel Corporation · Oct 9, 2014
Submission Summary (Full Text)
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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY
A. 510(k) Number:
k121942
B. Purpose for Submission:
This is a 510(k) application for a new qualitative Real-Time Polymerase Chain Reaction (PCR) assay used with the Cepheid SmartCycler II instrument for the *in vitro* qualitative detection of adenovirus DNA in nasopharyngeal swabs (NPS) and nasopharyngeal wash/aspirates (NPA) from symptomatic human patients.
C. Measurand:
Target DNA sequences for a region of the hexon gene of Adenovirus species
D. Type of Test:
Real-Time Polymerase Chain Reaction assay for the qualitative detection of adenovirus DNA in NPS and NPA samples. A bi-functional fluorescent probe-primer is used together with a reverse primer to amplify a specific target for the analyte and the RNA internal control. Amplification and detection is performed on the Cepheid SmartCycler II with Dx software version 1.7b or 3.0.
E. Applicant:
Argene SA
F. Proprietary and Established Names:
Adenovirus R-gene® US
G. Regulatory Information:
1. Regulation section:
21 CFR 866.3980 Respiratory viral panel multiplex nucleic acid assay
2. Classification:
Class II
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3. Product code:
OCC
4. Panel:
Microbiology (83)
H. Intended Use:
1. Intended use(s):
Adenovirus R-gene® US assay is a Real Time PCR in vitro diagnostic test for the rapid and qualitative detection of Adenovirus viral DNA isolated and purified from nasopharyngeal swab or nasopharyngeal wash/aspirate specimens obtained from individuals exhibiting signs and symptoms of acute respiratory infection. The intended use for this test is to aid in the diagnosis of respiratory Adenovirus infections in humans in conjunction with other clinical and laboratory findings. The test detects, but does not differentiate, Adenovirus species (A, B, C, D, E, F and G). Negative results do not preclude Adenovirus 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):
For prescription use only
4. Special instrument requirements:
To be used with the Cepheid SmartCycler II utilizing Dx software version 1.7b or 3.0
I. Device Description:
The test is a Taqman-based real-time PCR amplification and detection assay for the detection of human adenovirus DNA in nasopharyngeal swab and nasopharyngeal aspirate/wash specimens. The assay uses the bioMérieux NucliSENS easyMAG nucleic acid extraction system to extract adenovirus DNA from swab or wash specimens obtained from patients with signs and symptoms of acute respiratory infection. Extracted adenovirus DNA is then amplified using the Adenovirus R-gene US assay components. Amplification is performed on the Cepheid SmartCycler II using Dx software versions 1.7b or 3.0.
The Adenovirus R-gene US assay contains the reaction mix (R⁺10), internal control (IC2), molecular-grade water (W0), positive control (PC10), and Package Insert.
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Adenovirus R-gene US reaction mix contents and probe info:
| Reagents | Description | Composition | Quantity/tube | # of Reactions | Form |
| --- | --- | --- | --- | --- | --- |
| R^{3}10 | Adenovirus and IC2 amplification pre-mix | Adenovirus and IC2 primers and probes, Tris, MgCl_{2}, dNTP’s, Taq Polymerase, Rox Carboxy x Rhodamine, Ammonium Sulfide, | 450 μL | 90 | Liquid |
| IC2 | Internal Control 2 | Native Phix bacteriophage, Phast media, Glycerol | 1 mL | 100 | Liquid |
| WO | Water for extraction (molecular grade) | Water | 1.8 mL | 18 | Liquid |
| PC10 | Adenovirus Positive Control | Composite plasmid, Tris buffer, yeast tRNA | 300 μL | 30 | Liquid |
Materials required but not supplied: Polyester, rayon or nylon tipped nasopharyngeal swabs, Sterile suction catheter (#8) for washes/aspirates specimen, 1.5 mL polypropylene microcentrifuge tubes, Sterile filter or positive displacement micropipettor tips, EasyMAG™ System Disposables (Sample Vessels and Tips), Biohit Pipette Tips for use with easyMAG™ System, Greiner Break Four uncoated plates for use with easyMAG™ System, Cepheid 25 μL PCR reaction tubes, single use latex or similar gloves, bioMérieux NucliSENS® easyMAG™ reagents (Buffer 1 Cat.#280130, Buffer 2 Cat.#280131, Buffer 3 Cat.#280132, Magnetic Silica Cat. #280133, Lysis Buffer (bottles) Cat.# 280134, Universal Transport Medium from DHI or MicroTest™ M4RT Transport from Remel, Proteinase K Solution, 600 mAU/mL (Novagen – Merck – 71049-3/71049-4), Molecular grade water, Sterile physiologic buffer, -18°C/-22°C Freezer, bioMérieux NucliSENS® easyMAG™ System with Software version 2.0, Cepheid SmartCycler II instrument with Dx Software version 1.7b or 3.0, Micropipettors (range between 1-10 μL, 10-200μL and 100-1000μL), Mini-centrifuge with adapter for Cepheid Reaction Tubes, Cepheid cooling block, U.V. light, Workstation or Plexiglas screen for samples and premix distribution.
Assay Procedure:
1- Collect swab specimens from symptomatic patients using a polyester, rayon, nylon tipped swab or wash/aspirate specimens using a sterile suction catheter.
2- Add Internal Control (IC2) to every sample to monitor for the presence of inhibitors or lysis failure.
3- Perform isolation and purification of nucleic acids using the NucliSENS® easyMAG® System (bioMérieux) and the automated Magnetic Extraction Reagents (bioMérieux).
4- Perform real time amplification of extracted samples using the Adenovirus R-gene US kit on the Cepheid SmartCycler II instrument.
5- Interpretation.
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# Interpretation of Results:
There are five possible results reported by the SmartCycler II summarized in the table and below:
| Assay Result | IC Result | Warning/Error Code | Adenovirus Result | Interpretation |
| --- | --- | --- | --- | --- |
| Negative | Pass | | Negative | Adenovirus nucleic acid not detected |
| Positive | NA* | | Positive | Adenovirus nucleic acid detected |
| Unresolved | Fail | | ND | Unresolved; PCR inhibition or reagent failure. Repeat testing with purified nucleic acid or obtain new sample. |
| ND | ND | 3079 | ND | Not Determined; error code 3079 |
| Invalid | | 4098 | ND | Not Determined; error code 4098 |
Positive: Human adenovirus DNA was detected in the sample
Negative: Human adenovirus DNA was not detected in the sample
Unresolved: PCR inhibition or reagent failure. Repeat testing from the purified nucleic acid or collect and test a new sample.
Invalid: Not determined; error code 4098
ND: (Not Determined) if the specimen result is unavailable because the run is in progress or an error or warning occurred during the run.
* Detection of Internal Control in the Cy3 channel is not required for a positive result. High viral load can lead to reduced or absent Internal Control signal.
Error Code 3079 can be observed with positives (Adenovirus Positive Control, Adenovirus positive swab/wash samples) when the fluorescence signal is too high. In this case, all results for that sample are reported by the Dx software as ND (Not Determined). If a Ct value $\geq 5$ is reported in the Adenovirus Ct columns, the sample results can be recorded as positive for the specific analyte. There were no 3079 type errors reported among the 1576 prospective clinical study specimens analyzed.
Error Code 4098: An invalid assay run will display Error Code 4098. One of the controls has failed and repeat testing should be done starting from the purified nucleic acid and using a new aliquot of the Positive Control. If repeated results are still invalid, results should not be reported and testing should be repeated from the original sample or a new sample should be collected and tested. There were no 4098 type errors reported among the 1576 prospective clinical study specimens analyzed.
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J. Substantial Equivalence Information:
1. Predicate device name(s):
Prodesse® ProAdeno™+
2. Predicate 510(k) number(s):
k102952
3. Comparison with predicate:
| Features | Item | Adenovirus R-gene US | Prodesse ProAdeno™+ Assay |
| --- | --- | --- | --- |
| Intended Use | Adenovirus R-gene US is a Real Time PCR in vitro diagnostic test for the rapid and qualitative detection of Adenovirus viral DNA isolated and purified from nasopharyngeal specimens (swab or wash/aspirate) obtained from individuals exhibiting signs and symptoms of acute respiratory infection. The intended use for this test is to aid in the diagnosis of Adenovirus infections in humans.
Negative results do not preclude Adenovirus infection and should not be used as the sole basis for treatment or other management decisions. | | The ProAdeno™+ Assay is a multiplex Real Time PCR in vitro diagnostic test for the qualitative detection of human Adenovirus (HAdV) DNA isolated and purified from nasopharyngeal swab specimens obtained from individuals exhibiting signs and symptoms of acute respiratory infection. This test is intended for use to aid in the diagnosis of HAdV infections in humans in conjunction with other clinical and laboratory findings. The test detects, but does not differentiate, serotypes 1-51.
Negative results do not preclude HAdV infection and should not be used as the sole basis for treatment or other patient management decisions. |
| 510(k) | k121942 | | k102952 |
| Regulation | 21 CFR 866.3980 | | 21 CFR 866.3980 |
| Product Code | OCC | | OCC |
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| Features | | |
| --- | --- | --- |
| Item | Adenovirus R-gene US | Prodesse ProAdenoTM+Assay |
| Assay Targets | DNA from Human Adenovirus | DNA from Human Adenovirus |
| Sample Types | Nasopharyngeal swabs and Nasal aspirates/washes | Nasopharyngeal swabs |
| Assay Type | Real-Time PCR | Real-Time PCR |
| Assay Results | Qualitative | Qualitative |
| Detection | Instrument-based fluorogenic target-specific hybridization | Instrument-based fluorogenic target-specific hybridization |
| Gene Target | Hexon gene | Hexon gene |
| Collection and Transport Media | Universal Transport Medium (DHI), MicroTestTM M4RT Transport (Remel) | M4 and M5 Viral Transport Medium (Remel), UVT (Becton Dickinson), UTM (Copan) |
| Assay Instrumentation | Cepheid SmartCycler® II with Dx Software | Cepheid SmartCycler® II System |
| Nucleic Acid Isolation | NucliSENS® easyMAG™ (bioMérieux) | MagnaPure LC System (Roche) NucliSENS® easyMAG™ (bioMérieux) |
| Controls Included | Positive Control plasmid DNA Neg control (mol. grade water) Internal control (IC2) phage particle | Adenovirus positive DNA transcript control and Internal DNA/RNA control |
| Results | Positive Negative Unresolved | Positive Negative Unresolved |
# K. Standard/Guidance Document Referenced (if applicable):
None
# L. Test Principle:
The real-time PCR process simultaneously amplifies and detects nucleic acid targets in a single closed-tube reaction. The Adenovirus R-gene US assay enables simultaneous detection of AdV and Internal Control DNA. The whole process is based on two steps: nucleic acid isolation and Real Time PCR amplification/detection. Human respiratory specimens (nasopharyngeal swabs, washes/aspirates) from symptomatic patients are processed initially to isolate and purify viral nucleic acid from the cellular specimen matrix. Each purified nucleic
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acid sample is added to the Rx10 amplification premix (Ready to use, Taq polymerase included). The Rx10 amplification premix contains oligonucleotide primers complementary to a fragment of the Hexon gene region coding for the hexagonal capsomers for AdV and target-specific oligonucleotide probes dual-labeled with a reporter dye and a quencher dye. As the amplification proceeds, the probe anneals specifically to a region of the template between the forward and reverse primers. As primer extension and amplification occurs, the exonuclease activity of the Taq polymerase cleaves the probe separating the reporter dye away from the quencher. This generates an increase in fluorescent signal upon excitation from a LED light source of appropriate wavelength. With each cycle, additional reporter dye molecules are cleaved from their respective probes, yielding increased fluorescence signal. The amount of fluorescence at any given cycle is dependent on the amount of PCR product (amplicons) present at that time. Fluorescent intensity is monitored at each PCR cycle by fluorescent detection modules within the real-time instrument.
## M. Performance Characteristics (if/when applicable):
### 1. Analytical performance:
#### a. Precision/Reproducibility:
The study evaluated the device's inter-laboratory, inter-assay, and intra-assay reproducibility for high negative (0.01x LoD), low positive (2x LoD), and moderately positive (10x LoD) specimens. The precision (repeatability and reproducibility) of the Adenovirus R-gene US assay was evaluated using human nasal matrix spiked with 3 viral inputs (moderate positive sample, low positive sample, high negative sample). A within-laboratory (repeatability) precision study was performed at an internal site (5 days, 2 operators, 1 instrument, 1 lot of reagent) and a between laboratory (reproducibility) precision study was performed at 3 external sites in the U.S. (5 days, 2 operators, 1 instrument at each site, 1 lot of reagent. Test panels consisted of 14 members (12 Adenovirus positive specimens plus two negative controls). Samples were extracted each day by both operators (5 days x 2 operators = 10 tests per sample) and tested in triplicate. For the moderate positive samples, 5 samples were present in the panel for a total of 150 tests (5 samples x 10 tests per sample x 3 replicates = 150). For low positive samples, 4 samples were present in the panel for a total of 120 tests (4 samples x 10 tests x 3 replicates). For high negative samples, there were 3 samples per panel for a total of 90 tests (3 samples x 10 tests x 3 replicates). Combined results for all sites and results stratified by site are presented in the tables below.
Reproducibility – Within Laboratory
| Sample | % Agreement | 95% CI | Mean Ct | SD | %CV |
| --- | --- | --- | --- | --- | --- |
| Moderate Positive | 100% (150/150) | 97.6-100% | 28.73 | 0.47 | 1.6 |
| Low Positive | 95% (114/120) | 89.4-98.1% | 32.90 | 3.00 | 9.1 |
| High Negative | 100% (90/90) | 96-100% | 24.19* | 0.17 | 0.70 |
| Total Agreement | 98.3% (354/360) | 96.4-99.4% | | | |
* Mean Ct calculated from Internal Control
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Reproducibility – Between Laboratories
| Sample | % Agreement | | | Total % Agreement and 95% CI | Mean Ct | SD | %CV |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | Site 1 | Site 2 | Site 3 | | | | |
| Moderate Positive | 100% (150/150) | 100% (150/150) | 100% (150/150) | 100% (450/450)
99.2-100% | 28.78 | 1.35 | 4.7 |
| Low Positive | 99.2% (119/120) | 99.2% (119/120) | 97.5% (117/120) | 98.6% (355/360)
96.8-99.6% | 32.0 | 2.17 | 6.8 |
| High Negative | 100% (90/90) | 95.6% (86/90) | 100% (90/90) | 98.5% (266/270)
96.2-99.6% | 24.12* | 0.36 | 1.5 |
| Total Agreement | 99.7% (359/360) | 98.6% (355/360) | 99.2% (357/360) | 99.2% (1071/1080)
98.4-99.6% | | | |
* Mean Ct calculated from Internal Control
b. Linearity/assay reportable range:
Not applicable
c. Traceability, Stability, Expected values (controls, calibrators, or methods):
Adenovirus R-gene US Stability and Positive Control Inter-Lot Reproducibility
Argene assessed the reproducibility of three different lots of Positive Control (PC) by testing each lot of controls with one lot of reaction mix. The study yielded a total of seven replicates per lot of PC mix. Reproducibility was determined simultaneously with freeze/thaw stability for the PC mix. The table below shows consistent reproducibility between lots and also stability for up to 29 freeze/thaw cycles.
PC Freeze/Thaw Stability and Reproducibility
| | PC10 R3424 | | | PC10 R3642 | | | PC10 R4070 | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Freeze/Thaw Cycles | Ct | Mean Ct | SD | Ct | Mean Ct | SD | Ct | Mean Ct | SD |
| 1 | 28.6 | 28.43 | 0.24 | 28.3 | 28.43 | 0.14 | 28.3 | 28.26 | 0.22 |
| 5 | 28.3 | | | 28.3 | | | 28.5 | | |
| 10 | 28.3 | | | 28.4 | | | 28.3 | | |
| 15 | 28.9 | | | 28.6 | | | 28.3 | | |
| 20 | 28.4 | | | 28.3 | | | 28.4 | | |
| 25 | 28.2 | | | 28.6 | | | 28.2 | | |
| 30 | 28.3 | | | 28.5 | | | 27.8 | | |
| PC10 | 28.3 | n/a | n/a | 28.3 | n/a | n/a | 28.3 | n/a | n/a |
| IC2W0 | neg | n/a | n/a | neg | n/a | n/a | neg | n/a | n/a |
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The table below shows that reaction mix $(\mathrm{R}^{\mathrm{x}}10)$ and Internal Control are stable for up to 10 freeze/thaw cycles.
Reaction Mix Freeze/Thaw Stability
| | | Reference 1 Thaw | | 11 Freeze/Thaw cycles | | Delta CT after 11 cycles | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | Dilution | AdV 530nm | IC2 560nm | AdV 530nm | IC2 560nm | AdV 530nm | IC2 560nm |
| AdV12 | \( 10^{-4} \) | 26.3 | 24.4 | 26.1 | 24.5 | -0.2 | 0.1 |
| AdV3 | \( 10^{-7} \) | 29.2 | 24.3 | 29.1 | 24.4 | -0.1 | 0.1 |
| AdV11 | \( 10^{-4} \) | 26.0 | 24.8 | 25.5 | 24.2 | -0.5 | -0.6 |
| AdV5 | \( 10^{-5} \) | 30.0 | 24.4 | 30.5 | 24.3 | 0.5 | -0.1 |
| AdV8 | \( 10^{-5} \) | 27.2 | 24.3 | 27.0 | 24.1 | -0.2 | -0.2 |
| AdV4 | \( 10^{-6} \) | 31.7 | 24.1 | 32.9 | 24.3 | 1.2 | 0.2 |
| AdV40 | \( 10^{-5} \) | 29.9 | 24.1 | 29.7 | 24.1 | -0.2 | 0.0 |
| IC2W0 | - | neg | 24.6 | neg | 24.6 | | |
| PC10 | - | 28.0 | neg | 28.0 | neg | | |
# Adenovirus R-gene US Specimen Stability
Samples must be stored refrigerated at $+2^{\circ}\mathrm{C} / + 8^{\circ}\mathrm{C}$ for up to 24 hours prior to processing or stored at $-18^{\circ}\mathrm{C} / - 22^{\circ}\mathrm{C}$ for 4 days or $-78^{\circ}\mathrm{C} / - 82^{\circ}\mathrm{C}$ for longer period.
# Adenovirus R-gene US Quality Control Ranges
Quality control ranges have been established as indicated in the table below. If the controls are not within these parameters, patient results should be considered invalid and the assay repeated. Each laboratory should establish its own Quality Control ranges and frequency of QC testing based on applicable local laws, regulations, and standard good laboratory practice.
Adenovirus R-gene US Expected Control Results
| | IC2a + W0 | | IC2a + sample | | PC10b | |
| --- | --- | --- | --- | --- | --- | --- |
| | Detected (+) | Not Detected (-) | Detected (+) | Not Detected (-) | Detected (+) | Not Detected (-) |
| Cy3 | Pass | Fail | Pass or error 3079 | Fail or error 4098 or n/a* | n/a | n/a |
| FAM | Invalid; error 4098 | Valid | Positive or error 3079 | Negative or error 4098 or Unresolved | Valid or error 3079 | Invalid; error 4098 |
a Typical Ct values for the Internal Control (IC2) range between 13.0 and 45.0 cycles
b Typical Ct values for the Positive Control range between 26.0 and 33.0 cycles
Error Code 3079 can be observed with positives (Adenovirus Positive Control, Adenovirus positive NP swab samples) when the fluorescence signal is too high. In this
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case, all results for that sample are reported by the Dx software as ND (Not Determined). If a Ct value $\geq 5$ is reported in the Adenovirus Ct columns, the sample results can be recorded as positive for the specific analyte.
* Detection of Internal Control is not required in the Cy3 channel for a positive Adenovirus result. High viral load can lead to reduced or absent Internal Control signal. Detection of IC2 is required for a valid negative Adenovirus result.
# d. Detection limit
Prior to the LoD determination, a sequence homology study was performed to classify the 60 known Human Adenoviruses into 21 different groups. Homology groups were established according to the number and the position of base mismatches with the best primer (forward and reverse) and the best probe which comprise the Adenovirus R-gene US amplification/detection premix.
One Human Adenovirus (AdV) type was chosen from each group of homologies to represent each of the 21 different sequences. The LoD study was set up so that each virus was tested using 4, 5, or 6 different dilutions near the estimated LoD and 15 replicates per dilution. Each replicate was taken through the DNA extraction step to amplification and detection. A total of 22 LoD's were determined comprising the 21 selected Human Adenoviruses, plus Adenovirus serotype 2 (a member of adenovirus species C). The homology groups and Adenovirus subtypes/species are displayed in the table below:
| Homology Group | HAdV Type | Species |
| --- | --- | --- |
| 1 | AdV 12 | A |
| 2 | AdV 18 | |
| 3 | AdV 31 | |
| 4 | AdV 3 | B1 |
| 5 | AdV 7 | |
| 6 | AdV 11 | B2 |
| 7 | AdV 1 | C |
| 8 | AdV 2,5 | |
| 9 | AdV 6 | |
| 10 | AdV 39 | D |
| 11 | AdV 15 | |
| 12 | AdV 9 | |
| 13 | AdV 8 | |
| 14 | AdV 17 | |
| 15 | AdV 25 | |
| 16 | AdV 28 | |
| 17 | AdV 42 | |
| 18 | AdV 4 | E |
| 19 | AdV 40 | F |
| 20 | AdV 41 | |
| 21 | AdV 52 | G |
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The Limit of Detection (LoD) was determined for the Adenovirus R-gene US assay by limiting dilution analysis of representative Adenoviruses from each of the 21 homology groups listed above as well as Adenovirus serotype 2. Fifteen replicates of viral dilutions were tested for Adenovirus positivity over a wide range of dilutions that surround the empirical LoD. The LoD was established by selecting the highest dilution of virus which could be detected at least 95% of the time.
## Adenovirus R-gene US Limit of Detection Summary
| Homology Group | HAdV Species | HAdV Type | LoD 95% TCID_{50}/ml |
| --- | --- | --- | --- |
| 1 | A | HAdV 12 | .000416 |
| 2 | | HAdV 18 | 18 |
| 3 | | HAdV 31 | 10 |
| 4 | B1 | HAdV 3 | 0.00316 |
| 5 | | HAdV 7 | 0.0445 |
| 6 | B2 | HAdV 11 | 889 |
| 7 | C | HAdV 1 | 0.0209 |
| 8-1 | | HAdV 2 | 0.00625 |
| 8-2 | | HAdV 5 | 0.044 |
| 9 | | HAdV 6 | 0.00512 |
| 10 | D | HAdV 39 | 0.0158 |
| 11 | | HAdV 15 | 0.316 |
| 12 | | HAdV 9 | 0.1 |
| 13 | | HAdV 8 | 0.000812 |
| 14 | | HAdV 17 | 158 |
| 15 | | HAdV 25 | 0.05 |
| 16 | | HAdV 28 | 28.1 |
| 17 | | HAdV 42 | 0.05 |
| 18 | E | HAdV 4 | 0.183 |
| 19 | F | HAdV 40 | 0.0104 |
| 20 | | HAdV 41 | 0.158 |
| 21 | G | HAdV 52 | na* |
* HAdV 52 was not available via organism collections and therefore the LoD was determined using plasmid DNA. The LoD was calculated to be 5000 copies/ml by qPCR.
## e. Analytical reactivity
### Cross-Reactivity
A panel of 61 potentially cross-reacting microorganisms (34 viral samples and 27 bacterial samples), representing respiratory pathogens or flora commonly present in the nasopharynx, was evaluated for cross-reactivity. Each potential cross-reactant was individually spiked into an Adenovirus negative nasal wash/aspirate at a challenging concentration. Adenovirus positive samples at 100 and 3 times the limit of detection (100x LoD and 2x LoD) were also tested. The Adenovirus R-gene US assay did not cross-react with any of the cross-reactants tested.
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Adenovirus R-gene US Cross-Reactivity Results
| | | Adenovirus R-gene US | |
| --- | --- | --- | --- |
| Virus | Concentration tested | 530nm – ADV Ct (cycles) | 560nm – IC2 Ct (cycles) |
| Adenovirus 3 | 100x LoD | 27.9 | 26.6 |
| | 2x LoD | 34.1 | 25.4 |
| Cytomegalovirus | 10^{4} TCID_{50}/mL | negative | 26.0 |
| Epstein Barr Virus | 10^{7} cp/mL | negative | 25.5 |
| BK Virus | 10^{4} TCID_{50}/mL | negative | 26.6 |
| Herpes Simplex Virus 1 | 10^{4} TCID_{50}/mL | negative | 26.3 |
| Herpes Simplex Virus 2 | 10^{4} TCID_{50}/mL | negative | 26.5 |
| Varicella Zoster Virus | 10^{3} TCID_{50}/mL | negative | 26.5 |
| Human Herpes Virus 6 | >10^{3} TCID_{50}/mL | negative | 25.9 |
| Human Herpes Virus 7 | 10^{4} cp/mL | negative | 25.9 |
| Human Herpes Virus 8 | 10^{4} cp/mL | negative | 25.6 |
| Influenza A Virus | 10^{5} TCID_{50}/mL | negative | 26.3 |
| Influenza B Virus | 10^{4} TCID_{50}/mL | negative | 25.9 |
| Respiratory Syncytial Virus A | 10^{4} TCID_{50}/mL | negative | 25.8 |
| Respiratory Syncytial Virus B | 10^{4} TCID_{50}/mL | negative | 25.3 |
| Human Metapneumovirus A | 10^{4} TCID_{50}/mL | negative | 25.3 |
| Human Metapneumovirus B | 10^{4} TCID_{50}/mL | negative | 26.1 |
| Human Bocavirus 1 | 10^{6} cp/mL | negative | 26.2 |
| Parainfluenza Virus 1 | 10^{4} TCID_{50}/mL | negative | 25.9 |
| Parainfluenza Virus 2 | 10^{5} TCID_{50}/mL | negative | 26.2 |
| Parainfluenza Virus 3 | 10^{6} TCID_{50}/mL | negative | 26.1 |
| Parainfluenza Virus 4 | 10^{4} TCID_{50}/mL | negative | 26.3 |
| Coronavirus NL63 | 10^{4} TCID_{50}/mL | negative | 26.1 |
| Rhinovirus 14 | 10^{3} TCID_{50}/mL | negative | 26.3 |
| Rhinovirus 87 | 10^{3} TCID_{50}/mL | negative | 26.4 |
| Rhinovirus 1B | 10^{3} TCID_{50}/mL | negative | 26.2 |
| Echovirus 25 | 10^{5} TCID_{50}/mL | negative | 26.8 |
| Coxackie B2 | 10^{5} TCID_{50}/mL | negative | 26.2 |
| Coxsackie A9 | 10^{5} TCID_{50}/mL | negative | 25.6 |
| Echovirus 9 | 10^{5} TCID_{50}/mL | negative | 26.4 |
| Poliovirus S3 | 10^{4} TCID_{50}/mL | negative | 25.6 |
| Echovirus 30 | 10^{4} TCID_{50}/mL | negative | 25.9 |
| Parechovirus 1 | 10^{4} TCID_{50}/mL | negative | 25.3 |
| Parechovirus 2 | 10^{4} TCID_{50}/mL | negative | 26.1 |
| Measles | 10^{4} pfu/mL | negative | 25.9 |
| Mumps | 10^{4} pfu/mL | negative | 26.6 |
{12}
| | | Adenovirus R-gene US | |
| --- | --- | --- | --- |
| Bacteria | Concentration tested | 530nm – ADV Ct (cycles) | 560nm – IC2 Ct (cycles) |
| Bordetella pertussis | 10^{6} cfu/mL | negative | 26.0 |
| Bordetella parapertussis | 10^{6} cfu/mL | negative | 26.2 |
| Mycoplasma pneumoniae | 10^{6} ccu/mL | negative | 26.3 |
| Chlamydophila pneumoniae | 10^{3} TCID_{50}/mL | negative | 26.2 |
| Legionella pneumophila | 10^{6} cfu/mL | negative | 26.4 |
| Bordetella bronchiseptica | 10^{6} cfu/mL | negative | 26.3 |
| Escherichia coli | 10^{6} cfu/mL | negative | 26.2 |
| Staphylococcus epidermidis | 10^{6} cfu/mL | negative | 26.3 |
| Klebsiella pneumoniae | 10^{6} cfu/mL | negative | 26.6 |
| Haemophilus influenzae | 10^{6} cfu/mL | negative | 26.4 |
| Serratia marcescens | 10^{6} cfu/mL | negative | 26.4 |
| Proteus mirabilis | 10^{6} cfu/mL | negative | 26.8 |
| Pseudomonas aeruginosa | 10^{6} cfu/mL | negative | 26.5 |
| Stenotrophomonas maltophila | 10^{6} cfu/mL | negative | 26.1 |
| Citrobacter freundii | 10^{6} cfu/mL | negative | 26.4 |
| Citrobacter koseri | 10^{6} cfu/mL | negative | 26.0 |
| Enterobacter cloacae | 10^{6} cfu/mL | negative | 26.1 |
| Acinobacter baumannii | 10^{6} cfu/mL | negative | 26.5 |
| Streptococcus agalactiae | 10^{6} cfu/mL | negative | 26.5 |
| Staphylococcus aureus | 10^{6} cfu/mL | negative | 25.5 |
| Klebsiella oxytoca | 10^{6} cfu/mL | negative | 25.8 |
| Enterobacter kobei | 10^{6} cfu/mL | negative | 25.5 |
| Morganella morganii | 10^{6} cfu/mL | negative | 26.0 |
| Streptococcus constellatus | 10^{6} cfu/mL | negative | 25.6 |
| Raoultella ornithinolytica | 10^{6} cfu/mL | negative | 25.8 |
| Haemophilus parainfluenza | 10^{6} cfu/mL | negative | 25.6 |
| Branhamella catarrhalis | 10^{6} cfu/mL | negative | 25.8 |
## Reactivity
No additional strains beyond the 22 tested for LoD were tested for reactivity in analytical studies.
## f. Interference studies:
## Interfering Substances
Potentially interfering or inhibitory substances that may be present in nasopharyngeal swabs or interfere with the PCR reaction were evaluated for the viral strains indicated below. To determine the potential interference of endogenous and exogenous chemical PCR inhibitors on Adenovirus detection, clinically relevant amounts of interfering substances were added to nasopharyngeal samples spiked with Adenovirus at 2x LoD and 10x LoD. A reference sample with no interfering substance was included at both 10x and 2x LoD.
{13}
The following table shows the interfering substances used for this study. Ten percent of the recommended dose was added to the sample unless otherwise stated in the table. The substances consisted of liquid nasal sprays, ingestible pills and lozenges, and endogenous substances.
Interfering Substances
| Substance Name | Active Ingredient | Concentration Tested | Justification |
| --- | --- | --- | --- |
| Mucin (Bovine submaxillary gland type I-S) | Purified mucin protein | 60 μg/mL | 1000x maximum level present in serum |
| Blood (human) EDTA anticoagulated | n/a | 2% (v/v) | Same concentration tested for similar IVD’s |
| Tamiflu® 45 mg | Oseltamivir | 7.5 mg/mL | 10% of total recommended dose |
| Afrin nasal spray | Oxymetazoline HCl | 7.45% (v/v) | 10% of total recommended dose |
| Tobrex 0.3% | Tobramycin | 9% (v/v) = 0.27 mg/mL | 10% of total recommended dose |
| Synagis 50 mg | Palivizumab | 81.8% (v/v) = 18 mg | 10% of total recommended dose |
| Relenza | Zanamivir | 45% (v/v) = 1 mg | 10% of total recommended dose |
Detailed chemical interference results are presented in the following table. Note that the study was performed in two separate runs with half of the potential interferents tested in run 1 and the other half in run 2. "Reference run 1" is the Adenovirus only control for the first run. "Reference run 2" is the Adenovirus only control for the second run. The two Adenovirus only reference samples were tested at concentrations of 10x LoD and 2x LoD. The data in the column indicating delta Ct values were calculated by taking the difference in average Ct cycles between the sample containing the potential chemical interferent and the "reference run" associated with that test.
{14}
Adenovirus R-gene US Interfering Substances
| Substance Name | AdV 3 Concentration | AdV 3 Ct (avg cycles) | AdV Delta Ct (cycles) | Internal Control 2 Ct (cycles) | Internal Control 2 Delta Ct (cycles) |
| --- | --- | --- | --- | --- | --- |
| Reference run 1 | 10x LoD | 30.77 | n/a | 26.17 | n/a |
| | 2x LoD | 33.90 | n/a | 25.97 | n/a |
| Mucin | 10x LoD | 31.77 | 1.00 | 26.27 | 0.10 |
| | 2x LoD | 33.65 | -0.25 | 25.97 | 0.00 |
| Blood | 10x LoD | 32.00 | 1.23 | 26.23 | 0.07 |
| | 2x LoD | 33.63 | -0.27 | 25.87 | -0.10 |
| Tamiflu® | 10x LoD | 33.33 | 2.57 | 26.90 | 0.73 |
| | 2x LoD | 36.90 | 3.00 | 27.03 | 1.07 |
| Afrin Nasal Spray | 10x LoD | 32.30 | 1.53 | 26.33 | 0.17 |
| | 2x LoD | 34.40 | 0.50 | 26.40 | 0.43 |
| Tobrex 0.3% | 10x LoD | 31.53 | 0.77 | 26.27 | 0.10 |
| | 2x LoD | 34.27 | 0.37 | 25.97 | 0.00 |
| Synagis 50 mg | 10x LoD | 31.55 | 0.78 | 26.13 | 0.03 |
| | 2x LoD | 33.27 | -0.63 | 25.93 | -0.03 |
| Substance Name | AdV 3 Concentration | AdV 3 Ct (avg cycles) | AdV Delta Ct (cycles) | Internal Control 2 Ct (cycles) | Internal Control 2 Delta Ct (cycles) |
| Reference run 2 | 10x LoD | 30.87 | n/a | 25.30 | n/a |
| | 2x LoD | 32.80 | n/a | 25.17 | n/a |
| Relenza | 10x LoD | 30.50 | -0.37 | 25.60 | 0.30 |
| | 2x LoD | 34.40 | 1.60 | 25.13 | -0.03 |
| FluMist® | 10x LoD | 30.77 | -0.10 | 25.37 | 0.07 |
| | 2x LoD | 34.15 | 1.35 | 25.07 | -0.10 |
| NeoSynephrine® | 10x LoD | 31.50 | 0.63 | 25.83 | 0.53 |
| | 2x LoD | 34.10 | 1.30 | 25.57 | 0.40 |
| Walgreens Original Anefrin Nasal Spray | 10x LoD | 31.50 | 0.63 | 26.50 | 1.20 |
| | 2x LoD | 34.20 | 1.40 | 25.67 | 0.50 |
| Zicam Homeopathic Nasal Gel | 10x LoD | 30.70 | -0.17 | 26.03 | 0.73 |
| | 2x LoD | 34.47 | 1.67 | 25.57 | 0.40 |
| Walgreens Saline Nasal Spray | 10x LoD | 32.03 | 1.17 | 26.13 | 0.83 |
| | 2x LoD | 35.73 | 2.93 | 25.63 | 0.47 |
| Chloraseptic® Sore Throat Lozenges | 10x LoD | 30.37 | -0.50 | 25.27 | -0.03 |
| | 2x LoD | 34.47 | 1.67 | 25.80 | 0.63 |
{15}
# Microbial Interference
To determine the potential interference of common nasal microorganisms on qualitative Adenovirus detection, a panel of microorganisms was spiked into Adenovirus positive NP washes/aspirates containing Adenovirus at two times the limit of detection (2x LoD). Crossing threshold (Ct) values were compared with a reference sample with no additional microorganism.
## Potential Interferents Tested for Microbial Interference
| Microorganism | Source | Concentration Tested |
| --- | --- | --- |
| Adenovirus 3 (reference) | ATCC VR-3 virus cultured on MRC5 cells | ~2x LoD (0.0066 TCID_{50}/mL) |
| Staphylococcus aureus | ATCC 33497 | 10^{6} cfu/mL |
| Bordetella pertussis | ATCC 9797 | 10^{6} cfu/mL |
| Respiratory Syncytial Virus A | ATCC VR-26 virus cultured on MRC5 cells | 10^{4} TCID_{50}/mL |
| Escherichia coli | Zeptometrix, ref0801517 | 10^{6} cfu/mL |
| Cytomegalovirus | ATCC VR-977 | 10^{4} TCID_{50}/mL |
| Parainfluenza 3 | ATCC VR-93 virus cultured on MK2 cells | 10^{6} TCID_{50}/mL |
| Influenza A | A/PR/8/34 ATCC VR-1469 virus cultured on MDCK cells | 10^{5} TCID_{50}/mL |
| Influenza B | Strain and source not specified; cultured on MDCK cells | 10^{4} TCID_{50}/mL |
| Rhinovirus 1B | ATCC VR-1645 strain B632 virus cultured on MRC5 cells | 10^{3} TCID_{50}/mL |
| Coronavirus NL63 | Source not specified; cultured on MK2 cells | 10^{4} TCID_{50}/mL |
| Measles | NCPV ref0809213v strain Mvi/Nottingham/GBR/18.04 | 10^{3} pfu/mL |
| Mumps | NCPV, ref0809281v strain 20082 | 10^{4} pfu/mL |
As shown in the table below, Adenovirus was consistently detected with no significant change in the Adenovirus Ct count regardless of whether an additional microorganism was present; thus indicating that there was no interference caused by the microorganisms tested.
## Adenovirus R-gene US Microbial Interference
| Microorganism | AdV Ct (avg cycles) | AdV Delta Ct (cycles) | Internal Control 2 Ct (cycles) | Internal Control 2 Delta Ct (cycles) |
| --- | --- | --- | --- | --- |
| AdV 3 2x LoD (Run 1) | 32.17 | n/a | 26.20 | n/a |
| AdV 3 + E. coli | 31.73 | -0.43 | 26.13 | -0.07 |
| AdV 3 + CMV | 32.33 | 0.17 | 26.23 | 0.03 |
{16}
| AdV 3 + Parainfluenza 3 | 31.97 | -0.20 | 26.30 | 0.10 |
| --- | --- | --- | --- | --- |
| AdV 3 + Influenza A | 32.23 | 0.07 | 25.97 | -0.23 |
| AdV 3 + Influenza B | 33.03 | 0.87 | 25.80 | -0.40 |
| AdV 3 + Rhinovirus 1B | 31.60 | -0.57 | 25.80 | -0.40 |
| AdV 3 + Coronavirus NL63 | 32.07 | -0.10 | 26.40 | 0.20 |
| AdV 3 + Measles virus | 32.90 | 0.73 | 26.07 | -0.13 |
| AdV 3 + Mumps virus | 32.53 | 0.37 | 25.97 | -0.23 |
| AdV 3 2x LoD (Run 2) | 34.07 | n/a | 25.37 | n/a |
| AdV 3 + Staph. aureus | 32.90 | -1.17 | 25.37 | 0.00 |
| AdV 3 + B. pertussis | 32.77 | -1.30 | 25.53 | 0.17 |
| AdV 3 + RSV A | 34.93 | 0.87 | 25.67 | 0.30 |
# g. Assay cut-off:
The cut-off determination was performed through testing of 218 samples: 98 negative, 67 high negative, and 53 positive. Of the 98 negative samples, all but four were below 20 AFU, four were between 20 and 30 AFU and none were over 30. Of the 67 high negative samples, 31 were below 20 AFU and 36 were between 20 and 30 AFU. Of the 53 positive samples, all were over 40 AFU. No fluorescence values were obtained between 30 and 40 AFU among the 218 samples tested. The threshold default value (30 AFU) of the SmartCycler II was confirmed to be the correct cut-off value for the Adenovirus R-gene US assay.
This cut-off value was further confirmed in a preliminary clinical study, performed at Caen Hospital (France), on 184 clinical samples, prior to US clinical studies. The cutoff was then confirmed by analysis of the U.S. clinical studies results at the 3 sites, New York, Chicago and Memphis. The 30 AFU value was confirmed as the cut-off to achieve the best sensitivity and specificity.
# h. Carryover Contamination:
This study was performed to examine potential carry-over/cross-contamination with the Adenovirus R-gene US assay by testing simulated human adenovirus (HAdV) high positive samples run in series with HAdV negative samples. The high positive samples consisted of negative nasal washes/aspirates spiked with HAdV type 4 at four logs above the Limit of Detection (LoD). The samples were processed and extracted in a high positive/negative alternating fashion on the bioMérieux NucliSENS® easyMAG® extraction system and likewise processed and run on the Cepheid SmartCycler II
{17}
instrument in an alternating fashion. Eleven high positive and eleven negative samples and a negative control were extracted per run on the easyMAG for a total of 55 high positive samples and 55 negative samples over 5 extraction runs and 4 amplification runs. When tested with high positive samples, no carry-over contamination was seen for the Adenovirus R-gene US assay.
The data from the study are summarized in the table below.
| EasyMag NucliSENS Run | Run 1 | Run 2 | | Run 3 | | Run 4 | Run 5 | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| SmartCycler II Run | Run 1 | | Run 2 | | Run 3 | | | Run 4 | Total |
| High Positive sample | 11/11 | 4/4 | 7/7 | 8/8 | 3/3 | 11/11 | 1/1 | 10/10 | 55/55 (100%) |
| Mean Ct | 19.4 | | 19.5 | | 19.3 | | | 19.6 | |
| Negative sample | 11/11 | 4/4 | 7/7 | 8/8 | 3/3 | 11/11 | 1/1 | 10/10 | 55/55 (100%) |
# 2. Comparison studies:
a. Method comparison with predicate device:
Not applicable
b. Matrix comparison:
Not applicable
c. Transport media compatibility :
Both types of Viral Transport Media (Universal Transport Medium from DHI, MicroTest™ M4RT Transport from Remel) claimed in the package insert were used in the prospective clinical study and were compatible with the Adenovirus R-gene assay.
# 3. Clinical studies:
Prospective clinical study:
Performance characteristics of the Adenovirus R-gene US assay were determined in a multi-center prospective investigational study employing 3 geographically diverse institutions in the US. The study began with recruitment and testing commencing on September 28, 2010. Specimens used in the study represent excess nasal specimens (NPS and NPA) that were collected from symptomatic men and women of all ages suspected of respiratory infection and submitted for routine care or analysis. Age ranges for patients providing specimens in the study ranged from 1 month to 101 years. The database was frozen on November 21, 2011 after testing 1186 swab and 395 NP wash/aspirate specimens and this dataset was used in the analysis. Two NP aspirate/wash specimens and three NPS were excluded from the analysis due to
{18}
protocol deviations. The Adenovirus R-gene US assay was compared with rapid culture (shell vial) followed by direct fluorescent antibody (DFA) screening and identification using the D3Ultra™ Direct Fluorescent Antibody (DFA) Respiratory Virus screening & ID kit from Diagnostic Hybrids (DHI). Each US site analyzed around 280-650 samples (swabs and washes/aspirates), for a total of 1576 samples. The studies were performed on the remnant of the specimen collected from children and adults after the routine clinical care.
Each sample was tested by Adenovirus R-gene US assay and rapid culture (shell vial) followed by DFA screening and identification. The Argene and DHI test were performed according to the instructions provided by the manufacturer. The shell vial culture was performed following standard laboratory procedures. The chosen sites were hospital virology laboratories experienced in DFA, cell culture and real time PCR. Performances (sensitivity and specificity) of the Adenovirus R-gene US assay were calculated as compared to the method of viral culture followed by DFA staining with DHI test.
## Clinical Study Results – Swab Specimens
| | | Culture Result | | | |
| --- | --- | --- | --- | --- | --- |
| | | Detected | Not Detected | Total | |
| Adenovirus R-gene US | Detected | 44 | 43^{a} | 87 | Sensitivity: 91.7% (80.0%-97.7%) 95%CI |
| | Not Detected | 4^{b} | 1092 | 1096 | Specificity: 96.2% (94.9%-97.2%) 95%CI |
| | | 48 | 1135 | 1183 | |
a 42/43 samples confirmed as Adenovirus positive by quantitative PCR
b 1/4 samples confirmed as Adenovirus positive by quantitative PCR
## Clinical Study Results – Wash/Aspirate Specimens
| | | Culture Result | | | |
| --- | --- | --- | --- | --- | --- |
| | | Detected | Not Detected | Total | |
| Adenovirus R-gene US | Detected | 21 | 21^{a} | 42 | Sensitivity: 100% (86.7%-100%) 95%CI |
| | Not Detected | 0 | 351 | 351 | Specificity: 94.4% (91.5%-96.5%) 95%CI |
| | | 21 | 372 | 393 | |
a 19/21 samples confirmed as Adenovirus positive by quantitative PCR
{19}
4. Clinical cut-off:
Not applicable
5. Expected values/Reference range:
The general demographic data for all eligible prospective specimens are presented in the two following tables:
Prevalence (positives as determined by reference method) and Expected Value (positives as determined by Adenovirus R-gene US assay) observed during the clinical study in swab specimens
| Swab Prospective Study | | | | | |
| --- | --- | --- | --- | --- | --- |
| Subject Age (years) | Number | Total Adeno R-gene US positive | Expected Value | Total DHI positive | Observed Prevalence |
| < 2 | 519 | 45 | 8.7% | 25 | 4.8% |
| 2-5 | 280 | 26 | 9.3% | 14 | 5.0% |
| 6-19 | 229 | 11 | 4.8% | 5 | 2.2% |
| 19-64 | 112 | 5 | 4.5% | 4 | 3.6% |
| >65 | 43 | 0 | ND | 0 | ND |
| Total | 1183 | 87 | 7.35% | 48 | 4.06% |
Prevalence (positives as determined by reference method) and Expected Value (positives as determined by Adenovirus R-gene US assay) observed during the clinical study in wash/aspirate specimens
| Wash/Aspirate Prospective Study | | | | | |
| --- | --- | --- | --- | --- | --- |
| Subject Age (years) | Number | Total Adeno R-gene US positive | Expected Value | Total DHI positive | Observed Prevalence |
| < 2 | 275 | 33 | 12.0% | 17 | 6.2% |
| 2-5 | 57 | 7 | 12.3% | 2 | 3.5% |
| 6-19 | 55 | 2 | 3.6% | 2 | 3.6% |
| 19-64 | 5 | 0 | 0.0% | 0 | 0.0% |
| >65 | 1 | 0 | ND | 0 | ND |
| Total | 393 | 42 | 10.69% | 21 | 5.34% |
N. Instrument Name:
Cepheid SmartCycler II utilizing Dx software version 1.7b or 3.0
{20}
O. System Descriptions:
1. Modes of Operation:
The bioMérieux NucliSens easyMAG is an automated nucleic acid isolation and purification system that is based upon the silica extraction technology. The easyMAG is capable of processing a total of 24 reactions with variable sample types, sample volumes, and elution volumes within a single run. Nucleic acid is purified within a single cartridge by several steps that include lysis and binding of nucleic acid to high affinity magnetic silica beads, a series of wash steps and heated elution of purified nucleic acid from the silica beads.
The Cepheid SmartCycler II® Real Time instrument with Dx software version 1.7b or 3.0a is used to perform real time PCR amplification and detection of nucleic acid. The Cepheid SmartCycler II® instrument is an integrated nucleic acid amplification and detection instrument system based on Cepheid’s proprietary microprocessor-controlled I-CORE module. For purified DNA samples, the SmartCycler II® instrument enables polymerase chain reaction (PCR) for the amplification of DNA, and hybridization of fluorogenic target-specific probes for the detection of the amplified DNA.
2. Software:
FDA has reviewed applicant’s Hazard Analysis and software development processes for this line of product types:
Yes ☐ X or No ☐
3. Specimen Identification:
Specimens ID’s are manually entered into the user interface by the user.
4. Specimen Sampling and Handling:
Liquid samples from nasopharyngeal swabs and/or nasal wash/aspirates are manually transferred collected and transferred into tubes for nucleic acid extraction and purification.
5. Calibration:
Calibration is not recommended.
6. Quality Control:
The positive control plasmid enables the experiment to be properly validated. This positive control is amplified with the same primers as the viral DNA of any Adenovirus present in patient samples. A bacteriophage Internal Control (IC2) is added to every sample to detect the presence of inhibitors or lysis failure.
{21}
P. Other Supportive Instrument Performance Characteristics Data Not Covered In The "Performance Characteristics" Section above:
Not applicable
Q. Proposed Labeling:
The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.
R. Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.
