ID-TAG RESPIRATORY VIRAL PANEL
Device Facts
| Record ID | DEN070013 |
|---|---|
| Device Name | ID-TAG RESPIRATORY VIRAL PANEL |
| Applicant | Luminex Molecular Diagnostics, Inc. |
| Product Code | OCC · Microbiology |
| Decision Date | Jan 3, 2008 |
| Decision | DENG |
| Submission Type | Post-NSE |
| Regulation | 21 CFR 866.3980 |
| Device Class | Class 2 |
Intended Use
The xTAG™ Respiratory Viral Panel (RVP) is a qualitative nucleic acid multiplex test intended for the simultaneous detection and identification of multiple respiratory virus nucleic acids in nasopharyngeal swabs from individuals suspected of respiratory tract infections. The following virus types and subtypes are identified using RVP: Influenza A, Influenza A subtype H1, Influenza A subtype H3, Influenza B, Respiratory Syncytial Virus subtype A, Respiratory Syncytial Virus subtype B, Parainfluenza 1, Parainfluenza 2, and Parainfluenza 3 virus, Human Metapneumovirus, Rhinovirus, and Adenovirus. The detection and identification of specific viral nucleic acids from individuals exhibiting signs and symptoms of respiratory infection aids in the diagnosis of respiratory viral infection if used in conjunction with other clinical and laboratory findings. It is recommended that specimens found to be negative after examination using RVP be confirmed by cell culture. Negative results do not preclude respiratory virus infection and should not be used as the sole basis for diagnosis, treatment or other management decisions. Positive results do not rule out bacterial infection, or co-infection with other viruses. The agent detected may not be the definite cause of disease. The use of additional laboratory testing (e.g. bacterial culture, immunofluorescence, radiography) and clinical presentation must be taken into consideration in order to obtain the final diagnosis of respiratory viral infection.
Device Story
xTAG RVP is a multiplex RT-PCR and Target Specific Primer Extension (TSPE) assay for qualitative detection of 12 respiratory viral targets in nasopharyngeal swabs. Input: extracted viral RNA/DNA. Process: multiplex RT-PCR amplification; TSPE reaction using biotinylated dNTPs; hybridization to spectrally distinguishable Luminex microspheres (beads) coupled with anti-tags; reporter molecule (streptavidin-phycoerythrin) binding. Output: median fluorescence intensity (MFI) signals analyzed by proprietary software to report presence/absence of viral targets. Used in clinical laboratories; operated by trained personnel. Results aid diagnosis of respiratory viral infections when combined with clinical/laboratory findings. Benefits include simultaneous identification of multiple pathogens, facilitating faster clinical management.
Clinical Evidence
Prospective clinical study of 544 nasopharyngeal swabs collected during the 2005/06 season. Comparator methods included viral culture, DFA, and RT-PCR/sequencing. Sensitivity ranged from 78.3% (Adenovirus) to 100% (RSV A/B, Para 1/2, Rhinovirus). Specificity ranged from 91.3% (Rhinovirus) to 100% (Adenovirus, H1 subtype). Bench testing included analytical sensitivity (LoD), specificity (cross-reactivity), and reproducibility studies across three sites.
Technological Characteristics
Multiplex RT-PCR and TSPE assay. Uses Luminex xMAP instrument (100 IS/200 systems) for spectrofluorometric detection. Materials: oligonucleotide primers/probes, coupled microspheres (beads), streptavidin-phycoerythrin reporter. Connectivity: standalone instrument output (CSV file) processed by proprietary software. Software algorithm: qualitative threshold-based (MFI ≥ 300 positive, < 150 negative).
Indications for Use
Indicated for individuals of all ages exhibiting signs and symptoms of respiratory tract infection. For 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.
Related Devices
- K103776 — XTAG RESPIRATORY VIRAL PANEL FAST, XTAG DATA ANALYSIS SOFTWARE FOR RVP FAST · Luminex Molecular Diagnostics, Inc. · Jul 1, 2011
- K112199 — XTAG RESPIRATORY VIRAL PANEL, XTAG DATA ANALYSIS SOFTWARE (TDAS RVP-1) · Luminex Molecular Diagnostics, Inc. · Sep 1, 2011
- K112781 — TAG RESPIRATORY VIRAL PANEL TAG DATA ANALYSIS SOFTWARE (TDAS RVP-I) · Luminex Molecular Diagnostics, Inc. · Feb 17, 2012
- K091667 — XTAG RESPIRATORY VIRAL PANEL, MODELS I019A0110, I019C011, I019D0112, I019E0113, S019-0116 · Luminex Molecular Diagnostics, Inc. · Jun 25, 2009
- K242613 — NxTAG® Respiratory Pathogen Panel · Luminex Molecular Diagnostics, Inc. · Oct 2, 2024
Submission Summary (Full Text)
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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY TEMPLATE
# A. 510(k) Number:
k063765
# B. Purpose for Submission:
New device
# C. Measurand:
Respiratory specimen virus nucleic acid (RNA or DNA) target sequences. Viruses targeted have been associated with respiratory infections in adults and/or children. Viral types and subtypes detected:
Influenza A, Influenza A H1, Influenza A H3, Influenza B, Respiratory Syncytial Virus Type A, Respiratory Syncytial Virus Type B, Parainfluenza virus 1, Parainfluenza virus 2, Parainfluenza virus 3, Human Metapneumovirus, Rhinovirus, Adenovirus.
# D. Type of Test:
Multiplex nucleic acid assay, qualitative determination of 12 respiratory virus type and subtype target sequences in nasopharyngeal swabs using nucleic acid isolation, amplification and detection on the Luminex xMAP instrument, which generates signals based on the acquisition of spectrofluorometric data.
# E. Applicant:
Luminex Molecular Diagnostics Inc.
# F. Proprietary and Established Names:
xTAG™ RVP (Respiratory Viral Panel) Common Name: Respiratory Viral Panel (RVP) Multiplex Nucleic Acid Detection Assav
# G. Regulatory Information:
- 1. Regulation section:
21 CFR 866.3980, Respiratory viral panel multiplex nucleic acid assay
- 2. Classification:
- Class II 3.
- Product code: ОСС. ОЕМ, ОЕР
- 4. Panel: Microbiology (83)
# H. Intended Use:
- l. Intended use(s):
The xTAG™ Respiratory Viral Panel (RVP) is a qualitative nucleic acid multiplex test intended for the simultaneous detection and identification of multiple respiratory virus nucleic acids in nasopharyngeal swabs from individuals suspected of respiratory tract infections. The following virus types and subtypes are identified using RVP: Influenza A, Influenza A subtype H1, Influenza A subtype H3, Influenza B, Respiratory Syncytial Virus subtype A, Respiratory Syncytial Virus subtype B, Parainfluenza 1, Parainfluenza 2, and Parainfluenza 3 virus,
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Human Metapneumovirus, Rhinovirus, and Adenovirus. The detection and identification of specific viral nucleic acids from individuals exhibiting signs and symptoms of respiratory infection aids in the diagnosis of respiratory viral infection if used in conjunction with other clinical and laboratory findings. It is recommended that specimens found to be negative after examination using RVP be confirmed by cell culture. Negative results do not preclude respiratory virus infection and should not be used as the sole basis for diagnosis, treatment or other management decisions.
Positive results do not rule out bacterial infection, or co-infection with other viruses. The agent detected may not be the definite cause of disease. The use of additional laboratory testing (e.g. bacterial culture, immunofluorescence, radiography) and clinical presentation must be taken into consideration in order to obtain the final diagnosis of respiratory viral infection.
### Due to seasonal prevalence, performance characteristics for Influenza A/H1 were established primarily with retrospective specimens.
The RVP assay cannot adequately detect Adenovirus species C, or serotypes 7a and 41. The R VP primers for detection of rhinovirus cross-react with enterovirus. A rhinovirus reactive result should be confirmed by an alternate method (e.g. cell culture).
Performance characteristics for Influenza A Virus were established when Influenza A/H3 and A/H1 were the predominant Influenza A viruses in circulation. When other Influenza A viruses are emerging, performance characteristics may vary. If infections with a novel Influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent Influenza viruses and sent to a state or local health department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.
- 2. Indication(s) for use: Same as Intended Use
- 3. Special conditions for use statement(s): For prescription use only
- Special instrument requirements: 4. Luminex® Instrument (100 IS and 200 systems)
# I. Device Description:
The xTAG™ RVP is a PCR-based system for detecting the presence / absence of viral DNA / RNA in clinical specimens. The oligonucleotide primer / probe components of the xTAGTM RVP have been designed to specifically target unique regions in the RNA / DNA of each molecular species listed in the following Table:
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| Respiratory viral targets |
|-------------------------------------|
| Influenza A (Matrix Gene) |
| Influenza A H1 (Hemagglutinin Gene) |
| Influenza A H3 (Hemagglutinin Gene) |
| Influenza B |
| Respiratory Syncytial Virus Type A |
| Respiratory Syncytial Virus Type B |
| Parainfluenza virus 1 |
| Parainfluenza virus 2 |
| Parainfluenza virus 3 |
| Human Metapneumovirus |
| Rhinovirus |
| Adenovirus |
Amplified products are sorted and analyzed on the Luminex® xMAP instrument, which generates signals based on the acquisition of spectrofluorometric data. The raw signals are median fluorescence intensities (MFI) which are acquired in a Luminex® Output.csv file that is subsequently analyzed by the software component of the xTAG™ RVP to establish the presence or absence of all viral types / subtypes for which a Luminex® microsphere population has been dedicated. The xTAG™ RVP primary components are:
1. PCR Primer Mix. The oligonucleotide primers incorporated in this mix have been designed to amplify conserved regions of the viral types / subtypes listed in the Table above and an internal control. Reverse transcription / PCR amplification of cDNA / DNA is the first step in the RVP assay. The PCR amplification product is then subjected to a Target Specific Primer Extension (TSPE) reaction.
2. Target Specific (TS) Primer Mix. Each of the oligonucleotide primers incorporated in this mix has been designed to extend (in the presence of thermostable DNA polymerase) only if the targeted cDNA / DNA sequence is present in the PCR amplification product. If a TS primer is extended, it will incorporate biotinylated dNTPs. After this TSPE reaction is completed and the reaction mix is treated to remove free dNTPs, the biotin that has been incorporated into TSPE reaction products will conjugate to a streptavidin - phycoerythrin reporter molecule that is added to the reaction mix. If a TS primer does not undergo this TSPE reaction, it will not be conjugated to this reporter molecule. Each TS primer also contains a proprietary "tag", which is a short oligonucleotide sequence designed to hybridize with a high degree of specificity to its complementary "anti-tag". Each anti-tag is coupled to a specific Luminex® microsphere population ("beads"). The TSPE Primer Mix will include oligonucleotides designed to discriminate the viral types / subtypes listed in the Table above.
3. Coupled Bead Mix. This is a suspension containing a defined set of Luminex® microspheres. Each microsphere population in this set is spectrally distinguishable from all other microsphere populations in the set when read on the Luminex® xMAP system. This feature is the basis on which MFI signals recorded in the Luminex® Output.csv file are sorted. The intensity of each recorded signal (Note: one MFI signal is recorded for each bead population in the Bead Mix) is a function of the degree to which the
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streptavidin-phycoerythrin reporter molecule has been incorporated into the bead population. This, in turn, is a function of the highly specific tag-anti-tag hybridization between the coupled beads and the TS primers which have incorporated biotiny lated dNTPs.
4. Data Analysis Software. This is proprietary software designed and developed by Luminex Molecular Diagnostics Inc. The software component of the system applies analysis algorithms to the MFI signals recorded in the Luminex® Output.csv file and reports a qualitative result for each viral type / control discriminated by the assay.
Other reagents required to perform testing with the device include ancillary reagents for which specific lots have been qualified by Luminex Molecular Diagnostics (LMD) and incorporated in the LMD quality system, for use with the xTAG™ RVP. The xTAG™ RVP product performance requires that only qualified ancillary reagent lots be used with the device. Any lots not specifically qualified by LMD for use with xTAGTM RVP are not validated for use with this assay, and may cause erroneous results. To find an up to date list of Qualified Ancillary Reagents log onto Luminex website Support page https://oraweb.luminexcorp.com/OA HTML/itflogin.jsp and search "RVP". Ancillary reagents should be used only according to the instructions for use contained in the RVP package insert. Any assay problems or failures that are suspected to involve ancillary reagents should be reported to Luminex Molecular Diagnostics Inc. The following is a list of ancillary reagents that are not supplied and are included in LMD's reagent qualification program:
* these reagents are not part of the ancillary reagent qualification program, and are not supplied with the kit
The xTAG™ RVP has been designed to generate unique PCR products for each of the targets described above with the exception of RSV targets. RSV subtypes detected by the xTAG™ RVP are discriminated at the TSPE step. The discrimination of Parainfluenza subtypes occurs at both the PCR and TSPE step. The detection of Influenza A subtypes is achieved by amplifying conserved regions of the matrix gene common to all subtypes and target specific regions of the hemagglutinin gene (2 sets of
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PCR primers for the 2 listed subtypes).
# J. Substantial Equivalence Information:
- 1. Predicate device name(s): None
- 2. Predicate 510(k) number(s): None
- Comparison with predicate: 3. Not applicable
# K. Standard/Guidance Document Referenced (if applicable):
- Special controls guidance documents will be promulgated. ●
- Guidance on Class II Special Controls Guidance Document: Reagents for Detection ● of Specific Novel Influenza A Viruses (March 2006) http://www.fda.gov/cdrh/oivd/guidance/1596.pdf.
- . Guidance on In Vitro Diagnostic Devices to Detect Influenza A Viruses: Labeling and Regulatory Path (April 2006) - http://www.fda.gov/cdrh/oivd/guidance/1594.pdf.
- . Guidance on Informed Consent for In Vitro Diagnostic Device Studies Leftover Human Specimens that are Not Individually Identifiable (April 2006) http://www.fda.gov/cdrh/oivd/guidance/1588.pdf.
- . Draft Guidance on Nucleic Acid Based In Vitro Diagnostic Devices for Detection of Microbial Pathogens (Dec 2005) – http://www.fda.gov/cdrh/oivd/guidance/1560.html.
- . Software Guidance for the content of premarket submissions for software contained in medical devices (May 2005) – http://www.fda.gov/cdrh/ode/guidance/337.html.
- General Guidance on Software Validation (Jan 2002) http://www.fda.gov/cdrh/comp/guidance/938.html.
- . CLSI EP17-A: Guidance for Protocols for Determination of Limits of Detection and Limits of Quantitations (Vol. 2, No. 34) (Oct 2004).
- CLSI MM13-A: Guidance for the Collection. Transport. Preparation and Storage of Specimens for Molecular Methods (Vol. 25, No. 31) (Dec 2005).
- CLSI EP7-A2: Guidance for Interference Testing in Clinical Chemistry (Vol. 25. No.27 Second Ed) (Nov 2005).
- CLSI EP12-A: Guidance for User Protocol for Evaluation of Qualitative Test ● Performance (Vol. 22, No. 14) (Sept 2002).
- . CLSI MM6-A: Guidance for the Quantitative Molecular Methods for Infectious Diseases (Vol. 23. No.28) (Oct 2003).
- CLSI EP5-A2: Guidance for Evaluation of Precision Performance of Quantitative ● Measurement Methods (Vol. 24, No. 25 Second Ed.) (Aug 2004).
- .
# L. Test Principle:
xTAG™ RVP incorporates multiplex Reverse Transcription Polymerase Chain Reaction (RT-PCR) and multiplex Target Specific Primer Extension (TSPE) with Luminex Molecular Diagnostic's proprietary Universal Tag sorting system on the Luminex® xMAP® platform (see figure below). XTAG™ RVP is compatible with both the Luminex® 100 IS and 200 systems.
Summary of Steps in Assay Performed Using XTAGTM RVP:
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Image /page/5/Figure/0 description: This image shows a flowchart of a laboratory process. The first step is "Sample Prep", followed by "Multiplex RT-PCR (25 μL)". The next steps are "SAP-EXO", "Multiplex TSPE using 5 µL treated RT-PCR", "Hybridization using 3.5 µL TSPE with 20 µL Bead Mix", "Add Reporter, Incubation", "Detection on Luminex®", and finally "Data Analysis by TDAS RVP-I".
- Viral nucleic acids are extracted from the sample, and a multiplex RT-PCR reaction is carried out under optimized conditions in a single multiplex PCR resulting in amplicons for each of the viruses/subtypes present in the sample. The amplimer sizes range from 107 bp to 402 bp to enable efficient incorporation of biotin-dCTP during the Target Specific Primer Extension (TSPE) reaction. Each PCR product is treated with Shrimp Alkaline Phosphatase (SAP) to inactivate any remaining nucleotides (especially dCTP), and with Exonuclease I (EXO) to degrade any primers left over from the PCR reaction.
- Multiplex Target Specific Primer Extension (TSPE) is then used to detect viral DNA present in the sample. In this step, each virus is detected by a Target-Specific Primer (TSP) with a unique DNA tag. For each TSP, the 3' end of the primer is a perfect match for its target, but will have a 3' mismatch on any other target. A DNA polymerase is used that will only extend the primer when there is a perfect match on the 3' end. so that the primer is only extended if its target DNA is present in the sample. Biotin-dCTP is incorporated into the extending chain if extension occurs.
- . After TSPE. the reaction is added directly to microwells containing bead-immobilized anti-tags, which are the complements of the DNA tags on the primers. The beads which contain the anti-tags are spectrally distinguishable from each other. A fluorescent reporter molecule (streptavidin - phycoerythrin) is bound to the biotin on the extended primers. Each tagged primer hybridizes only to its unique anti-tag complement;
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therefore, each colored bead represents a specific virus, through the bead/anti-tag/tagged primer association. The beads are then analyzed by the Luminex® instrument (100 IS and 200 systems). The Luminex® 100 IS and 200 systems contain two lasers: one identifies the color-coded bead, and the other identifies the presence or absence of extended primer through the phycoerythrin reporter. Thus, the presence of a virus in a sample is identified by the presence of phycoerythrin signal attached to the TSP for that virus.
- All viruses are identified in a single multiplex reaction. The data generated by the . Luminex® 100 IS and 200 systems is analyzed by the Software component of the kit (TDAS RVP-I) to provide a summary report summarizing of viruses present in the sample, if any. This summary report contains the qualitative output of the test (i.e. calls for each of the 12 analytes + 2 controls probed in each sample). Detailed reports including median fluorescence intensity (MFI) values are also available.
### Interpretation of Results:
TDAS RVP-I will display, for each sample, the calls for each target. Possible calls for a given target of a specific sample are:
- POS: the viral target is detected (i.e. analyte signal falls within the positive zone: MFI . >300)
- . NEG: the viral target is not detected (i.e. analyte signal falls within the negative zone: MFI <150)
- . *No Call: there is a failure in one or more assay parameters / controls.
Similarly, TDAS RVP-I will display, for each sample, the call for the Internal Control target and the Run Control target:
- PRES: the recommended Internal / Run Control is detected (MFI ≥ 300) .
- . ABS: the recommended Internal / Run Control is not detected (MFI < 300)
- . *No Call: inability to determine presence or absence of the Internal / Run Control due to an assay-specific criterion not being met.
- *The distinction between a "No Call" resulting from a target / assay / control failure or ambiguous result ("Invalid Result"), and a "No Call" resulting from an "Equivocal Signal" for a particular target is made in the TDAS RVP-I "Notes and Explanations" column that accompanies each sample output. Scenarios resulting in either of these 2 categories of "No Calls" are summarized in the table below:
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| Scenario resulting in a TDAS “No Call”<br>output for any given viral target | TDAS Warning<br>Message(s) in<br>summary view* | Reason for<br>Viral<br>Target “No<br>Call” | Re-test<br>Recommendations |
|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------|
| Signal for viral target falls within the equivocal<br>zone ( $150 ≤ MFI < 300$ ) and internal control<br>call is PRES. | “Target(s) failed:<br>value(s) not within<br>predefined ranges” | equivocal<br>signal | Re-run from RNA<br>step (or re-extract or<br>obtain new specimen<br>at laboratory’s<br>discretion) |
| Signal for viral target falls within the equivocal<br>zone ( $150 ≤ MFI < 300$ ) and internal control<br>call is ABS and at least one other target has a<br>signal in the positive zone ( $MFI ≥ 300$ ). | Target(s) failed:<br>value(s) not within<br>predefined ranges” | equivocal<br>signal | Re-run from RNA<br>step (or re-extract or<br>obtain new specimen<br>at laboratory’s<br>discretion) |
| Signal for one or both Influenza A subtypes (H1<br>and H3) falls within the positive zone ( $MFI ≥ 300$ ) and Influenza A matrix signal falls within<br>the negative zone ( $MFI < 150$ ). This results in a<br>No Call for both matrix signal and subtype* | “Targets failed:<br>incompatible signals<br>between targets” | invalid<br>result | Re-run from RNA<br>step (or re-extract or<br>obtain new specimen<br>at laboratory’s<br>discretion) |
| None of the viral target signals fall within the<br>positive zone ( $MFI ≥ 300$ ) and internal control<br>call is ABS. | “Sample failed:<br>unexpected control<br>call(s)” | invalid<br>result | Re-extract (or obtain<br>new specimen at<br>laboratory’s<br>discretion) |
| One or more viral targets or controls with low<br>bead count. | “Assay failed: low<br>bead count(s) for<br>negative control<br>sample”<br>“Sample failed: low<br>bead count for internal<br>control”<br>“Target failed: low<br>bead count” | invalid<br>result | Re-run from bead<br>hybridization step (or<br>re-run from RNA step<br>or re-extract or obtain<br>new specimen at<br>laboratory’s<br>discretion) |
| Plate failure due to unexpected signals in the last<br>position on the assay plate (reserved by TDAS<br>for the negative control).<br>Note: Signal > $150$ MFI units in any negative | “Assay failed:<br>unexpected value(s)<br>encountered or sample<br>is empty for negative<br>control sample” | invalid<br>result | Re-run from RNA<br>step |
| control sample on a plate, for one or more viral<br>analytes, is indicative of carryover<br>contamination of the plate. In such a case, it is<br>strongly recommended that the samples on that<br>plate be rerun, starting from the PCR step. | “Assay failed: a<br>negative control signal<br>exceeds acceptable<br>value”. | | Re-run from RNA<br>step or re-extract all<br>samples at<br>laboratory’s<br>discretion |
*RVP detection of Influenza A, subtypes H1 and H3 is achieved through (1) detection of the Flu A matrix gene which is common to all subtypes, and also (2) detection of subtype-specific regions of the hemagglutinin gene. Interpretation of results is discussed further below, using the example of Influenza A.
** Re-test Recommendations: It is recommended that the sample be re-tested once according to the instructions provided in the table. If a re-test needs to be carried out due to a "No Call" (due to either an equivocal or invalid result) being returned for a particular sample or target, the re-test results should be considered the final RVP result for that analyte. For detection of Influenza A H1 and H3 subtypes, there are specific precautions that must be followed which are described below. For all other analytes, if the final RVP result is a "no call" then follow-up testing is recommended.
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Any assay problems or failures that are suspected to involve ancillary reagents should be reported to Luminex Molecular Diagnostics Inc.
NOTE: if the influenza A matrix signal falls within the positive zone (MFI ≥ 300) and all subtype (H1 and H3) signals fall within the negative zone (MFI < 150), a POS call will be generated for influenza A and a NEG call will be generated for each of the H1, H3 subtypes. This is not considered an "Ambiguous Result". It may be indicative of an atypical variant of influenza A. See Interpretation and Reporting of Influenza A results below.
### Interpretation of Influenza A Results:
The RVP assay has been designed to probe for 3 distinct analytes associated with Influenza A virus: 1) a conserved sequence in the matrix gene (Influenza A target); 2) a conserved sequence specific to the H1 subtype of the hemagglutinin gene and 3) a conserved sequence specific to the H3 subtype of the hemagglutinin gene. A clear positive signal (MFI greater than or equal to 300) in the matrix gene is establishing an Influenza A infection. A clear negative signal (MFI less than 150) for each of the listed Influenza A analytes (Influenza A matrix, H1 and H3) should be interpreted as negative for Influenza A. A sample result that involves any other combination of signals for these 3 Influenza A analytes should be considered either equivocal or ambiguous. Further investigation of such equivocal / ambiguous results is recommended. In the particular case where the Influenza A target is detected with no clear positive result for either hemagglutinin target, special precautions must be followed (see reporting below).
### Reporting Influenza A Results:
- . Report negative test results for Influenza A as "Matrix gene target not detected, and hemagglutinin gene targets not detected. It is recommended that specimens found to be negative after examination using a respiratory viral panel nucleic acid detection assay be confirmed by cell culture. Negative results do not preclude respiratory virus infection and should not be used as the sole basis for diagnosis, treatment or other management decisions."
- . Report positive test results as "Positive for matrix gene target - Influenza A positive, and (where applicable) hemagglutinin gene target (specify hemagglutinin target detected, e.g. H1, or H3). This result does not rule out co-infections with pathogens that were not screened for by RVP. A positive result for a hemagglutinin gene target does not identify a specific influenza A strain (e.g. H1N1). The agent detected may not be the definite cause of disease. Results should be used in conjunction with other clinical and laboratory findings."
- . When Influenza A target is detected with no clear positive result for either H1 or H3, the sample should be re-tested from the extraction step together with positive controls for these two analytes. Extract prepared from the sample should be run in duplicate. In the case where the re-test on both replicates does not type for H1 or H3 and analyte controls are properly typed, necessitates notification of appropriate local, state or federal public health authorities to determine necessary measures for verification of results in accordance with the MMWR notice (http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5613a4.htm and http://www.cste.org/ps/2007pdfs/novelfluanndssjan10final23.pdf}. The purpose of the surveillance program described in these documents is to determine whether untypeable Influenza A specimens represent novel strains of Influenza A. In the event that remnant
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sample is not available, then extracted material should be forwarded to CDC per the procedures outlined above.
- . A "No Call" due to an equivocal or invalid result as shown in the table above, should not be reported but re-tested as per recommendations in this Table.
### M. Performance Characteristics (if/when applicable):
- 1. Analytical performance:
- a. Precision/Reproducibility:
Three separate precision studies were performed to assess the following:
- a) Reproducibility of the assay in the specimens near the clinical cutoff of the assav
- b) Reproducibility of the assay using virus concentrations expected to be found in clinical specimens (clinically significant concentration)
- c) Reproducibility in dual co-infected specimens.
a) Reproducibility near the assay cut-offs was assessed across 3 sites using replicates of samples containing viral material from culture-derived isolates in the matrix simulating intended use specimen type. The panel contained samples prepared to represent low positive (LP) and high negative (HN) analyte levels relative to the RVP cut-offs. Each simulated sample within the panel was divided into aliquots, blinded and stored frozen (-70°C) prior to testing. Thus, aliquots of the same blinded panel of samples were tested at the three different sites. Each site used a different extraction method and for each of the 3 extraction methods evaluated. 2 aliquots of a given sample dilution were extracted per day, for each of 3 days (i.e. a total of six extractions per site). At each site, both extracts from a given day were assayed in singlicate on the same RVP run. Calls (Positive, Equivocal, Negative) generated for the viral analyte in question are summarized in Tables below.
| Virus / Titer | All Days (3 extraction days x 2 extractions per day) | | | | | | | |
|---------------------------------------------------------------------|------------------------------------------------------|------------|-------------|------------|---------------------------|---------------|---------------------------|-------|
| Flu A-H3 (Strain:<br>A/Victoria/3/75<br>(H3N2), DHI Lot<br>#121106) | Site | # Positive | # Equivocal | # Negative | 25th<br>Percentile<br>MFI | Median<br>MFI | 75th<br>Percentile<br>MFI | %CV* |
| Flu A<br>Low Positive<br>(LP) (2 TCID50<br>per reaction) | Site 1 | 6/6 | 0/6 | 0/6 | 1531.38 | 1731.75 | 1969.25 | 29.25 |
| | Site 2 | 6/6 | 0/6 | 0/6 | 1428.75 | 1746.75 | 1828.88 | 40.89 |
| | Site 3 | 6/6 | 0/6 | 0/6 | 541.50 | 800.00 | 899.13 | 39.94 |
| | Total | 18 / 18 | 0 / 18 | 0 / 18 | 870.38 | 1463.00 | 1814.38 | 48.45 |
| H3<br>Low Positive<br>(LP) (200 TCID50<br>per reaction) | | 6 / 6 | 0 / 6 | 0 / 6 | 1679.00 | 1767.00 | 2017.75 | 14.89 |
| | Site 2 | 6 / 6 | 0 / 6 | 0 / 6 | 1567.38 | 1718.25 | 1912.63 | 23.70 |
| | Site 3 | 6 / 6 | 0 / 6 | 0 / 6 | 902.88 | 1077.25 | 1243.00 | 18.44 |
| | Total | 18 / 18 | 0 / 18 | 0 / 18 | 1256.00 | 1661.00 | 1793.75 | 30.04 |
| Flu A<br>High Negative (HN)<br>(0.2 TCID50<br>per reaction) | Site 1 | 0/6 | 2/6 | 4 / 6 | 76.75 | 133.00 | 157.00 | N/A** |
| | Site 2 | 0/6 | 6 / 6 | 0/6 | 180.00 | 192.50 | 200.88 | N/A |
| | Site 3 | 0/6 | 0/6 | 6 / 6 | 12.63 | 40.00 | 66.25 | N/A |
| | Total | 0 / 18 | 8 / 18 | 10 / 18 | 56.75 | 133.00 | 176.25 | N/A |
| H3<br>High Negative (HN)<br>(2 TCID50<br>per reaction) | Site 1 | 0 / 6 | 0 / 6 | 6 / 6 | 64.25 | 68.00 | 73.25 | N/A |
| | Site 2 | 0 / 6 | 0 / 6 | 6 / 6 | 100.00 | 119.50 | 127.75 | N/A |
| | Site 3 | 0 / 6 | 0 / 6 | 6 / 6 | 15.13 | 32.50 | 49.50 | N/A |
| | Total | 0 / 18 | 0 / 18 | 18 / 18 | 46.88 | 68.00 | 95.50 | N/A |
| Summary of Flu A and H3 calls in simulated Influenza A-H3 samples |
|-------------------------------------------------------------------|
|-------------------------------------------------------------------|
For reproducibility Tables. %CV = Standard Deviation / Mean*100
For reproducibility Tables. N/A = not applicable.
{10}------------------------------------------------
| Virus / Titer | All Days (3 extraction days x 2 extractions per day) | | | | | | | |
|-----------------------------------------------------------------------|------------------------------------------------------|------------|-------------|------------|---------------------------|---------------|---------------------------|-------|
| Flu A-H1 (Strain:<br>A/PR/8/34 (H1N1),<br>Zeptometrix lot<br>#303543) | Site | # Positive | # Equivocal | # Negative | 25th<br>Percentile<br>MFI | Median<br>MFI | 75th<br>Percentile<br>MFI | %CV |
| | Site 1 | 6 | 0 | 0 | 465.00 | 660.50 | 802.38 | 29.41 |
| Flu A<br>Low Positive | Site 2 | 5 | 1 | 0 | 391.25 | 433.50 | 503.50 | 27.06 |
| (LP) (0.02TCID50<br>per reaction) | Site 3 | 2 | 3 | 1 | 216.75 | 241.75 | 479.75 | 60.22 |
| | Total | 13 / 18 | 4 / 18 | 1 / 18 | 288.38 | 433.50 | 570.38 | 45.13 |
| H1<br>Low Positive<br>(LP) (0.06 TCID50<br>per reaction) | Site 1 | 6 | 0 | 0 | 1038.50 | 1151.50 | 1324.13 | 18.24 |
| | Site 2 | 6 | 0 | 0 | 697.13 | 938.00 | 1088.13 | 36.99 |
| | Site 3 | 6 | 0 | 0 | 666.88 | 890.50 | 933.00 | 33.31 |
| | Total | 18/ 18 | 0 / 18 | 0 / 18 | 826.63 | 990.5 | 1110.75 | 33.00 |
| Flu A<br>High Negative (HN)<br>(0.001 TCID50<br>per reaction) | Site 1 | 0 | 0 | 6 | 37.75 | 59.00 | 83.63 | N/A |
| | Site 2 | 0 | 0 | 6 | 92.38 | 98.00 | 101.75 | N/A |
| | Site 3 | 0 | 0 | 6 | 4.00 | 10.25 | 22.50 | N/A |
| | Total | 0 / 18 | 0 / 18 | 18 / 18 | 20.50 | 59.00 | 94.13 | N/A |
| H1<br>High Negative (HN)<br>(0.004 TCID50<br>per reaction) | Site 1 | 0 | 1 | 5 | 58.00 | 95.50 | 135.25 | N/A |
| | Site 2 | 0 | 3 | 3 | 102.50 | 136.00 | 175.50 | N/A |
| | Site 3 | 0 | 0 | 6 | 22.50 | 50.25 | 77.25 | N/A |
| | Total | 0 / 18 | 4 / 18 | 14 / 18 | 52.50 | 87.00 | 140.00 | N/A |
### Summary of Flu A and H1 calls in simulated Influenza A-H1 samples:
Summary of Flu B calls in simulated Influenza B samples:
| Virus / Titer | All Days (3 extraction days x 2 extractions per day) | | | | | | | |
|--------------------------------------------------------|------------------------------------------------------|------------|-------------|------------|---------------------------|---------------|---------------------------|-------|
| Flu B (Strain:<br>Influenza<br>B/Malaysia/2506/04) | Site | # Positive | # Equivocal | # Negative | 25th<br>Percentile<br>MFI | Median<br>MFI | 75th<br>Percentile<br>MFI | %CV |
| Low Positive<br>(LP) (0.001 TCID50<br>per reaction) | Site 1 | 6 / 6 | 0 / 6 | 0 / 6 | 1272.00 | 1440.00 | 1684.13 | 20.83 |
| | Site 2 | 6 / 6 | 0 / 6 | 0 / 6 | 1009.00 | 1258.00 | 1528.00 | 41.44 |
| | Site 3 | 6 / 6 | 0 / 6 | 0 / 6 | 918.75 | 1036.50 | 1201.50 | 18.78 |
| | Total | 18 / 18 | 0 / 18 | 0 / 18 | 1034.25 | 1263.00 | 1528.00 | 31.11 |
| High Negative (HN)<br>(0.00002 TCID50<br>per reaction) | Site 1 | 0 / 6 | 0 / 6 | 6 / 6 | 18.50 | 22.00 | 26.25 | N/A |
| | Site 2 | 0 / 6 | 1 / 6 | 5 / 6 | 76.63 | 93.25 | 120.38 | N/A |
| | Site 3 | 0 / 6 | 0 / 6 | 6 / 6 | 4.00 | 31.00 | 81.25 | N/A |
| | Total | 0 / 18 | 1 / 18 | 17 / 18 | 18.50 | 55.00 | 90.88 | N/A |
# Summary of hMPV calls in simulated hMPV samples:
| Virus / Titer | All Days (3 extraction days x 2 extractions per day) | | | | | | | |
|-------------------------------------------------------|------------------------------------------------------|------------|-------------|------------|---------------------------|---------------|---------------------------|-------|
| hMPV (CAN 97-83;<br>in-house) | Site | # Positive | # Equivocal | # Negative | 25th<br>Percentile<br>MFI | Median<br>MFI | 75th<br>Percentile<br>MFI | %CV |
| Low Positive<br>(LP) (0.002 TCID50<br>per reaction) | Site 1 | 6 / 6 | 0 / 6 | 0 / 6 | 662.38 | 701.25 | 757.38 | 82.67 |
| | Site 2 | 5 / 6 | 1 / 6 | 0 / 6 | 377.25 | 601.50 | 742.50 | 63.98 |
| | Site 3 | 6 / 6 | 0 / 6 | 0 / 6 | 538.88 | 646.00 | 690.50 | 24.50 |
| | Total | 17 / 18 | 1 / 18 | 0 / 18 | 523.88 | 662.00 | 757.38 | 69.75 |
| High Negative (HN)<br>(0.0001 TCID50<br>per reaction) | Site 1 | 0 / 6 | 0 / 6 | 6 / 6 | 20.25 | 30.00 | 55.13 | N/A |
| | Site 2 | 0 / 6 | 0 / 6 | 6 / 6 | 76.00 | 82.00 | 89.13 | N/A |
| | Site 3 | 0 / 6 | 0 / 6 | 6 / 6 | 31.75 | 46.00 | 54.63 | N/A |
| | Total | 0 / 18 | 0 / 18 | 18 / 18 | 30.00 | 59.00 | 80.00 | N/A |
{11}------------------------------------------------
#### Summary of RSV A calls in simulated RSV A samples:
| Virus / Titer | All Days (3 extraction days x 2 extractions per day) | | | | | | | |
|----------------------------------------------------|------------------------------------------------------|------------|-------------|------------|---------------------------|---------------|---------------------------|-------|
| RSV A (Strain: A2,<br>Zeptometrix lot<br>303544) | Site | # Positive | # Equivocal | # Negative | 25th<br>Percentile<br>MFI | Median<br>MFI | 75th<br>Percentile<br>MFI | %CV |
| Low Positive<br>(LP) (10 TCID50<br>per reaction) | Site 1 | 6 / 6 | 0 / 6 | 0 / 6 | 2171.38 | 3150.00 | 3990.63 | 40.60 |
| | Site 2 | 6 / 6 | 0 / 6 | 0 / 6 | 1004.00 | 1291.25 | 1442.00 | 32.56 |
| | Site 3 | 6 / 6 | 0 / 6 | 0 / 6 | 834.00 | 1193.00 | 1507.00 | 64.94 |
| | Total | 18 / 18 | 0 / 18 | 0 / 18 | 1067.00 | 1509.25 | 2721.13 | 65.22 |
| High Negative (HN)<br>(0.8 TCID50<br>per reaction) | Site 1 | 1 / 6 | 2 / 6 | 3 / 6 | 110.50 | 144.00 | 158.00 | N/A |
| | Site 2 | 1 / 6 | 2 / 6 | 3 / 6 | 104.00 | 145.25 | 255.50 | N/A |
| | Site 3 | 0 / 6 | 0 / 6 | 6 / 6 | 21.50 | 25.50 | 39.25 | N/A |
| | Total | 2 / 18 | 4 / 18 | 12 / 18 | 52.00 | 111.00 | 153.88 | N/A |
#### Summary of RSV B calls in simulated RSV B samples:
| Virus / Titer | All Days (3 extraction days x 2 extractions per day) | | | | | | | |
|-------------------------------------------------------------|------------------------------------------------------|------------|-------------|------------|---------------------------|---------------|---------------------------|-------|
| RSV B (Strain: B<br>WV/14617/ '85 [B-1<br>wild type], ATCC) | Site | # Positive | # Equivocal | # Negative | 25th<br>Percentile<br>MFI | Median<br>MFI | 75th<br>Percentile<br>MFI | %CV |
| Low Positive<br>(LP) (0.1 TCID50<br>per reaction) | Site 1 | 6 / 6 | 0 / 6 | 0 / 6 | 639.75 | 818.50 | 962.38 | 45.96 |
| | Site 2 | 5 / 6 | 1 / 6 | 0 / 6 | 474.00 | 602.00 | 814.75 | 45.05 |
| | Site 3 | 6 / 6 | 0 / 6 | 0 / 6 | 609.50 | 735.50 | 968.75 | 29.21 |
| | Total | 17 / 18 | 1 / 18 | 0 / 18 | 556.75 | 683.00 | 926.13 | 41.49 |
| High Negative (HN)<br>(0.0008 TCID50<br>per reaction) | Site 1 | 0 / 6 | 1 / 6 | 5 / 6 | 61.50 | 91.75 | 110.75 | N/A |
| | Site 2 | 0 / 6 | 0 / 6 | 6 / 6 | 72.63 | 87.00 | 100.63 | N/A |
| | Site 3 | 0 / 6 | 0 / 6 | 6 / 6 | 22.25 | 39.75 | 56.13 | N/A |
| | Total | 0 / 18 | 1 / 18 | 17 / 18 | 53.13 | 71.75 | 95.00 | N/A |
#### Summary of Para 1 calls in simulated Parainfluenza-1 samples:
| Virus / Titer | All Days (3 extraction days x 2 extractions per day) | | | | | | | |
|---------------------------------------------------|------------------------------------------------------|------------|-------------|------------|---------------------------|---------------|---------------------------|-------|
| Para 1 (Strain: C-<br>35, DHI Lot<br>081006B) | Site | # Positive | # Equivocal | # Negative | 25th<br>Percentile<br>MFI | Median<br>MFI | 75th<br>Percentile<br>MFI | %CV |
| Low Positive<br>(LP) (100 TCID50<br>per reaction) | Site 1 | 5 / 6 | 0 / 6 | 1 / 6 | 863.00 | 924.50 | 1099.25 | 50.83 |
| | Site 2 | 5 / 6 | 1 / 6 | 0 / 6 | 347.13 | 502.75 | 633.63 | 65.52 |
| | Site 3 | 5 / 6 | 0 / 6 | 1 / 6 | 769.50 | 798.25 | 848.38 | 73.11 |
| | Total | 15 / 18 | 1 / 18 | 2 / 18 | 482.88 | 798.25 | 940.25 | 63.02 |
| High Negative (HN)<br>(2 TCID50<br>per reaction) | Site 1 | 0 / 6 | 0 / 6 | 6 / 6 | 35.00 | 45.00 | 64.75 | n/a |
| | Site 2 | 0 / 6 | 0 / 6 | 6 / 6 | 68.00 | 83.00 | 95.75 | n/a |
| | Site 3 | 0 / 6 | 0 / 6 | 6 / 6 | 2.13 | 11.50 | 20.50 | n/a |
| | Total | 0 / 18 | 0 / 18 | 18 / 18 | 21.50 | 52.00 | 70.88 | n/a |
#### Summary of Para 2 calls in simulated Parainfluenza-2 samples:
| Virus / Titer | All Days (3 extraction days x 2 extractions per day) | | | | | | | |
|-------------------------------------------------|------------------------------------------------------|------------|-------------|------------|---------------------------|---------------|---------------------------|-------|
| Para 2 (Strain:<br>Greer, DHI Lot<br>062706) | Site | # Positive | # Equivocal | # Negative | 25th<br>Percentile<br>MFI | Median<br>MFI | 75th<br>Percentile<br>MFI | %CV |
| Low Positive<br>(LP) (6 TCID50<br>per reaction) | Site 1 | 6 / 6 | 0 / 6 | 0 / 6 | 600.25 | 726.50 | 1116.00 | 44.73 |
| | Site 2 | 3 / 6 | 1 / 6 | 2 / 6 | 179.00 | 308.50 | 387.75 | 83.16 |
| | Site 3 | 5 / 6 | 1 / 6 | 0 / 6 | 453.50 | 595.50 | 910.00 | 50.25 |
{12}------------------------------------------------
| | Total | 14 / 18 | 2 / 18 | 2 / 18 | 332.75 | 544.50 | 930.75 | 59.26 |
|----------------------------------------------------|--------|---------|--------|---------|--------|--------|--------|-------|
| High Negative (HN)<br>(0.4 TCID50<br>per reaction) | Site 1 | 0 / 6 | 1 / 6 | 5 / 6 | 54.50 | 69.00 | 112.38 | N/A |
| | Site 2 | 0 / 6 | 0 / 6 | 6 / 6 | 78.50 | 86.50 | 96.38 | N/A |
| | Site 3 | 0 / 6 | 0 / 6 | 6 / 6 | 18.25 | 52.50 | 67.63 | N/A |
| | Total | 0 / 18 | 1 / 18 | 17 / 18 | 51.50 | 73.25 | 96.38 | N/A |
#### Summary of Para 3 calls in simulated Parainfluenza-3 samples:
| Virus / Titer | All Days (3 extraction days x 2 extractions per day) | | | | | | | | |
|-----------------------------------------------------|------------------------------------------------------|------------|-------------|------------|---------------------------|---------------|---------------------------|-------|--|
| Para 3 (Strain: C-<br>243, DHI Lot<br>052506) | Site | # Positive | # Equivocal | # Negative | 25th<br>Percentile<br>MFI | Median<br>MFI | 75th<br>Percentile<br>MFI | %CV | |
| Low Positive<br>(LP) (0.2 TCID50<br>per reaction) | Site 1 | 4 / 6 | 2 / 6 | 0 / 6 | 293.88 | 405.50 | 543.00 | 43.89 | |
| | Site 2 | 3 / 6 | 3 / 6 | 0 / 6 | 239.50 | 291.00 | 348.50 | 54.53 | |
| | Site 3 | 3 / 6 | 2 / 6 | 1 / 6 | 200.00 | 285.00 | 461.88 | 50.45 | |
| | Total | 10 / 18 | 7 / 18 | 1 / 18 | 236.00 | 327.00 | 482.63 | 47.44 | |
| High Negative (HN)<br>(0.02 TCID50<br>per reaction) | Site 1 | 0 / 6 | 0 / 6 | 6 / 6 | 21.25 | 22.25 | 27.38 | N/A | |
| | Site 2 | 0 / 6 | 0 / 6 | 6 / 6 | 63.88 | 70.75 | 75.00 | N/A | |
| | Site 3 | 0 / 6 | 0 / 6 | 6 / 6 | 6.25 | 19.00 | 25.75 | N/A | |
| | Total | 0 / 18 | 0 / 18 | 18 / 18 | 21.25 | 27.50 | 62.38 | N/A | |
#### Summary of Rhino calls in simulated Rhinovirus samples:
| Virus / Titer | All Days (3 extraction days x 2 extractions per day) | | | | | | | | |
|--------------------------------------------------------|------------------------------------------------------|------------|-------------|------------|---------------------------|---------------|---------------------------|-------|--|
| Rhinovirus (Type<br>54: ATCC) | Site | # Positive | # Equivocal | # Negative | 25th<br>Percentile<br>MFI | Median<br>MFI | 75th<br>Percentile<br>MFI | %CV | |
| | Site 1 | 6 / 6 | 0 / 6 | 0 / 6 | 775.25 | 906.50 | 972.50 | 27.97 | |
| Low Positive<br>(LP) (0.0006 TCID50<br>per reaction) | Site 2 | 6 / 6 | 0 / 6 | 0 / 6 | 512.00 | 670.00 | 769.50 | 33.92 | |
| | Site 3 | 6 / 6 | 0 / 6 | 0 / 6 | 827.38 | 1215.25 | 1283.25 | 30.47 | |
| | Total | 18 / 18 | 0 / 18 | 0 / 18 | 666.00 | 827.00 | 1049.38 | 35.55 | |
| | Site 1 | 0 / 6 | 0 / 6 | 6 / 6 | 36.25 | 50.00 | 54.75 | N/A | |
| High Negative (HN)<br>(0.00004 TCID50<br>per reaction) | Site 2 | 0 / 6 | 0 / 6 | 6 / 6 | 78.75 | 94.00 | 96.88 | N/A | |
| | Site 3 | 0 / 6 | 0 / 6 | 6 / 6 | 24.88 | 67.50 | 121.75 | N/A | |
| | Total | 0 / 18 | 0 / 18 | 18 / 18 | 36.25 | 60.75 | 96.88 | N/A | |
#### Summary of Adeno Calls in simulated Adenovirus samples:
| Virus / Titer | All Days (3 extraction days x 2 extractions per day) | | | | | | | |
|------------------------------------------------------------|------------------------------------------------------|------------|-------------|------------|---------------------------|---------------|---------------------------|-------|
| Adenovirus<br>(cultured patient<br>isolate - Species<br>C) | Site | # Positive | # Equivocal | # Negative | 25th<br>Percentile<br>MFI | Median<br>MFI | 75th<br>Percentile<br>MFI | %CV |
| Low Positive<br>(LP) (0.8 TCID50<br>per reaction) | Site 1 | 6 / 6 | 0 / 6 | 0 / 6 | 865.63 | 925.25 | 992.75 | 9.52 |
| (LP) (0.8 TCID50<br>per reaction) | Site 2 | 5 / 6 | 0 / 6 | 1 / 6 | 708.75 | 834.00 | 905.63 | 44.23 |
| (LP) (0.8 TCID50<br>per reaction) | Site 3 | 6 / 6 | 0 / 6 | 0 / 6 | 758.88 | 971.00 | 1204.50 | 41.80 |
| | Total | 17 / 18 | 0 / 18 | 1 / 18 | 813.63 | 888.25 | 1007.50 | 36.16 |
| High Negative<br>(HN) (0.05 TCID50<br>per reaction) | Site 1 | 0 / 6 | 2 / 6 | 4 / 6 | 121.00 | 126.75 | 178.63 | N/A |
| | Site 2 | 2 / 6 | 3 / 6 | 1 / 6 | 162.75 | 225.00 | 303.75 | N/A |
| | Site 3 | 1 / 6 | 3 / 6 | 2 / 6 | 146.75 | 222.00 | 263.50 | N/A |
| | Total | 3 / 18 | 8 / 18 | 7 / 18 | 124.50 | 189.00 | 259.00 | N/A |
{13}------------------------------------------------
For all analytes assessed in the reproducibility study described above, a total of 55 (out of 468) replicates were miscalled. Of these 55 missed calls, 23 were from low positive samples which generated either an equivocal (n=16/23) or negative (n=7/23) call for the analyte in question. The remaining 32 missed calls were from high negative samples for which 27/32 generated equivocal calls and 5/32 generated positive calls.
b) Reproducibility of the assay using virus concentrations expected to be found in clinical specimens (clinically significant concentration). A separate reproducibility study was carried out on simulated samples prepared at titers representative of what is typically encountered in clinical samples. An aliquot of each sample was extracted once and 6 replicates were prepared from each extract for evaluation by RVP. Median MFI values across all extractions methods for each viral analyte evaluated in this study (excluding adenovirus) ranged from 1140 to 7381. The strain of adenovirus evaluated in this study (Type 5, Adenoid 75, ATCC VR-5) is a member of species C with a median MFI value (387) which was significantly lower than that observed for other analytes. Results of this study are summarized in Tables below.
| Virus /<br>TCID50<br>per<br>reaction | Site | # Positive | # Equivocal | # Negative | 6 replicates prepared from each extract (1 extract per method)<br>25th<br>Percentile<br>MFI | Median<br>MFI | 75th<br>Percentile<br>MFI | CV |
|----------------------------------------------|----------------------------------------------------------------|------------|-------------|------------|---------------------------------------------------------------------------------------------|---------------|---------------------------|-------|
| Flu A /<br>10 | Site 1 | 6 | 0 | 0 | 6707.5 | 7005 | 7288.25 | 13.1 |
| | Site 2 | 6 | 0 | 0 | 4178.5 | 4438.5 | 4876.625 | 22.0 |
| | Site 3 | 6 | 0 | 0 | 1789.25 | 2640.75 | 3215.875 | 33.1 |
| | Total | 18 / 18 | 0 / 18 | 0 / 18 | 3071.625 | 4438.5 | 6623 | 46.4 |
| H1 /10 | Site 1 | 6 | 0 | 0 | 4444.75 | 4824 | 4961.375 | 13.9 |
| | Site 2 | 6 | 0 | 0 | 2080 | 3152.75 | 3343.875 | 33.6 |
| | Site 3 | 6 | 0 | 0 | 1362.625 | 2168 | 2549.625 | 35.6 |
| | Total | 18 / 18 | 0 / 18 | 0 / 18 | 2106.75 | 3152.75 | 4351 | 46.2 |
| Flu A /<br>100 | Site 1 | 6 | 0 | 0 | 5415.375 | 5704.25 | 5885.5 | 5.6 |
| | Site 2 | 6 | 0 | 0 | 7350.125 | 7768.75 | 8105.25 | 6.1 |
| | Site 3 | 6 | 0 | 0 | 6374.75 | 7430 | 7634.375 | 18.6 |
| | Total | 18 / 18 | 0 / 18 | 0 / 18 | 5836.5 | 7305.5 | 7634.375 | 17.4 |
| H3 / 100 | Site 1 | 6 | 0 | 0 | 907.75 | 990 | 1050.125 | 8.7 |
| | Site 2 | 6 | 0 | 0 | 2570.75 | 2809 | 3039 | 13.9 |
| | Site 3 | 6 | 0 | 0 | 654 | 946 | 1238 | 43.9 |
| | Total | 18 / 18 | 0 / 18 | 0 / 18 | 907.75 | 1140.5 | 2467 | 61.9 |
| Flu B /<br>0.5 | Site 1 | 6 | 0 | 0 | 4234.5 | 4283 | 4354.375 | 2.9 |
| | Site 2 | 6 | 0 | 0 | 762.875 | 1267 | 1829.25 | 52.8 |
| | Site 3 | 6 | 0 | 0 | 5897 | 6460 | 7067.25 | 16.5 |
| | Total | 18 / 18 | 0 / 18 | 0 / 18 | 1996.375 | 4283 | 5542.125 | 54.7 |
| Virus /<br>TCID50<br>per<br>reaction | Site | # Positive | # Equivocal | # Negative | 25th<br>Percentile<br>MFI | Median<br>MFI | 75th<br>Percentile<br>MFI | CV |
| | Site 1 | 6 | 0 | 0 | 5726.875 | 5946.25 | 6102.25 | 6.3 |
| RSV A<br>/100 | Site 2 | 6 | 0 | 0 | 3360.375 | 3599 | 3690.25 | 10.1 |
| | Site 3 | 6 | 0 | 0 | 2821.25 | 4090.5 | 4653.25 | 33.8 |
| | Total | 18 / 18 | 0 / 18 | 0 / 18 | 3544.5 | 4339.5 | 5681.75 | 30.5 |
| | Site 1 | 6 | 0 | 0 | 4549.375 | 4706 | 4815.375 | 5.3 |
| RSV B<br>/100 | Site 2 | 6 | 0 | 0 | 2907 | 2964.75 | 2980.125 | 8.9 |
| | Site 3 | 6 | 0 | 0 | 3661.5 | 4563.25 | 4638.5 | 17.1 |
| | Total | 18 / 18 | 0 / 18 | 0 / 18 | 3010.5 | 4467.75 | 4655.875 | 23.1 |
| | Site 1 | 6 | 0 | 0 | 3190 | 3209.5 | 3244 | 3.6 |
| Para 1<br>/100 | Site 2 | 6 | 0 | 0 | 970.5 | 1548.5 | 1835.5 | 48.7 |
| | Site 3 | 6 | 0 | 0 | 1751.75 | 1826.5 | 1875 | 6.2 |
| | Total | 18/ 18 | 0 / 18 | 0 / 18 | 1635 | 1881.5 | 3185.375 | 42.7 |
| | Site 1 | 6 | 0 | 0 | 5359.25 | 5425.5 | 5638.75 | 6.3 |
| Para 2<br>/100 | Site 2 | 6 | 0 | 0 | 1539.125 | 1859.25 | 2237.5 | 42.7 |
| | Site 3 | 6 | 0 | 0 | 2179.875 | 2257.5 | 2307.75 | 10.1 |
| | Total | 18 / 18 | 0 / 18 | 0 / 18 | 2048.625 | 2311 | 5314 | 53.0 |
| | Site 1 | 6 | 0 | 0 | 2951.375 | 3075.25 | 3135.75 | 4.3 |
| Para 3<br>/25 | Site 2 | 3 | 0 | 3 | 116.75 | 234.5 | 600.5 | 87.2 |
| | Site 3 | 6 | 0 | 0 | 5977.75 | 6785.5 | 8248.75 | 35.5 |
| | Total | 15/ 18 | 0 / 18 | 3 / 18 | 723.125 | 2988.75 | 5106.875 | 87.5 |
| | Site 1 | 2 | 4 | 0 | 247.375 | 284.75 | 300 | 13.9 |
| Adeno<br>/5000 | Site 2 | 6 | 0 | 0 | 362.625 | 387 | 484.5 | 20.0 |
| | Site 3 | 6 | 0 | 0 | 546.625 | 684 | 719 | 20.7 |
| | Total | 14 / 18 | 4 / 18 | 0 / 18 | 303.875 | 387 | 541.375 | 41.0 |
| | Site 1 | 6 | 0 | 0 | 4764.875 | 4958 | 5072.375 | 13.4 |
| hMPV<br>/0.5 | Site 2 | 6 | 0 | 0 | 7405.125 | 7670.5 | 7717.25 | 5.6 |
| | Site 3 | 6 | 0 | 0 | 7583.75 | 8175.25 | 8743.875 | 20.6 |
| | Total | 18/18 | 0 / 18 | 0 / 18 | 5033.125 | 7381.25 | 7788 | 24.0 |
| | Site 1 | 6 | 0 | 0 | 2787.125 | 2836 | 2890.5 | 2.4 |
| Rhino<br>/100 | Site 2 | 6 | 0 | 0 | 3133.25 | 3219 | 3413.5 | 6.5 |
| | Site 3 | 6 | 0 | 0 | 3366.25 | 3631 | 4159.75 | 26.1 |
| | Total | 18/18 | 0 / 18 | 0 / 18 | 3162.25…
