JBAIDS INFLUENZA A SUBTYPING KIT

K111778 · U.S Army Medical Materiel Development Activity · OQW · Sep 13, 2011 · Microbiology

Device Facts

• Record ID: K111778
• Device Name: JBAIDS INFLUENZA A SUBTYPING KIT
• Applicant: U.S Army Medical Materiel Development Activity
• Product Code: OQW · Microbiology
• Decision Date: Sep 13, 2011
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3332
• Device Class: Class 2

Intended Use

The Joint Biological Agent Identification and Diagnostic System (JBAIDS) Influenza A Subtyping Kit is intended for the in vitro qualitative detection and differentiation of seasonal Influenza A/H1, seasonal Influenza A/H3, and 2009 H1N1Influenza viral nucleic acids isolated and purified from nasopharyngeal swab (NPS) and nasopharyngeal wash (NPW) specimens from human patients with signs and symptoms of respiratory infection, in conjunction with clinical and epidemiological risk factors. The JBAIDS Influenza A Subtyping Kit contains reverse transcriptase real-time polymerase chain reaction (rRT-PCR) assays for use on the JBAIDS instruments. The Flu A H1, Flu A H3, and Flu A H1 2009 assays of the JBAIDS Influenza A Subtyping Kit target a region of the hemagglutinin (HA) gene of the respective Influenza A virus. The Flu A Sw assay of the JBAIDS Influenza A Subtyping Kit targets a region of the nucleocapsid protein (NP) gene of the 2009 H1N1 Influenza virus, as well as some other Influenza A viruses of swine lineage. This kit is not intended to detect Influenza B or Influenza C viruses. A negative result for all assays in the JBAIDS Influenza A Subtyping Kit is a presumptive negative result for Influenza A. These results should be confirmed using the JBAIDS Influenza A & B Detection Kit. Test results are to be used in conjunction with other clinical and epidemiological information. Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions. Due to low seasonal prevalence, performance characteristics for detection of seasonal Influenza A/H1 were established primarily with retrospective and contrived clinical specimens. All users, analysts, and any person reporting diagnostic results from use of this device should be trained to perform and interpret the results from this procedure by JBAIDS instructors or designees prior to use. Use of this device is limited to designated Department of Defense (DoD) laboratories equipped with the JBAIDS instruments. If infection with a novel influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to state or local health departments for testing. Viral culture should not be attempted in these cases unless a biosafety laboratory (BSL) 3+ facility is available to receive and culture specimens.

Device Story

JBAIDS Influenza A Subtyping Kit uses rRT-PCR to detect and differentiate Influenza A subtypes (H1, H3, 2009 H1N1) from purified NPS/NPW specimens. Input: viral RNA isolated via IT 1-2-3 Platinum Path or Roche MagNA Pure Compact kits. Operation: freeze-dried assays with hydrolysis probes (6-FAM/TAMRA) target HA or NP genes; JBAIDS instrument performs thermal cycling and fluorescence detection. Output: qualitative results (positive/negative/uncertain) generated by JBAIDS software. Used in designated DoD laboratories by trained personnel. Results support clinical/epidemiological assessment; negative results are presumptive and require confirmation via JBAIDS Influenza A & B Detection Kit. Benefits: rapid subtyping of influenza A strains to inform patient management and public health surveillance.

Clinical Evidence

Prospective study (2010-2011) at 5 DoD sites analyzed 795 specimens (312 NPS, 483 NPW). Comparator: CDC rRT-PCR Flu Panel with sequencing. PPA/NPA for 2009 H1N1: 100%/99.3%; Seasonal H3: 100%/100%. Seasonal H1 performance supplemented by 51 NPS archived samples (100% PPA/NPA) and 136 contrived specimens (100% PPA/NPA). Reproducibility study across 3 sites showed ≥98% detection rate for samples ≥LoD.

Technological Characteristics

Real-time RT-PCR assay using hydrolysis probes (6-FAM/TAMRA). Reagents provided as freeze-dried, single-use vials. Requires JBAIDS instrument and software. Nucleic acid extraction via IT 1-2-3 Platinum Path or Roche MagNA Pure Compact. Storage at room temperature. Targets HA gene (H1, H3, 2009 H1) and NP gene (2009 H1/swine lineage).

Indications for Use

Indicated for in vitro qualitative detection and differentiation of seasonal Influenza A/H1, A/H3, and 2009 H1N1 viral nucleic acids in nasopharyngeal swab (NPS) and nasopharyngeal wash (NPW) specimens from human patients with respiratory infection symptoms. Limited to designated DoD laboratories.

Regulatory Classification

Identification

Reagents for detection of specific novel influenza A viruses are devices that are intended for use in a nucleic acid amplification test to directly detect specific virus RNA in human respiratory specimens or viral cultures. Detection of specific virus RNA aids in the diagnosis of influenza caused by specific novel influenza A viruses in patients with clinical risk of infection with these viruses, and also aids in the presumptive laboratory identification of specific novel influenza A viruses to provide epidemiological information on influenza. These reagents include primers, probes, and specific influenza A virus controls.

Special Controls

The device is classified as Class II under regulation 21 CFR 866.3332 with special controls. The special control guidance document "Reagents for Detection of Specific Novel Influenza A viruses" will be available shortly.

*Classification.* Class II (special controls). The special controls are:(1) FDA's guidance document entitled “Class II Special Controls Guidance Document: Reagents for Detection of Specific Novel Influenza A Viruses.” See § 866.1(e) for information on obtaining this document. (2) The distribution of these devices is limited to laboratories with experienced personnel who have training in standardized molecular testing procedures and expertise in viral diagnosis, and appropriate biosafety equipment and containment.

Predicate Devices

• CDC Human Influenza Virus real-time RT-PCR Detection and Characterization Panel (K080570)
• CDC Influenza 2009 A(H1N1)pdm Real-Time RT-PCR Panel (K101564)

Reference Devices

• JBAIDS instrument (K051713)

Submission Summary (Full Text)

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K 111778

# SEP 1 3 2011 510(k) Summary JBAIDS Influenza A Subtyping Kit

- According to the requirements of 21 CFR 807.92, the following information Introduction: provides sufficient detail to understand the basis for a determination of substantial equivalence.
- Submitted by: U.S. Army Medical Materiel Development Activity Division of Regulated Activities and Compliance 1430 Veterans Drive Fort Detrick, MD
- Robert Miller Ph.D., RAC Primary Contact: Director, Division of Regulated Activities and Compliance Telephone: [phone] Facsimile: 301-619-0197 e-mail: usamrmcregulatoryaffairs(@amedd.army.mil
- Secondary Contact: Patricia Beverly, RAC Division of Regulated Activities and Compliance Telephone: [phone] Facsimile: 301-619-0197

e-mail: [e-mail] or [e-mail]

- Beth Lingenfelter Technical Contact: Director of Regulatory Affairs, Idaho Technology, Inc. Telephone: [phone], ext 407 Facsimile: 801-588-0507 e-mail: [e-mail]
Date Prepared: August, 2011

Device Name: Trade Name:

JBAIDS Influenza A Subtyping Kit

Common Name:

Real-time PCR assay for differentiation of influenza A subtypes

Classification Name:

Reagents for Detection of Specific Novel Influenza A Viruses (CFR 866.3332)

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## Intended Use

The Joint Biological Agent Identification and Diagnostic System (JBAIDS) Influenza A Subtyping Kit is intended for the in vitro qualitative detection and differentiation of seasonal Influenza A/H1, seasonal Influenza A/H3, and 2009 H1N1Influenza viral nucleic acids isolated and purified from nasopharyngeal swab (NPS) and nasopharyngeal wash (NPW) specimens from human patients with signs and symptoms of respiratory infection, in conjunction with clinical and epidemiological risk factors. The JBAIDS Influenza A Subtyping Kit contains reverse transcriptase real-time polymerase chain reaction (rRT-PCR) assays for use on the JBAIDS instruments. The Flu A H1, Flu A H3, and Flu A H1 2009 assays of the JBAIDS Influenza A Subtyping Kit target a region of the hemagglutinin (HA) gene of the respective Influenza A virus. The Flu A Sw assay of the JBAIDS Influenza A Subtyping Kit targets a region of the nucleocapsid protein (NP) gene of the 2009 H1N1 Influenza virus, as well as some other Influenza A viruses of swine lineage. This kit is not intended to detect Influenza B or Influenza C viruses.

A negative result for all assays in the JBAIDS Influenza A Subtyping Kit is a presumptive negative result for Influenza A. These results should be confirmed using the JBAIDS Influenza A & B Detection Kit.

Test results are to be used in conjunction with other clinical and epidemiological information. Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions.

Due to low seasonal prevalence, performance characteristics for detection of seasonal Influenza A/H1 were established primarily with retrospective and contrived clinical specimens.

All users, analysts, and any person reporting diagnostic results from use of this device should be trained to perform and interpret the results from this procedure by JBAIDS instructors or designees prior to use. Use of this device is limited to designated Department of Defense (DoD) laboratories equipped with the JBAIDS instruments.

If infection with a novel influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to state or local health departments for testing. Viral culture should not be attempted in these cases unless a biosafety laboratory (BSL) 3+ facility is available to receive and culture specimens.

## Device Description

The JBAIDS Influenza A Subtyping Kit is a real time RT-PCR test kit, which, when used with the JBAIDS instrument and software, allows the qualitative in vitro detection and identification of influenza A subtypes H1, H3, and H1 2009 (swine lineage) viral RNA. The assays have been optimized as freeze-dried assays with primer and fluorescent-probe sets for the detection of

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influenza A/H1, A/H3, and A/2009 H1 viral RNA. In particular, the Flu A H1, Flu A H3, and Flu A H1 2009 assays target distinct regions of the hemagglutinin gene specific to those subtypes, and the Flu A Sw assay targets a region of the nucleocapsid protein gene as a secondary target for the influenza A/2009 H1(swine lineage) virus. The tests are performed using the JBAIDS instrument and software.

## Assay Principle

Before testing, NPS or NPW specimens are purified using Technology's 1-2-3TM Platinum Path Sample Purification Kit or the Roche MagNA Pure Compact Nucleic Acid Isolation Kit I. The resulting purified sample is added to Unknown reagent vials along with reconstitution buffer. When viral RNA is present, a fragment of influenza A viral RNA is transcribed and amplified. The amplicon is detected by fluorescence using a specific hydrolysis probe. Each probe is labeled on one end with a fluorescent reporter moiety (6-carboxyfluorescein (6-FAM)) and elsewhere with a quencher moiety (carboxy tetramethylrhodamine (TAMRA)). When the probe is intact, the quencher absorbs the light emitted by the reporter moiety. During PCR, the probe hybridizes to the target sequence before the exonuclease activity of Taq polymerase hydrolyzes the probe, separating the fluorophore from the quencher and permitting detection of the fluorescent signal generated by the reporter. The fluorescent signal increases as additional templates are amplified and more probes are hydrolyzed.

JBAIDS Software analyzes the fluorescence amplification curves and reports results as positive, negative, or uncertain. A failure of the Positive or Negative Control will result in the entire run being called invalid. Retesting is required to resolve uncertain or invalid results.

## Substantial Equivalence

The JBAIDS Influenza A Subtyping System is substantially equivalent to other products in commercial distribution intended for similar use. The JBAIDS instrument has been previously cleared under K051713.

The JBAIDS Influenza A Subtyping System is substantially equivalent to the CDC Human Influenza Virus real-time RT-PCR Detection and Characterization Panel, which was cleared on September 30, 2008 under 510(k) # K080570, and the CDC Influenza 2009 A(H1N1)pdm Real-Time RT-PCR Panel, which was cleared on June 22, 2010 under 510(k) #101564.

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| Element | JBAIDS Influenza A Subtyping Kit | CDC rRT-PCR Flu Panel (K080570) | CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel (K101564) |
|---------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Intended Use | Qualitative <i>in vitro</i> detection and differentiation of seasonal Influenza A/H1, seasonal Influenza A/H3 and Influenza A/2009 H1N1 viral nucleic acids from nasopharyngeal swab (NPS) and nasopharyngeal wash (NPW) specimens on the JBAIDS instrument after obtaining an influenza A positive test from the JBAIDS Influenza A & B Detection Kit. | Qualitative <i>in vitro</i> detection of influenza virus type A or B and for determination of the subtype of seasonal human influenza A virus, as seasonal A/HI or A/H3, if present, from viral RNA in nasopharyngeal and/or nasal swab specimens, for presumptive identification of virus in patients who may be infected with influenza A subtype A/H5 (Asian lineage) from viral RNA in human respiratory specimens and viral culture on an ABI 7500 Fast Dx Real-time PCR instrument | Qualitative <i>in vitro</i> detection of influenza virus type A and 2009(H1N1 influenza viral RNA from nasopharyngeal swabs (NPS), nasal swabs (NS), throat swabs (TS), nasal aspirates (NA), nasal washes (NW), dual nasopharyngeal / throat swabs (NPS/TS) and lower respiratory tract specimens (LRTS) from human patients with signs and symptoms of respiratory infection and/or from viral culture, on the Applied Biosystems (ABI) 7500 Fast Dx Real-Time PCR instrument. |
| Technology | Real-time PCR using hydrolysis probes | Real-time PCR using hydrolysis probes | Real-time PCR using hydrolysis probes |
| Assay Results | Qualitative | Qualitative | Qualitative |
| Nucleic Acid Extraction | Yes | Yes | Yes |
| Table 2. Differences Between the JBAIDS Influenza A Subtyping Kit and its Predicate Devices | | | |
| Element | JBAIDS Influenza A<br>Subtyping Kit | CDC rRT-PCR Flu Panel<br>(K080570) | CDC rRT-PCR 2009<br>A(H1N1)pdm Flu Panel<br>(K101564) |
| Viruses Detected | Influenza A/H1, Influenza A/H3<br>and Influenza A/2009 H1N1 | Influenza A, Influenza B,<br>Influenza A/H1, Influenza A/H3<br>and Influenza A/H5 | Influenza A and Influenza A<br>2009 H1N1 |
| Specimen types | Nasopharyngeal swabs,<br>nasopharyngeal washes | Nasopharyngeal swabs, nasal<br>swabs, and virus culture | Nasopharyngeal swabs, nasal<br>swabs, nasal aspirates, nasal<br>washes, dual nasopharyngeal /<br>throat swabs, broncheoalveolar<br>lavage, tracheal aspirate,<br>bronchial wash and viral culture |
| Required<br>Instrumentation | JBAIDS instrument | Applied Biosystems 7500 Fast<br>Dx Real-time PCR instrument<br>with SDS software v 1.4 | Applied Biosystems 7500 Fast<br>Dx Real-time PCR instrument |
| Interpretation of<br>Test Results | Automated analysis of test<br>results and controls | User required to interpret test<br>and control results | User required to interpret test<br>and control results |
| Enzyme Master<br>Mix | Assays come in freeze-dried<br>single use vials that include all<br>components of master mix | Invitrogen SuperScript™ III<br>Platinum® One-Step<br>Quantitative RT-PCR Kits | Invitrogen SuperScript™ III<br>Platinum® One-Step<br>Quantitative RT-PCR Kits |
| Reagent Storage | Reagents are stored at room<br>temperature | Reagents are stored at ≤ -20°C | Reagents are stored at ≤ -20°C |
| Extraction<br>Methods | • IT <i>1</i> - <i>2</i> -3™ Platinum Path<br>Sample Purification Kit | • Qiagen QIAamp® Viral RNA<br>Mini Kit | • Qiagen QIAamp® Viral RNA<br>Mini Kit |
| | • Roche MagNA Pure Compact<br>Nucleic Acid Isolation Kit I | • Qiagen RNeasy® Mini Kit<br>• Roche MagNA Pure TNA Kit | • Roche MagNA Pure Compact<br>Nucleic Acid Isolation Kit I<br>Roche MagNA Pure TNA Kit |
| | | • Roche MagNA Pure LC RNA<br>Isolation Kit II | • Roche MagNA Pure LC RNA<br>isolation Kit II |
| | | | • Qiagen QIAcube with<br>QIAamp viral RNA mini kit<br>• bioMerieux NucliSENS<br>easyMAG |

:

.

## Table 1. Similarities Between the JBADS Influenza A Subtyping Kit and its Predicate Devices

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.

,

·

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# 2. Differences Between the IRAIDS Influenza A Subtyning Kit and its Predicate Devices

## Summary of Performance Data

## Clinical Performance

The clinical performance of the JBAIDS Influenza A Subtyping Kit was evaluated during a prospective study at 5 geographically separated military clinical sites over the 2010-2011 influenza season (December 2010 to April 2011). Subjects with signs and/or symptoms of influenza-like illness were enrolled. Upon obtaining informed consent,

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NPS and NPW specimens were collected for JBAIDS and comparator testing. A total of 795 valid specimens were analyzed at the five study sites: 312 NPS and 483 NPW specimens. Table 3 provides a summary of demographic information for the 795 subjects for which valid specimen results were obtained in the prospective study.

| | | Overall | Site 1 | Site 2 | Site 3 | Site 4 | Site 5 |
|---------------|--------|-------------|-------------|-------------|------------|------------|-------------|
| NPS | | 312 | રે રેપ | 206 | રેર | 0 | 0 |
| NPW | | 483 | 320 | 0 | 0 | 118 | વર્ષ રે |
| | Total | 795 | 370 | 206<br>ર્સ | | 118 | 45 |
| | Female | 405 (50.9%) | 188 (50.8%) | 122 (59.2%) | 23 (41.1%) | 56 (47.5%) | 16 (35.6%) |
| Sex | Male | 390 (49.1%) | 182 (49.2%) | 84 (40.8%) | 33 (58.9%) | 62 (52.5%) | 29 (64.4%) |
| | Mean | 26.4 | 23.3 | 24.5 | 30.3 | 23.1 | 30.8 |
| | Median | 24.0 | 24.0 | 18.0 | 27.5 | 17.0 | 26.0 |
| Ageª | Min | 0.5 | 0.5 | 0.5 | 2.0 | 0.5 | 18.0 |
| | Max | 92.0 | 92.0 | 69.0 | 81.0 | 68.0 | 62.0 |
| | ્રર | 149 (18.7%) | 88 (23.8%) | 40 (19.4%) | 4 (7.1%) | 17 (14.4%) | 0 (0%) |
| Age<br>Range® | 6-21° | 229 (28.8%) | 79 (21.4%) | 74 (35.9%) | 10 (17.9%) | 54 (45.8%) | 12° (26.7%) |
| | 22-49 | 331 (41.6%) | 178 (48.1%) | 54 (26.2%) | 35 (62.5%) | 34 (28.8%) | 30 (66.7%) |
| | >50 | 86 (10.8%) | 25 (6.8%) | 38 (18.4%) | 7 (12.5%) | 13 (11%) | 3 (6.7%) |

Table 3. Demographic Summary for the JBAIDS Influenza A Subtyping Kit Prospective Study.

4 0.5 was used for all ages under 1 year for these calculations.

b The age groups ≤ 5 years and ≥ 50 years correspond to high risk groups for which the CDC strongly recommends seasonal influenza vaccination (http://www.cdc.goy/flu//flu/protect/keyfacts.htm). 6 Site 5 enrolled adults only; this category reflects participants 18 to 21 years of age

Of the 795 prospective specimens, successful results were obtained for 94% (751/795) of these specimens on the first attempt (Site 1: 359/370 =97%; Site 2: 191/206 =93%; Site 3: 54/56 =96%; Site 4: 110/118 =93%; Site 5: 37/45 =98%). The remaining 6% (44/795) required retesting: "Invalid" (32/44), "Inconclusive" (1/44), and "Unsubtypeable" (1/44) (11 samples from Site 1; 15 samples from Site 2; 2 samples from Site 3; 8 sample from Site 4: and 8 sample from Site 5). Forty (40) out of 44 samples resolved upon a 1st retest and the remaining 4 samples required a re-extraction and retest and resolved.

Nucleic acid from each specimen was isolated using either the IT I-2-3 Platinum Path Sample Purification Kit (manual sample processing) or the Roche MagNA Pure Compact Nucleic Acid Isolation Kit I (automated sample processing) and tested with the JBAIDS Influenza A Subtyping Kit. The performance of the JBAIDS Influenza A Subtyping Kit was evaluated by comparing the JBAIDS test results with a comparator/reference method. The reference method was the CDC rRT-PCT Flu Panel influenza A and influenza B assays, followed by subtype-specific PCR and bi-directional sequencing of amplicons. Clinical sensitivity was calculated as Positive Percent Agreement (PPA) and specificity was calculated as Negative Percent Agreement (NPA). The exact binomial

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two-sided 95% confidence interval was calculated. The results are summarized in Table 4.

| Influenza A<br>Strain | Sample<br>Matrix | Purification<br>Kit | PPA | | | NPA | | |
|-----------------------|------------------|---------------------|---------------|---------|-----------|------------|---------|------------|
| | | | TP/(TP+FN) | Percent | 95% CI | TN/(TN+FP) | Percent | 95% CI |
| | Seasonal H1 | NPW | Platinum Path | 0/0 | - | - | 277/278 | 99.6% |
| | NPW | MagNA Pure | 0/0 | - | - | 205/205 | 100.0% | 98.2-100% |
| | | Combined | 0/0 | - | - | 482/483 | 99.8% | 98.9-100% |
| | NPS | Platinum Path | 0/0 | - | - | 132/132 | 100.0% | 97.2-100% |
| | NPS | MagNA Pure | 0/0 | - | - | 180/180 | 100.0% | 98.0-100% |
| | | Combined | 0/0 | - | - | 312/312 | 100.0% | 98.8-100% |
| | NPW | Platinum Path | 50/50 | 100.0% | 92.9-100% | 227/228 | 99.6% | 97.6-100% |
| 2009 H1N1 | | MagNA Pure | 16/16 | 100.0% | 79.4-100% | 187/189 | 98.9% | 96.2-99.9% |
| | | Combined | 66/66 | 100.0% | 94.6-100% | 414/417 | 99.3% | 97.9-99.9% |
| | NPS | Platinum Path | 24/24 | 100.0% | 85.8-100% | 108/108 | 100.0% | 96.6-100% |
| | | MagNA Pure | 10/10 | 100.0% | 69.2-100% | 169/170 | 99.4% | 96.8-100% |
| | | Combined | 34/34 | 100.0% | 89.7-100% | 277/278 | 99.6% | 98.0-100% |
| | NPW | Platinum Path | 14/14 | 100.0% | 76.8-100% | 264/264 | 100.0% | 98.6-100% |
| Seasonal H3 | | MagNA Pure | 19/19 | 100.0% | 82.4-100% | 186/186 | 100.0% | 98.0-100% |
| | | Combined | 33/33 | 100.0% | 89.4-100% | 450/450 | 100.0% | 99.2-100% |
| | NPS | Platinum Path | 18/18 | 100.0% | 81.5-100% | 115/115 | 100.0% | 96.8-100% |
| | | MagNA Pure | 8/8 | 100.0% | 63.1-100% | 171/171 | 100.0% | 97.9-100% |
| | | Combined | 26/26 | 100.0% | 86.8-100% | 286/286 | 100.0% | 98.7-100% |

Table 4. JBAIDS Influenza A Subtyping Kit Prospective Clinical Performance Summary

Seasonal influenza A/H1 virus was not circulating during the 2010-2011 influenza season (http://www.cdc.gov/flu/) and was not detected during the prospective clinical study of the JBAIDS Influenza A Subtyping Kit. To supplement the results of the clinical study, an evaluation of preselected archived samples was performed. Due to the limited availability of archived specimens, the clinical study was further supplemented with surrogate clinical contrived specimens.

#### Testing of Preselected Archived Specimens

Additional testing of pre-selected archived clinical NPS specimens was performed at two different clinical study sites to supplement the prospective clinical testing data. Because it is possible that the archived samples had been misidentified or had degraded during storage or previous handling, the presence of Influenza A/H1 viral RNA was confirmed using "validation" PCR assays. The validation PCR assays were identical to the comparator assays that were used for the prospective clinical evaluation study. A total of 51 NPS specimens were obtained and confirmed for testing: 30 known to be positive seasonal Influenza A/H1 specimens and 21 influenza-negative specimens. Validated samples were purified using either the IT 1-2-3 Platinum Path Sample Purification Kit or

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the Roche MagNA Pure Compact Nucleic Acid Isolation Kit I and then tested with the Flu A and human sample control (Flu SC) assay from the JBAIDS Influenza A & B Detection Kit and the Flu A H1 assay from the JBAIDS Influenza A Subtyping Kit. The specimens were split evenly for purification with the Platinum Path or MagNA Pure purification kits and then randomized such that the users performing the JBAIDS testing were blinded as to the expected test result.

Table 5 presents the PPA and NPA for the archived clinical specimens. Data from both extraction kits are combined due to identical performance.

| Influenza<br>Assay | Sample<br>Type | PPA | Percent | 95% CI | NPA | Percent | 95% CI |
|--------------------|----------------|-------|---------|-----------|-------|---------|-----------|
| Flu A H1 | NPS | 29/29 | 100% | 88.1-100% | 21/21 | 100% | 83.4-100% |

Table 5, Performance Summary of Seasonal Influenza A/H1 Archived Clinical Specimens

Due to the absence of seasonal Influenza A/H1 virus in circulation during the 2010-2011 influenza seasonal (http://www.cdc.gov/flu/) and lack of availability of archived NPW specimens for seasonal Influenza A/H1, contrived clinical samples (residual influenza negative NPS and NPW samples spiked with a known concentration of seasonal Influenza A/H1 virus) were used as a surrogate to further evaluate the performance of the JBAIDS Influenza A Subtyping Kit.

## Testing of Surrogate Clinical Specimens

A total of 136 individual influenza-negative clinical specimens (68 NPS samples and 68 NPW samples) were spiked at a range of concentrations, including near the system limit of detection (LoD), as well as un-spiked, then randomized, and sent to two different clinical trial sites for testing. Of the 136 surrogate samples included in this study, a valid JBAIDS test result was obtained for 128 samples (62 NPW and 66 NPS). The remaining 8 samples with invalid results could not be retested due to insufficient sample volume, and were not included in the analysis.

Table 6 presents the PPA and NPA for the surrogate clinical specimens. Half of the samples were extracted using the Platinum Path purification kit and half using the MagNA pure kit. Performance from both extraction kits was identical, so results are combined.

| Assay | Sample Type | PPA | | | NPA | | |
|----------------------------|-------------|------------|---------|-----------|------------|---------|-----------|
| | | TP/(TP+FN) | Percent | 95% CI | TN/(TN+FP) | Percent | 95% CI |
| Flu A H1 | NPW | 54/54 | 100% | 93.4-100% | 8/8 | 100.0% | 63.1-100% |
| Flu A H1 | NPS | 59/59 | 100% | 93.9-100% | 7/7 | 100.0% | 59.0-100% |
| Flu A H1 2009/<br>Flu A Sw | NPW | 0/0 | - | - | 62/62 | 100.0% | 94.2-100% |
| Flu A H1 2009/<br>Flu A Sw | NPS | 0/0 | - | - | 66/66 | 100.0% | 94.6-100% |
| Flu A H3 | NPW | 0/0 | - | - | 62/62 | 100.0% | 94.2-100% |
| Flu A H3 | NPS | 0/0 | - | - | 66/66 | 100.0% | 94.6-100% |

Table 6. Performance Summary of Seasonal Influenza A/H1 Surrogate Clinical Specimens

U.S. Army Office of the Surgeon General. 510(k) June, 2011 JBAIDS Influenza A Subtyping System

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Analyses of the clinical data set, preselected archived specimens, and surrogate clinical specimens demonstrate that the JBAIDS Influenza A Subtyping Kit is a sensitive and specific test system for the differentiation of influenza A/H1, influenza A/H3, and influenza A/2009 H1 virus subtypes.

## Selected Analytic Studies

#### Limit of Detection

The analytical sensitivity or Limit of Detection (LoD) for each target assay (Flu A H1, Flu A H3, Flu A H1 2009, and Flu A Sw) was determined using both NPS and NPW samples spiked with quantified virus strains. The LoD was the lowest concentration where ≥ 95% of samples yielded positive results. Twenty (20) independent specimens from 20 unique donors were spiked with each virus strain for each sample type/purification kit combination and tested at the LoD concentration. The LoD values for representative virus strains detected by the JBAIDS Influenza A Subtyping Kit are listed in Table 7.

| Subtyping Kit | | | |
|-------------------------------|---------------------|-------------------------|-------------------|
| Assay(s) | Influenza Type | Strain | LoD<br>(EID50/mL) |
| Flu A H1 | Influenza A H1N1 | A/New Caledonia/20/1999 | 50a |
| | | A/Hawaii/15/2001 | 5,000 |
| Flu A H3 | Influenza A H3N2 | A/New York/55/2004 | 5 |
| | | A/Wisconsin/67/2005 | 10 |
| Flu A H1 2009<br>and Flu A Sw | 2009 H1N1 Influenza | A/New York/18/2009 | 1,500 |
| | | A/California/7/2009 | 5,000 |

| Table 7. LoD Concentrations for Representative Virus Strains Detected by the JBAIDS Influenza A | |
|-------------------------------------------------------------------------------------------------|--|
| Subtyping Kit | |

4 The aliquot of A/New Caledonia/20/1999 tested contains 17-45 times more PCR target copies per E/Dg than the aliquot of A/Hawaii/15/2001 tested.

#### Inclusivity

The analytical reactivity of the JBAIDS Influenza A Subtyping Kit assays was evaluated with inclusivity panels consisting of eight seasonal influenza A H1N1 strains (Table 8), 10 seasonal influenza A H3N2 strains (Table 9), and 11 2009 H1N1 influenza strains (Table 10) that represent the genetic, temporal, and geographic diversity of the influenza analytes. Each organism was tested in a simulated NPS sample matrix at or near the system LoD (5, 50, and 500 EID50/mL or TCID50/mL for H1N1 strains; 0.5, 5 and 50 EID50/mL or TCID50/mL for H3N2 strains; and 150, 1,500 and 15,000 EID50/mL or TCIDsofmL for H1N1 2009 strains). Higher concentrations were tested if the analyte was not detected at the initial test concentrations. Four (4) of the 29 influenza strains tested in this study were not detected with the appropriate JBAIDS influenza A subtyping assays.

{9}------------------------------------------------

There was considerable variability in the ability of the Flu A H1 assay to detect strains (lowest detected concentrations ranged from 5-500,000 TCID30/mL). Sequence alignments of tested strains indicate variability potentially due to mismatches under the primers and probe. The influenza A/1/Denver/1/57 strain was not detected at a final concentration of 5,000 TCID50/mL.

For the Flu A H3 assay, the following strains were not detected at the following concentration: A/Aichi/2/68 at 114,000 TCID50/mL, A/Hong Kong/8/68 at 137,000 TCID50/mL, and A/MRC-2 recomb at 7,350 TCID30/mL. Sequence alignments indicate that the A/Aichi/2/68 and A/Hong Kong/8/68 isolates have mismatches in the probe sequence. The sequence for A/MRC-2 recomb was not available.

For the Flu A H1 and Flu A H3 assays, in silico evaluation of contemporary strains (2006-2011) indicate that there are few mismatches, and strains should be detected.

| Strain | Lowest<br>Concentration<br>Detected |
|--------------------------|-------------------------------------|
| A/PR/8/34 | 500,000 TCID50/mL |
| A/NWS/33 | 50 TCID50/mL |
| A/Weiss/43 | 500 TCID50/mL |
| A1/FM/1/47 | 5 TCID50/mL |
| A/Mal/302/54 | 5,000 TCID50/mL |
| A1/Denver/1/57 | NDª |
| A/Solomon Islands/3/2006 | 5 TCID50/mL |
| A/Brisbane/59/07 | 50 TCID50/mL |

| | Table 8. Results of Influenza A H1N1 Inclusivity | |
|--|--------------------------------------------------|--|
| | | |

8 ND stands for 'not detected'

| Strain | Lowest Concentration<br>Detected |
|----------------------|----------------------------------|
| A/Aichi/2/68 | NDa |
| A/Hong Kong/8/68 | NDa |
| A/Port Chalmers/1/73 | 5 TCID50/mL |
| A/Victoria/3/75 | 50 TCID50/mL |
| A/Brisbane/10/07 | 5 TCID50/mL |
| A/Taiwan/760/2007 | 0.5 TCID50/mL |
| A/Uruguay/716/2007 | 0.5 EID50/mL |
| A/Perth/16/09 | 5 TCID50/mL |
| A/Alice | 0.5 TCID50/mL |
| A/MRC-2 recomb | NDa |

#### Table 9. Results of Influenza A H3N2 Inclusivity

4 ND stands for 'not detected'

June, 2011

{10}------------------------------------------------

| Strain | Lowest Concentration<br>Detected |
|--------------------------|----------------------------------|
| A/California/4/2009 | 1,500 TCID50/mL |
| A/California/8/2009 | 150 EID50/mL |
| A/England/195/2009 | 150 TCID50/mL |
| A/Mexico/4108/2009 | 150 EID50/mL |
| A/North Carolina/18/2009 | 1,500 TCID50/mL |
| A/South Carolina/18/2009 | 150 TCID50/mL |
| A/SwineNY/01/2009 | 150 TCID50/mL |
| A/SwineNY/02/2009 | 150 TCID50/mL |
| A/SwineNY/03/2009 | 150 TCID50/mL |
| A/Texas/48/2009 | 1,500 TCID50/mL |
| A/Washington/29/2009 | 150 TCID50/mL |

#### Table 10. Results of 2009 H1N1 Influenza Inclusivity

### Exclusivity

The potential for cross-reactivity between JBAIDS influenza assays was evaluated by testing simulated NPS samples containing high concentrations of influenza viruses (tens to thousands-fold higher than LoD). Table 11 lists all of the non-target influenza strains tested at high concentrations with the Flu A H1, Flu A H3, Flu A Sw and Flu A H1 2009 assays. In all cases, the assays gave the expected negative results with the non-target influenza assays.

Three (3) Influenza A H1N1 strains, A/Maryland/12/1991, A/Iowa/1/2006, and A/swine/Wisconsin/125/1997, were detected by the Flu A Sw assay: These results were not unexpected since the first two of these strains were isolated from humans but have an origin of swine lineage and the third was isolated from swine. In addition, the Influenza A H3N2 virus strain A/SW/IA/1/99 (swine origin) was detected by both the Flu A H3 and Flu A Sw assays. Detection of swine Influenza A/H3 viruses by the Flu A H3 assay is not unexpected as the hemagglutinin sequences for swine and human isolates are very similar.

| | Type/Subtype | Strain | Concentration<br>Tested | Assays<br>Tested |
|-------------|-----------------------------------|--------------------------------------|-------------------------|------------------|
| Influenza A | H2N2 (Avian) | A/chicken/Pennsylvania/298101-4/2004 | 3.16E+07 TCID50/mL | Flu A H1 |
| | H3N8 (Avian) | A/MAL/ALB/16/87 | 1.72E+03 TCID50/mL | Flu A H3 |
| | H4N8 (Avian) | A/chicken/Alabama/1975 | 1.00E+08 EID50/mL | Flu A Sw |
| | H5N1 (Avian-Human<br>Recombinant) | A/Vietnam/1203/2004(H5N1)-PR8 | 3.16E+07 EID50/mL | Flu A H1<br>2009 |
| | H5N1 (Avian) | A/DK/PA/4560069-9/06 | 1.00E+05 TCID50/mL | |
| | H7N3 (Avian) | A/TY/UT/24721-10/95 | 3.06E+04 TCID50/mL | |

| Table 11. Results of Testing for Cross-Reactivity with Influenza A Subtyping Assay | | | | | |
|------------------------------------------------------------------------------------|----------------|---------------------|-----------------|------------------|---------------------|
| <b>Virus</b> | <b>Subtype</b> | <b>Assay Result</b> | <b>Virus</b> | <b>Subtype</b> | <b>Assay Result</b> |
| Adenovirus | Type 1 | Negative | Influenza B | Victoria Lineage | Negative |
| Adenovirus | Type 3 | Negative | Influenza B | Yamagata Lineage | Negative |
| Coronavirus | 229E | Negative | Metapneumovirus | | Negative |
| Coronavirus | HKU1 | Negative | Parainfluenza | Type 1 | Negative |
| Coronavirus | NL63 | Negative | Parainfluenza | Type 2 | Negative |
| Coronavirus | OC43 | Negative | Parainfluenza | Type 3 | Negative |
| Enterovirus | EV-D68 | Negative | Rhinovirus | | Negative |
| Enterovirus | | Negative | RSV | | Negative |

{11}------------------------------------------------

| Type/Subtype | Strain | Concentration<br>Tested | Assays<br>Tested |
|---------------------------------------------|----------------------------|--------------------------------------------------|------------------------------|
| H6N2 (Avian) | A/Chicken/CA/32213-1/2000 | 1.26E+07 EID50/mL | |
| H9N2 (Avian) | A/Turkey/Wisconsin/1966 | 5.60E+07 EID50/mL | |
| H3N8 (Canine) | A/canine/Florida/43/2004 | 1.00E+05 TCID50/mL | |
| H3N8 (Equine) | A/Equine/Ohio/01/2009 | 1.00E+05 TCID50/mL | |
| H1N1 (Swine) | A/swine/Wisconsin/125/1997 | 1.00E+05 TCID50/mL | |
| H1N1 (Swine) | A/SW/GB/19582/92 | 5.64E+03 TCID50/mL | |
| H3N2 (Swine) | A/SW/IA/1/99 | 1.41E+03 TCID50/mL | |
| H1N1 (Human of<br>swine lineage) | A/Maryland/12/1991 | 1.00E+05 TCID50/mL | |
| H1N1 (Human of<br>swine lineage) | A/Iowa/1/2006 | 1.00E+05 TCID50/mL | |
| H7N2 (Human) | A/New York/107/2003 | 30 µl of an unknown<br>concentration into<br>1mL | |
| | B/Lee/40 | 7.36E+03 TCID50/mL | |
| | B/Allen/45 | 1.00E+05 TCID50/mL | |
| | B/GL/1739/54 | 7.36E+03 TCID50/mL | Flu A H1 |
| | B/Maryland/1/59 | 7.36E+03 TCID50/mL | Flu A H3 |
| Influenza B | B/Taiwan/2/62 | 4.54E+04 TCID50/mL | Flu A Sw |
| | B/Hong Kong/5/72 | 7.36E+03 TCID50/mL | Flu A H1<br>2009 |
| | B/Malaysia/2506/04 | 5.09E+03 TCID50/mL | |
| | B/FL/04/06 | 1.50E+04 TCID50/mL | |
| | B/Brigit | 3.14E+04 TCID50/mL | |
| | A/Brisbane/59/07 | 1.00E+05 TCID50/mL | |
| | A1/FM/1/47 | 4.24E+03 TCID50/mL | |
| | A/PR/8/34 | 1.00E+05 TCID50/mL | Flu A H3 |
| Influenza A<br>H1N1 | A/NWS/33 | 4.24E+03 TCID50/mL | Flu A Sw |
| | A1/Denver/1/57 | 4.24E+03 TCID50/mL | Flu A H1<br>2009 |
| | A/Solomon Islands/3/2006 | 1.25E+04 TCID50/mL | |
| | A/Weiss/43 | 4.24E+03 TCID50/mL | |
| | A/Mal/302/54 | 1.25E+04 TCID50/mL | |
| | A/Port Chalmers/1/73 | 5.10E+03 TCID50/mL | |
| | A/Victoria/3/75 | 4.24E+03 TCID50/mL | |
| | A/Aichi/2/68 | 1.00E+05 TCID50/mL | Flu A H1 |
| Influenza A<br>H3N2 | A/Hong Kong/8/68 | 1.00E+05 TCID50/mL | Flu A Sw<br>Flu A H1<br>2009 |
| | A/Alice (VR-776) | 4.24E+03 TCID50/mL | |
| | A/MRC-2 recomb (VR-777) | 7.36E+03 TCID50/mL | |
| | A/Brisbane/10/07 | 7.36E+03 TCID50/mL | |
| Influenza A<br>(swine lineage)<br>H1N1 2009 | Swine NY/02/2009 | 1.25E+04 TCID50/mL | Flu A H1 |
| | Swine NY/03/2009 | 7.36E+03 TCID50/mL | Flu A H3 |
| | Swine NY/01/2009 | 3.78E+04 TCID50/mL | |
| | A/Mexico/4108/2009 | 1.00E+05 EID50/mL | |
| | A/California/8/2009 | 1.00E+05 TCID50/mL | |
| | A/California/04/2009 | 1.00E+05 TCID50/mL | |
| Type/Subtype | Strain | Concentration<br>Tested | Assays<br>Tested |
| | A/Washington/29/2009 | 1.00E+05 TCID50/mL | |
| | A/South Carolina/18/2009 | 1.00E+05 TCID50/mL | |
| | A/England/195/2009 | 4.74E+04 TCID50/mL | |
| | A/North Carolina/39/2009 | 1.00E+05 TCID50/mL | |

{12}------------------------------------------------

The non-influenza exclusivity panel consisted of 17 bacteria, 18 viruses, and one fungus, which were selected based on the relatedness to JBAIDS influenza analytes, clinical relevance (cause respiratory symptoms or represent nasopharyngeal flora), or high prevalence within the population (e.g. Herpes Simplex Virus). Simulated NPS samples were spiked with bacteria or fungi at a concentration of 10° CFU/mL or TCID30/mL and viruses at a concentration between 103 - 10' copies/mL or TCID50/mL. The JBAIDS Influenza A subtyping assays did not cross-react with the exclusivity panel organisms at the test concentrations listed in Table 12.

| Virus | Concentration Tested | Bacteria/Fungi | Concentration Tested |
|-----------------------------|----------------------|----------------------------|----------------------|
| Adenovirus | 1.00E+05 TCID50/mL | Bordetella pertussis | 1.00E+06 CFU/mL |
| Bocavirus | 4.20E+07 copies/mL | Candida albicans | 1.00E+06 CFU/mL |
| Coronavirus 229E | 7.35E+03 TCID50/mL | Corynebacterium diptheriae | 1.00E+06 CFU/mL |
| Coronavirus OC43 | 6.57E+04 TCID50/mL | Escherichia coli | 1.00E+06 CFU/mL |
| Coronavirus NL63 | 5.10E+03 TCID50/mL | Haemophilus influenza | 7.80E+04 CFU/mL |
| Coronavirus HKU1 | 1.00E+05 copies/mL | Lactobacillus plantarum | 1.00E+06 CFU/mL |
| Cytomegalovirus (CMV) | 1.50E+04 TCID50/mL | Legionella pneumophila | 1.00E+06 TCID50/mL |
| Enterovirus | 1.00E+05 TCID50/mL | Moraxella catarrhalis | 1.00E+06 CFU/mL |
| Epstein-Barr Virus (EBV) | 1.00E+05 copies/mL | Mycobacterium tuberculosis | 1.00E+06 CFU/mL |
| Human Metapneumovirus | 7.35E+03 TCID50/mL | Mycoplasma pneumonia | 1.69E+05 TCID50/mL |
| Human Rhinovirus | 5.10E+03 TCID50/mL | Neisseria elongata | 1.00E+06 CFU/mL |
| Measles Virus (Rubeola) | 1.00E+05 TCID50/mL | Neisseria meningitidis | 1.00E+06 CFU/mL |
| Mumps | 4.53E+04 TCID50/mL | Pseudomonas aeruginosa | 1.00E+06 CFU/mL |
| Parainfluenza virus 1 | 1.25E+04 TCID50/mL | Staphylococcus aureus | 1.00E+06 CFU/mL |
| Parainfluenza virus 2 | 1.50E+04 TCID50/mL | Staphylococcus epidermidis | 1.00E+06 CFU/mL |
| Parainfluenza virus 3 | 1.00E+05 TCID50/mL | Streptococcus pneumonia | 1.00E+06 CFU/mL |
| Parainfluenza virus 4 | 1.00E+05 TCID50/mL | Streptococcus pyogenes | 1.00E+06 CFU/mL |
| Respiratory Syncytial Virus | 1.25E+04 TCID50/mL | Streptococcus salivarius. | 7.59E+05 CFU/mL |

Table 12. Non-Influenza Exclusivity Panel

## Reproducibility

A multicenter study was performed to determine overall system reproducibility. Reproducibility testing occurred at three test sites utilizing six total panels of NPS and NPW samples, each, were spiked with a representative seasonal influenza A H1N1 virus (A/New Caledonia/20/1999). Panels of simulated NPS and simulated NPW samples, each, were spiked with a representative seasonal influenza A H3N2 virus (A/New York/55/2004). Finally, panels of simulated NPS and simulated NPW samples,

{13}------------------------------------------------

each, were spiked with a representative 2009 H1N1 influenza virus (A/New York/18/2009). Samples in each panel consisted of three samples spiked below LoD (high negative, LoD/20), three samples spiked with a low concentration of virus (low positive, LoD), and three samples spiked at a medium concentration of virus (medium positive, 3×LoD) for a total of nine samples per panel. Each panel was tested twice daily at each site for a total of 30 results per sample and 90 results per spike level. The detection rate was ≥ 98% for samples containing influenza virus ≥ LoD. As expected, samples spiked below LoD have variable results. Results are shown in Table 13, Table 14 and Table 15.

| Sample<br>Type | Virus<br>Spike<br>Level | IT 1-2-3 Platinum Path<br>Sample Purification Kit | | | Roche MagNA Pure Compact<br>Nucleic Acid Isolation Kit I | | | | Both Kits,<br>All Sites<br>(% Pos.) | 95% CI | |
|----------------|-------------------------|---------------------------------------------------|-----------------|-----------------|----------------------------------------------------------|-----------------|-----------------|-----------------|-------------------------------------|------------------|-----------|
| | | Site 1 | Site 2 | Site 3 | Overall<br>for All<br>Sites | Site 1 | Site 2 | Site 3 | Overall<br>for All<br>Sites | | |
| NPS | 3xLoD | 15/15<br>(100%) | 14/15<br>(93%) | 15/15<br>(100%) | 44/45<br>(98%) | 15/15<br>(100%) | 14/15<br>(93%) | 15/15<br>(100%) | 44/45<br>(98%) | 88/90<br>(98%) | 92.2-99.7 |
| | LoD | 15/15<br>(100%) | 15/15<br>(100%) | 15/15<br>(100%) | 45/45<br>(100%) | 15/15<br>(100%) | 15/15<br>(100%) | 15/15<br>(100%) | 45/45<br>(100%) | 90/90<br>(100%) | 96.7-99.9 |
| | Detection<br>≥ LoD | 30/30<br>(100%) | 29/30<br>(97%) | 30/30<br>(100%) | 89/90<br>(99%) | 30/30<br>(100%) | 29/30<br>(97%) | 30/30<br>(100%) | 89/90<br>(99%) | 178/180<br>(99%) | 96.0-99.9 |
| | LoD/20 | 15/15<br>(100%) | 13/15<br>(87%) | 8/15<br>(53%) | 36/45<br>(80%) | 13/15<br>(87%) | 11/15<br>(73%) | 14/15<br>(93%) | 38/45<br>(84%) | 74/90<br>(82%) | 72.7-89.4 |
| | Detection<br>all Levels | 45/45<br>(100%) | 42/45<br>(93%) | 38/45<br>(84%) | 125/135<br>(93%) | 43/45<br>(96%) | 40/45<br>(89%) | 44/45<br>(98%) | 127/135<br>(94%) | 252/270<br>(93%) | 90.0-96.0 |
| NPW | 3xLoD | 15/15<br>(100%) | 15/15<br>(100%) | 15/15<br>(100%) | 45/45<br>(100%) | 15/15<br>(100%) | 15/15<br>(100%) | 15/15<br>(100%) | 45/45<br>(100%) | 90/90<br>(100%) | 96.7-99.9 |
| | LoD | 14/15<br>(93%) | 15/15<br>(100%) | 15/15<br>(100%) | 44/45<br>(98%) | 15/15<br>(100%) | 14/15<br>(93%) | 15/15<br>(100%) | 44/45<br>(98%) | 88/90<br>(98%) | 92.2-99.7 |
| | Detection<br>≥ LoD | 29/30<br>(97%) | 30/30<br>(100%) | 30/30<br>(100%) | 89/90<br>(99%) | 30/30<br>(100%) | 29/30<br>(97%) | 30/30<br>(100%) | 89/90<br>(99%) | 178/180<br>(99%) | 96.0-99.9 |
| | LoD/20 | 13/15<br>(87%) | 15/15<br>(100%) | 9/15<br>(60%) | 37/45<br>(82%) | 14/15<br>(93%) | 14/15<br>(93%) | 15/15<br>(100%) | 43/45<br>(96%) | 80/90<br>(89%) | 80.5-94.5 |
| | Detection<br>all Levels | 42/45<br>(93%) | 45/45<br>(100%) | 39/45<br>(87%) | 126/135<br>(93%) | 44/45<br>(98%) | 43/45<br>(96%) | 45/45<br>(100%) | 132/135<br>(98%) | 258/270<br>(96%) | 92.4-97.7 |

Table 13. Summary of Reproducibility Testing for the Flu A H1 Assay (Agreement with Expected Positive Results)

{14}------------------------------------------------

| | | IT 1-2-3 Platinum Path<br>Sample Purification Kit | | | | Roche MagNA Pure Compact<br>Nucleic Acid Isolation Kit I | | | | | |
|----------------|-------------------------|---------------------------------------------------------------------|-----------------|-----------------|-----------------------------|---------------------------------------------------------------------|-----------------|-----------------|-----------------------------|-------------------------------------|-----------|
| Sample<br>Type | Virus<br>Spike<br>Level | Number Positive Samples/<br>Total Samples<br>(% Positive Detection) | | | | Number Positive Samples/<br>Total Samples<br>(% Positive Detection) | | | | Both Kits,<br>All Sites<br>(% Pos.) | 95% CI |
| | | Site 1 | Site 2 | Site 3 | Overall<br>For All<br>Sites | Site 1 | Site 2 | Site 3 | Overall<br>For All<br>Sites | | |
| sNPS | 3×LoD | 15/15<br>(100%) | 15/15<br>(100%) | 15/15<br>(100%) | 45/45<br>(100%) | 15/15<br>(100%) | 15/15<br>(100%) | 13/15<br>(87%) | 43/45<br>(96%) | 88/90<br>(98%) | 92.2-99.7 |
| | LoD | 15/15<br>(100%) | 15/15<br>(100%) | 15/15<br>(100%) | 45/45<br>(100%) | 15/15<br>(100%) | 15/15<br>(100%) | 15/15<br>(100%) | 45/45<br>(100%) | 90/90<br>(100%) | 96.7-99.9 |
| | Detection<br>≥ LoD | 30/30<br>(100%) | 30/30<br>(100%) | 30/30<br>(100%) | 90/90<br>(100%) | 30/30<br>(100%) | 30/30<br>(100%) | 28/30<br>(93%) | 88/90<br>(98%) | 178/180<br>(99%) | 96.0-99.9 |
| | LoD/20 | 12/15<br>(80%) | 10/15<br>(67%) | 9/15<br>(60%) | 31/45<br>(69%) | 14/15<br>(93%) | 13/15<br>(87%) | 14/15<br>(93%) | 41/45<br>(91%) | 72/90<br>(80%) | 70.2-87.7 |
| | Detection<br>all Levels | 42/45<br>(93%) | 40/45<br>(89%) | 39/45<br>(87%) | 121/135<br>(90%) | 44/45<br>(98%) | 43/45<br>(96%) | 42/45<br>(93%) | 129/135<br>(96%) | 250/270<br>(93%) | 88.8-95.4 |
| sNPW | 3×LoD | 15/15<br>(100%) | 15/15<br>(100%) | 14/15<br>(93%) | 44/45<br>(98%) | 15/15<br>(100%) | 15/15<br>(100%) | 15/15<br>(100%) | 45/45<br>(100%) | 89/90<br>(99%) | 94.0-99.9 |
| | LoD | 14/15<br>(93%) | 15/15<br>(100%) | 15/15<br>(100%) | 44/45<br>(98%) | 15/15<br>(100%) | 15/15<br>(100%) | 15/15<br>(100%) | 45/45<br>(100%) | 89/90<br>(99%) | 94.0-99.9 |
| | Detection<br>≥ LoD | 29/30<br>(97%) | 30/30<br>(100%) | 29/30<br>(97%) | 88/90<br>(98%) | 30/30<br>(100%) | 30/30<br>(100%) | 30/30<br>(100%) | 90/90<br>(100%) | 178/180<br>(99%) | 96.0-99.9 |
| | LoD/20 | 11/15<br>(73%) | 10/15<br>(67%) | 9/15<br>(60%) | 30/45<br>(67%) | 15/15<br>(100%) | 14/15<br>(93%) | 15/15<br>(100%) | 44/45<br>(98%) | 74/90<br>(82%) | 72.7-89.5 |
| | Detection<br>all Levels | 40/45<br>(89%) | 40/45<br>(89%) | 38/45<br>(84%) | 118/135<br>(87%) | 45/45<br>(100%) | 44/45<br>(98%) | 45/45<br>(100%) | 134/135<br>(99%) | 252/270<br>(93%) | 89.7-96.0 |

Table 14. Summary of Reproducibility Testing for the Flu A H3 Assay (Agreement with Expected Positive Results)

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{15}------------------------------------------------

| Sample Type | Virus Spike Level | IT 1-2-3 Platinum Path Sample Purification Kit | | | | Roche MagNA Pure Compact Nucleic Acid Isolation Kit I | | | | Both Kits, All Sites | 95% CI |
|-------------|----------------------|---------------------------------------------------------------|--------------|--------------|-----------------------|---------------------------------------------------------------|--------------|--------------|-----------------------|----------------------|-----------|
| | | Number Positive Samples/ Total Samples (% Positive Detection) | | | | Number Positive Samples/ Total Samples (% Positive Detection) | | | | | |
| | | Site 1 | Site 2 | Site 3 | Overall for All Sites | Site 1 | Site 2 | Site 3 | Overall for All Sites | | |
| sNPS | 3×LoD | 15/15 (100%) | 15/15 (100%) | 15/15 (100%) | 45/45 (100%) | 15/15 (100%) | 15/15 (100%) | 15/15 (100%) | 45/45 (100%) | 90/90 (100%) | 96.7-99.9 |
| | LoD | 15/15 (100%) | 15/15 (100%) | 15/15 (100%) | 45/45 (100%) | 15/15 (100%) | 15/15 (100%) | 15/15 (100%) | 45/45 (100%) | 90/90 (100%) | 96.7-99.9 |
| | Detection ≥ LoD | 30/30 (100%) | 30/30 (100%) | 30/30 (100%) | 90/90 (100%) | 30/30 (100%) | 30/30 (100%) | 30/30 (100%) | 90/90 (100%) | 180/180 (100%) | 98.3-99.9 |
| | LoD/20 | 15/15 (100%) | 15/15 (100%) | 15/15 (100%) | 45/45 (100%) | 15/15 (100%) | 15/15 (100%) | 15/15 (100%) | 45/45 (100%) | 90/90 (100%) | 96.7-99.9 |
| | Detection all Levels | 45/45 (100%) | 45/45 (100%) | 45/45 (100%) | 135/135 (100%) | 45/45 (100%) | 45/45 (100%) | 45/45 (100%) | 135/135 (100%) | 270/270 (100%) | 98.9-99.9 |
| sNPW | 3×LoD | 15/15 (100%) | 15/15 (100%) | 15/15 (100%) | 45/45 (100%) | 15/15 (100%) | 15/15 (100%) | 15/15 (100%) | 45/45 (100%) | 90/90 (100%) | 96.7-99.9 |
| | LoD | 15/15 (100%) | 15/15 (100%) | 15/15 (100%) | 45/45 (100%) | 15/15 (100%) | 15/15 (100%) | 15/15 (100%) | 45/45 (100%) | 90/90 (100%) | 96.7-99.9 |
| | Detec…