CDC INFLUENZA 2009 A(H1N1)PDM REAL-TIME RT-PCR PANEL

K101564 · Centers for Disease Control and Prevention · OQW · Jun 22, 2010 · Microbiology

Device Facts

• Record ID: K101564
• Device Name: CDC INFLUENZA 2009 A(H1N1)PDM REAL-TIME RT-PCR PANEL
• Applicant: Centers for Disease Control and Prevention
• Product Code: OQW · Microbiology
• Decision Date: Jun 22, 2010
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3332
• Device Class: Class 2

Indications for Use

The CDC Influenza 2009 A(H1N1)pdm Real-Time RT-PCR Panel (CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel) is intended for use in real-time RT-PCR assays on an Applied Biosystems (ABI) 7500 Fast Dx Real-Time PCR Instrument in conjunction with clinical and epidemiological information: - For the qualitative detection of influenza virus type A viral RNA from nasopharyngeal swabs (NPS), nasal swabs (NS), nasal aspirates (NA), nasal washes (NW), dual nasopharyngeal / throat swabs (NPS/TS), bronchoalveolar lavage (BAL), tracheal aspirate (TA), and bronchial wash (BW), collected from the respiratory tract of human patients with signs and symptoms of respiratory infection and/or from viral culture. - For differentiation of 2009 H1N1 influenza virus RNA from nasopharyngeal swabs (NPS), nasal swabs (NS), nasal aspirates (NA), nasal washes (NW), dual nasopharyngeal / throat swabs (NPS/TS), bronchoalveolar lavage (BAL), tracheal aspirate (TA), and bronchial wash (BW) collected from the respiratory tract of human patients with signs and symptoms of respiratory infection and/or from viral culture. - To provide epidemiologic information for surveillance of the 2009 H1N1 influenza virus. Performance characteristics for influenza A were established during the 2009-2010 influenza season when 2009 H1N1 influenza virus was the predominant influenza A virus in circulation. Performance characteristics may vary against other emerging influenza A viruses. A negative test result for the bronchoalveolar lavage (BAL), tracheal aspirate (TA), and bronchial wash (BW) is presumptive and it is recommended these results be confirmed by viral culture. Negative results do not preclude influenza virus infection and should not be used as the sole basis for treatment or other patient management decisions. 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 department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens. All users, analysts, and any person reporting results from use of this device should be trained to perform and interpret the results from this procedure by a competent instructor prior to use. CDC Influenza Division will limit the distribution of this device to only those users who have successfully completed a training course provided by CDC instructors or designees.

Device Story

In vitro diagnostic real-time RT-PCR assay; detects influenza A (M gene), 2009 H1N1 (NP gene), and 2009 H1N1 subtype (HA gene) RNA. Input: RNA extracted from respiratory specimens (swabs, aspirates, washes, BAL). Operation: RT-PCR amplification using oligonucleotide primers and TaqMan hydrolysis probes on ABI 7500 Fast Dx instrument. Output: Qualitative detection/differentiation of influenza A and 2009 H1N1. Used in public health laboratories by trained personnel. Results interpreted via cycle threshold (Ct) values compared to controls (NTC, positive control, human RNase P extraction control). Aids clinical decision-making and epidemiological surveillance. Benefits: rapid identification of 2009 H1N1 pandemic strain.

Clinical Evidence

Prospective study at 9 sites (8 public health, 1 DoD) during 2009-2010 season; 1901 specimens (NPS, NS, throat swabs, nasal washes/aspirates, lower respiratory). Compared to virus culture/IFA/DFA and bi-directional sequencing. Sensitivity/specificity >96% for upper respiratory specimens; >83% for lower respiratory specimens. Positive percent agreement >96% (upper) and 100% (lower).

Technological Characteristics

Real-time RT-PCR assay; TaqMan hydrolysis probes; ABI 7500 Fast Dx instrument; requires specific qualified RNA extraction kits (Qiagen/Roche/bioMérieux); includes internal human RNase P control; qualitative output.

Indications for Use

Indicated for qualitative detection and differentiation of influenza A and 2009 H1N1 influenza viral RNA in respiratory specimens (NPS, NS, NA, NW, NPS/TS, BAL, TA, BW) from symptomatic human patients or viral culture.

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

• Focus Simplexa™ Influenza A H1N1 (2009) assay (K100148)
• CDC Human Influenza Virus Real-Time RT-PCR Detection and Characterization Panel (K080570)

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

A. 510(k) Number:
K101564

B. Purpose for Submission:
New Device

C. Measurand:
Specific influenza virus nucleic acid target sequences. Influenza types and subtypes detected:
1) Highly conserved region of the matrix (M) gene from influenza A viruses;
2) Highly conserved region of the nucleoprotein (NP) gene specific for 2009 H1N1 influenza type A;
3) Highly conserved region of hemagglutinin (HA) gene specific for 2009 H1N1 influenza A subtype H1.

D. Type of Test:
Real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for the qualitative detection and differentiation of influenza A and 2009 H1N1 influenza viral RNA in nasopharyngeal swabs (NPS), nasal swabs (NS), throat swabs (TS), nasal aspirates (NPA), nasal washes (NW), dual nasopharyngeal/throat swabs (NPS/TS), and bronchoalveolar lavage [BAL], bronchial wash [BW], and tracheal aspirate [TA] from human patients with signs and symptoms of respiratory infection and/or from viral culture. The isolation and purification of the nucleic acids is performed using one of the six recommended methods listed below in section "I. Device Description". Amplification and detection is performed on the Applied Biosystems 7500 Fast Dx instrument (K082562) with Sequence Detection Software version 1.4.

E. Applicant:
Centers for Disease Control and Prevention, Atlanta, GA

F. Proprietary and Established Names:
Proprietary: The CDC Influenza 2009 A(H1N1)pdm Real-Time RT-PCR Panel
Generic: CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel

G. Regulatory Information:
1. Regulation section: 866.3332

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2. Classification: Class II
3. Product code: OQW, NSU
4. Panel: Microbiology (83)

H. Intended Use:

1. Intended use(s):

The CDC Influenza 2009 A(H1N1)pdm Real-Time RT-PCR Panel (CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel) is intended for use in real-time RT-PCR assays on an Applied Biosystems (ABI) 7500 Fast Dx Real-Time PCR Instrument in conjunction with clinical and epidemiological information:

- For the qualitative detection of influenza virus type A viral RNA from nasopharyngeal swabs (NPS), nasal swabs (NS), nasal aspirates (NA), nasal washes (NW), dual nasopharyngeal / throat swabs (NPS/TS), bronchoalveolar lavage (BAL), tracheal aspirate (TA), and bronchial wash (BW), collected from the respiratory tract of human patients with signs and symptoms of respiratory infection and/or from viral culture.
- For differentiation of 2009 H1N1 influenza virus RNA from nasopharyngeal swabs (NPS), nasal swabs (NS), nasal aspirates (NA), nasal washes (NW), dual nasopharyngeal / throat swabs (NPS/TS), bronchoalveolar lavage (BAL), tracheal aspirate (TA), and bronchial wash (BW) collected from the respiratory tract of human patients with signs and symptoms of respiratory infection and/or from viral culture.
- To provide epidemiologic information for surveillance of the 2009 H1N1 influenza virus.

Performance characteristics for influenza A were established during the 2009-2010 influenza season when 2009 H1N1 influenza virus was the predominant influenza A virus in circulation. Performance characteristics may vary against other emerging influenza A viruses.

A negative test result for the bronchoalveolar lavage (BAL), tracheal aspirate (TA), and bronchial wash (BW) is presumptive and it is recommended these results be confirmed by viral culture.

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

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 department for testing. Viral culture

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should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.

All users, analysts, and any person reporting results from use of this device should be trained to perform and interpret the results from this procedure by a competent instructor prior to use. CDC Influenza Division will limit the distribution of this device to only those users who have successfully completed a training course provided by CDC instructors or designees.

2. Indication(s) for use:

Same as intended use

3. Special conditions for use statement(s):

For prescription use only

4. Special instrument requirements:

To be used with the Applied Biosystems ABI 7500 Fast Dx Real-Time PCR Instrument with Sequence Detection Software version 1.4 and one of the six recommended RNA extraction methods.

I. Device Description:

The CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel is a real-time RT-PCR test that uses oligonucleotide primers and dual-labeled hydrolysis (TaqMan®) probes for the in vitro qualitative detection and differentiation of human influenza A and the 2009 H1N1 influenza viral RNA on the ABI 7500 Fast Dx Real-Time PCR instrument. Viral RNA can be detected from nasopharyngeal swabs (NPS), nasal swabs (NS), nasal aspirates (NA), nasal washes (NW), dual nasopharyngeal/throat swabs (NPS/TS, bronchoalveolar lavage (BAL), tracheal aspirate (TA), and bronchial wash (BW) collected from patients with signs and symptoms of respiratory infection and from viral culture isolates. CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel also includes control materials.

The use of the CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel requires ancillary reagents for which specific lots have been qualified by the CDC Influenza Division and incorporated in the CDC quality system. Any lots not specifically qualified by CDC for use with the CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel are not validated for use with this assay and may affect device performance. A supplemental cumulative list of qualified ancillary reagents lots for use with the CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel is provided with each shipment or can be requested by sending an email to [e-mail].

The following is a list of ancillary reagents that are not supplied with the CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel and are included in CDC's reagent qualification program.

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| | Reagent | Quantity | Catalog No. |
| --- | --- | --- | --- |
| rRT-PCR Enzyme Mastermix Options | Invitrogen SuperScript™ III Platinum® One-Step Quantitative RT-PCR System (without Rox) | 100 reactions | 11732-020 |
| | | 500 reactions | 11732-088 |
| | Invitrogen SuperScript™ III Platinum® One-Step Quantitative RT-PCR System (with Rox) | 100 reactions | 11745-100 |
| | | 500 reactions | 11745-500 |
| Nucleic Acid Purification Kit Options | Qiagen QIAamp® Viral RNA Mini Kit | 50 extractions | 52904 |
| | | 250 extractions | 52906 |
| | Roche MagNA Pure Compact Nucleic Acid Isolation Kit I | 32 extractions | 03 730 964 001 |
| | Roche MagNA Pure LC 2.0 with Total Nucleic Acid (TNA) Kit | 192 extractions | 03 038 505 001 |
| | Roche MagNA Pure Compact RNA Isolation Kit | 32 extractions | 04 802 993 001 |
| | bioMérieux NucliSENS® easyMAG® (Automated magnetic extraction reagents sold separately) | | |

J. Substantial Equivalence Information:

1. Predicate device name(s): Focus Simplexa™ Influenza A H1N1 (2009) assay and CDC Human Influenza Virus Real-Time RT- PCR Detection and Characterization Panel
2. Predicate 510(k) number(s): K0100148 and K080570
3. Comparison with predicate:

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Similarities

| Device Characteristics | CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel (New Device) | Simplexa™ Influenza A H1N1 (2009) (K100148) | CDC Human Influenza Virus Real-Time RT-PCR Detection and Characterization Panel (K080570) |
| --- | --- | --- | --- |
| Intended Use | Qualitative in vitro 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 | Qualitative in vitro detection and differentiation of influenza A and 2009 H1N1 influenza viral RNA in nasopharyngeal swabs (NPS), nasal swabs (NS), and nasopharyngeal aspirates (NPA) from human patients with signs and symptoms of respiratory infection on the 3M Integrated Cycler | Qualitative in vitro detection of influenza virus type A or B and for determination of the subtype of seasonal human influenza A virus, as seasonal A/H1 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 |
| Identification of Inf A | Yes (Universal A) | Yes | Yes |
| Assay Results | Qualitative | Qualitative | Qualitative |
| Nucleic Acid Extraction | Yes | Yes | Yes |
| Assay Type | Real-time RT-PCR | Real-time PCR | Real-time RT-PCR |

Differences

| Device Characteristics | CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel (New Device) | Simplexa™ Influenza A H1N1 (2009) (New Device) | CDC Human Influenza Virus Real-Time RT-PCR Detection and Characterization Panel (Predicate Device #2) |
| --- | --- | --- | --- |
| Sample types | NPS, NS, NA, NW, NPS/TS, BAL, TA, BW | NPS, NS, NPA | NPS, NS |
| Extraction Methods | • QIAamp® Viral RNA Mini Kit, Qiagen Inc. • MagNA Pure Compact - Total Nucleic Acid Kit, Roche Applied Science • MagNA Pure Compact - RNA Isolation Kit, Roche Applied Science • MagNA Pure LC - RNA Isolation Kit II, Roche Applied Science • Qiagen QIAcube with QIAamp® Viral RNA Mini Kit, Qiagen Inc. • NucliSENS® easyMAG®, bioMerieux | • QIAamp® Viral RNA Mini Kit, Qiagen Inc. • MagNA Pure Total Nucleic Acid Isolation Kit (Roche) • MagNA Pure LC Instrument (Roche) | • QIAamp® Viral RNA Mini Kit, Qiagen Inc. • RNeasy® Mini Kit, Qiagen, Inc. • MagNA Pure LC RNA Isolation Kit II, Roche Applied Science • MagNA Pure Total Nucleic Acid Kit, Roche Applied Science |

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| Identification of 2009 H1N1 Subtype | Yes | Yes | No |
| --- | --- | --- | --- |
| Required Instrumentation | Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument with SDS software version 1.4 | Integrated cycler with Integrated Cycler Studio software v. 2.0 | Applied Biosystems 7500 Fast Dx Real- Time PCR Instrument with SDS software version 1.4 |
| Multiplex Capability | No (Modular Use with CDC rRT-PCR Flu Panel (K08570) | Yes | No |

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

- Guidance for Industry and FDA Staff - Class II Special Controls Guidance Document: Reagents for Detection of Specific Novel Influenza A Viruses, March 22, 2006
http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/ucm078583.htm
- Guidance for Industry and FDA Staff - Class II Special Controls Guidance Document: Testing for Detection and Differentiation of Influenza A Virus Subtypes Using Multiplex Assays October 9, 2009
http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/ucm180307.htm
- Draft Guidance for Industry and FDA Staff - Establishing Performance Characteristics of In Vitro Diagnostic Devices for Detection or Detection and Differentiation of Influenza Viruses, February 15, 2008
http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/ucm079171.htm
- Guidance for Industry and FDA Staff, In Vitro Diagnostic Devices to Detect Influenza A Viruses: Labeling and Regulatory Path, May 1, 2007
http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/ucm078538.htm

## L. Test Principle:

The CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel includes a set of oligonucleotide primers and dual-labeled hydrolysis (Taqman®) probes for use in real-time RT-PCR assays on an ABI 7500 Fast Dx Real-Time PCR instrument for the in vitro qualitative detection of viral nucleic acids from human influenza A and 2009 H1N1 influenza in respiratory specimens. The assay consists of reagents to detect the matrix (M) gene RNA from influenza A virus, nucleoprotein (NP) gene and hemagglutinin (HA) gene RNA from 2009 H1N1 influenza virus, and the human RNase P gene RNA from the human specimen extraction control (HSC). An extraction control (HSC) is added to each batch of specimens to be extracted to monitor the quality of the extraction procedure, overall reagent integrity, and presence of PCR-inhibitory substances. A no template control (NTC) is included on each plate to serve as a negative control. Positive controls for each

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target are also provided and are to be included in each assay run. The targeted regions of viral RNA are transcribed into complimentary DNA (cDNA) and amplified by the polymerase chain reaction (PCR). The fluorescently labeled probes anneal to amplified DNA fragments and the fluorescent signal intensity is monitored by the ABI 7500 Fast Dx instrument during each PCR cycle. Amplification of target is recorded as increase of fluorescence over time in comparison to background signal.

## Interpretation of Results

### Extraction and Control Results and Interpretation

#### No Template Control (NTC)

The NTC consists of using nuclease-free water in the rRT-PCR reactions instead of RNA. The NTC reactions for all primer and probe sets should not exhibit fluorescence growth curves that cross the threshold line. If any of the NTC reactions exhibit a growth curve that crosses the cycle threshold, sample contamination may have occurred. Invalidate the run and repeat the assay with strict adherence to the guidelines.

#### Influenza 2009 A(H1N1)pdm Positive Control

The positive control consists of an influenza 2009 H1N1 influenza virus and cultured human cells (A549). Purified RNA from the positive control will yield a positive result with the following primer and probe sets: InfA, pdm InfA, pdm H1, and RP.

#### Human Specimen Control (HSC) (Extraction Control)

The HSC control consists of noninfectious cultured human cell (A549) material. The HSC is used as a RNA extraction procedural control to demonstrate successful recovery of RNA as well as extraction reagent integrity. Purified RNA from the HSC should yield a positive result with the RP primer and probe set and negative results with all influenza specific markers.

### Specimen Results and Interpretation

#### RNase P (RP)

- All clinical samples should exhibit fluorescence growth curves in the RP reaction that cross the threshold line within 38 cycles (< 38 Ct), thus indicating the presence of the human RNase P gene. Failure to detect RP in any clinical specimens may indicate:
- Improper extraction of nucleic acid from clinical materials resulting in loss of RNA and/or RNA degradation
- Absence of sufficient human cellular material due to poor collection or loss of specimen integrity
- Improper assay set up and execution
- Reagent or equipment malfunction

- If the RP assay does not produce a positive result for human clinical specimens, interpret as follows:
- If the InfA, pdm InfA, and pdm H1 are positive, even in the presence of a negative RP, the influenza result should be considered valid. It is possible, that some samples may fail to exhibit RP growth curves due to low cell numbers in the

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original clinical sample. A negative RP signal does not preclude the presence of influenza virus RNA in a clinical specimen.

- If all influenza markers AND RP are all negative for the specimen, the assay is "inconclusive" for the specimen. If residual specimen is available, repeat the extraction procedure and repeat the test. If all markers remain negative after re-test, report the results as "inconclusive" and a new specimen should be collected if possible.

> The RP assay may be negative when testing virus culture samples.

# Influenza Markers (InfA, pdm InfA, and pdm H1)

> When all controls exhibit the expected performance, a specimen is considered negative if influenza marker growth curves DO NOT cross the threshold line within 38 cycles (< 38 Ct) and RP growth curve does cross the threshold line within 38 cycles (< 38 Ct).

> When all controls exhibit the expected performance, a specimen is considered positive for influenza A if the influenza marker (InfA) growth curve crosses the threshold line within 38 cycles (<38 Ct) and the pdm InfA, and pdm H1 markers DO NOT cross the threshold line within 38 cycles. RP may be positive or negative as described above.

> When all controls exhibit the expected performance and none of the growth curves for the influenza markers or the RP marker cross the threshold line within 38 cycles (<38 Ct), the result is inconclusive. The extracted RNA from the specimen should be re-tested. If residual RNA is not available, re-extract RNA from residual specimen and re-test. If the re-tested sample is negative for all markers and all controls exhibit the expected performance, the result is "Inconclusive."

> When all controls exhibit expected performance and growth curves for InfA, pdm InfA, and pdm H1 markers cross the threshold line within 38 cycles (<38 Ct), report the specimen to be "Positive for Influenza 2009 A/H1N1."

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CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel Users Guide for Interpretation of Results – Quick Reference and Reporting

| InfA Target | pdm InfA Target | pdm H1 Target | RP Target | Report |
| --- | --- | --- | --- | --- |
| - | - | - | + | “Influenza A Not Detected” |
| + | - | - | ± | “Positive for Influenza A, Influenza 2009 A/H1 Virus not detected” |
| + | + | + | ± | “Positive for Influenza A, Positive for Influenza 2009 A/H1 Virus” |
| + | - | + | ± | “Positive for Influenza A, Inconclusive for Influenza 2009 A/H1 Virus” |
| + | + | - | ± | “Positive for Influenza A, Inconclusive for Influenza 2009 A/H1 Virus”
*Reflex Testing with the CDC rRT-PCR Flu Panel IVD Required |
| - | - | + | ± | “Negative for Influenza A, *Inconclusive for 2009 A/H1 Influenza Virus” |
| - | + | - | ± | “Negative for Influenza A, *Inconclusive for Influenza 2009 A/H1 Virus” |
| - | - | - | - | Inconclusive Test
*Likely Poor Extraction or Specimen Quality |

* When an inconclusive result is obtained, re-extract the specimen and test the newly extracted RNA (recommended) or re-test the extracted RNA

## M. Performance Characteristics:

**Analytical performance:**

a) Precision/Reproducibility:

The precision and reproducibility of the CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel were evaluated at six laboratory sites. Each testing site assessed a panel of four simulated samples at relative moderate, low (near the assay lower limit of detection range), and "high negative" viral RNA concentration, and a negative sample. The

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panels and assay controls were tested at each site by two operators on 5 different days within a 10-day period. Each site tested one of the six extraction methods associated with this device. Simulated samples in the qualification panel used in the reproducibility evaluation were:

Sample #1 Influenza 2009 H1N1 influenza moderate viral RNA titer range
Sample #2 Influenza 2009 H1N1 influenza low viral RNA titer range
Sample #3 Influenza 2009 H1N1 influenza "high negative" RNA titer range
Sample #4 Influenza Negative (uninfected A549 cells)

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Summary of Reproducibility Study for the CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel

| N=10 tests | bioMerieux NucliSENS easyMAG | | | Roche MagNA Pure LC TNA | | | Roche MagNA Pure Compact NA | | | Qiagen QIAcube Viral RNA | | | Qiagen Manual Viral RNA | | | Roche MagNA Pure Compact RNA | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | Agreement with expected result | Avg. Ct | % CV | Agreement with expected result | Avg. Ct | % CV | Agreement with expected result | Avg. Ct | % CV | Agreement with expected result | Avg. Ct | % CV | Agreement with expected result | Avg. Ct | % CV | Agreement with expected result | Avg. Ct | % CV | Total Agreement | 95% CI |
| No Template Control | | 10/10 | NA | NA | 10/10 | NA | NA | 10/10 | NA | NA | 10/10 | NA | NA | 10/10 | NA | NA | 10/10 | NA | NA | 60/60 | 94.0-100 |
| Sample 1 moderate | InfA | 10/10 | 28.52 | 2.59 | 10/10 | 30.53 | 2.55 | 10/10 | 31.99 | 4.23 | 10/10 | 27.92 | 2.06 | 10/10 | 28.85 | 2.00 | 10/10 | 30.54 | 2.12 | 60/60 | 94.0-100 |
| | pdm InfA | 10/10 | 27.49 | 2.10 | 10/10 | 29.98 | 2.69 | 10/10 | 30.63 | 3.10 | 10/10 | 27.68 | 2.25 | 10/10 | 28.72 | 1.59 | 10/10 | 29.81 | 2.57 | 60/60 | 94.0-100 |
| | pdm H1 | 10/10 | 30.21 | 2.12 | 10/10 | 32.23 | 3.17 | 10/10 | 32.98 | 2.31 | 10/10 | 30.63 | 1.48 | 10/10 | 31.09 | 1.69 | 10/10 | 32.38 | 2.57 | 60/60 | 94.0-100 |
| | RP | 10/10 | 28.78 | 1.33 | 10/10 | 30.79 | 2.65 | 10/10 | 32.27 | 2.51 | 10/10 | 27.55 | 2.43 | 10/10 | 27.82 | 2.45 | 10/10 | 29.43 | 1.77 | 60/60 | 94.0-100 |
| Sample 2 low | InfA | 10/10 | 33.10 | 3.26 | 8/10 | 31.75 | 35.61 | 8/10 | 36.12 | 4.11 | 10/10 | 32.95 | 1.04 | 10/10 | 33.49 | 3.14 | 10/10 | 34.48 | 2.88 | 58/60 | 88.5-99.6 |
| | pdm InfA | 10/10 | 32.50 | 2.48 | 9/10 | 30.42 | 35.27 | 9/10 | 31.50 | 35.27 | 10/10 | 32.91 | 3.32 | 10/10 | 33.89 | 3.27 | 10/10 | 34.48 | 2.92 | 58/60 | 88.5-99.6 |
| | pdm H1 | 10/10 | 35.19 | 3.10 | 7/10 | 28.85 | 52.94 | 7/10 | 29.34 | 52.77 | 10/10 | 34.66 | 1.27 | 10/10 | 36.12 | 2.18 | 10/10 | 36.57 | 3.70 | 54/60 | 79.5-96.2 |
| | RP | 10/10 | 28.44 | 1.34 | 10/10 | 30.33 | 2.78 | 10/10 | 31.44 | 3.27 | 10/10 | 27.41 | 1.95 | 10/10 | 28.34 | 1.55 | 10/10 | 28.57 | 5.11 | 60/60 | 94.0-100 |
| Sample 3 "high negative" | InfA | 8/10 | NA | NA | 10/10 | NA | NA | 9/10 | NA | NA | 10/10 | NA | NA | 7/10 | NA | NA | 10/10 | NA | NA | 54/60 | 79.5-96.2 |
| | pdm InfA | 10/10 | NA | NA | 10/10 | NA | NA | 10/10 | NA | NA | 9/10 | NA | NA | 8/10 | NA | NA | 10/10 | NA | NA | 57/60 | 86.1-99.0 |
| | pdm H1 | 10/10 | NA | NA | 9/10 | NA | NA | 10/10 | NA | NA | 9/10 | NA | NA | 10/10 | NA | NA | 10/10 | NA | NA | 58/60 | 88.5-99.6 |
| | RP | 10/10 | 28.04 | 2.13 | 10/10 | 30.08 | 4.92 | 10/10 | 31.12 | 2.60 | 10/10 | 26.89 | 2.09 | 10/10 | 28.33 | 1.41 | 10/10 | 28.45 | 3.31 | 60/60 | 94.0-100 |
| Sample 4 A549 cells | RP | 10/10 | 28.68 | 2.75 | 10/10 | 30.20 | 5.68 | 10/10 | 32.10 | 1.74 | 10/10 | 27.06 | 2.65 | 10/10 | 28.60 | 2.29 | 10/10 | 29.33 | 4.00 | 60/60 | 94.0-100 |
| HSC | RP | 10/10 | 28.51 | 2.30 | 10/10 | 29.80 | 4.48 | 10/10 | 29.55 | 3.11 | 10/10 | 25.89 | 2.04 | 10/10 | 27.30 | 1.61 | 10/10 | 27.35 | 4.98 | 60/60 | 94.0-100 |
| Influenza 2009 A(H1N1) pdm Positive Control | InfA | 10/10 | 23.71 | 2.10 | 10/10 | 21.95 | 2.97 | 10/10 | 23.54 | 4.10 | 10/10 | 21.62 | 1.39 | 10/10 | 23.66 | 2.63 | 10/10 | 24.95 | 2.21 | 60/60 | 94.0-100 |
| | pdm InfA | 10/10 | 22.81 | 2.68 | 10/10 | 22.27 | 1.91 | 10/10 | 23.79 | 2.78 | 10/10 | 21.82 | 2.62 | 10/10 | 23.63 | 1.57 | 10/10 | 24.93 | 2.03 | 60/60 | 94.0-100 |
| | pdm H1 | 10/10 | 25.76 | 1.55 | 10/10 | 24.35 | 4.78 | 10/10 | 26.36 | 2.42 | 10/10 | 25.31 | 1.19 | 10/10 | 25.89 | 2.13 | 10/10 | 27.52 | 2.53 | 60/60 | 94.0-100 |
| | RP | 10/10 | 28.84 | 1.66 | 10/10 | 27.44 | 3.23 | 10/10 | 29.03 | 4.03 | 10/10 | 27.35 | 1.84 | 10/10 | 28.59 | 2.35 | 10/10 | 28.82 | 3.26 | 60/60 | 94.0-100 |

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b) Linearity/reportable range:

Not applicable

c) Traceability, stability, expected values (controls calibrators or methods)

Before analyzing the samples tested using the CDC Influenza 2009 A(H1N1)pdm Real-Time RT-PCR Panel on the ABI 7500 Fast Dx Real-Time PCR instrument, the ABI instrument must be prepared following the procedures described in the Package Insert for the CDC Influenza 2009 A(H1N1)pdm Real-Time RT-PCR Panel and the ABI 7500 Fast Dx Real-Time PCR instrument User Manual.

## Assay Controls

Quality Control procedures must be followed in conformance with local, state, and/or federal regulations or accreditation requirements and a laboratory’s standard quality control procedures. It is recommended that the user refer to CLSI document C24-A2.

The following control material should be used when testing with the CDC Influenza 2009 A(H1N1)pdm Real-Time RT-PCR Panel:

- Internal positive control, the human RNase P (RP) primer and probe set detects human RP and is used with each clinical specimen to indicate that adequate isolation of nucleic acid resulted from the extraction of the specimen. A positive RP assay result also ensures that common reagents and equipment are functioning properly, and demonstrates the absence of inhibitory substances.
- Influenza 2009 A(H1N1)pdm Positive Control (pdmPC) contains noninfectious (beta-propiolactone inactivated influenza 2009 H1N1 influenza virus and cultured human cells (A549). The positive control will yield a positive result with the following primer and probe sets: InfA, pdm InfA, pdm H1, and RP to ensure the detection of influenza 2009 H1N1 influenza virus.
- A Human Specimen Control (HSC) is a noninfectious cultured human cell material for use as a RNA extraction procedural control. It is added to each batch of specimens to be extracted to monitor the successful recovery of RNA as well as extraction reagent integrity and presence of PCR-inhibitory substances. It should yield a positive result with the RP primer and probe set.
- A no template control (NTC) consists of nuclease-free water in the rRT-PCR reactions instead of RNA. The NTC reactions for all primer and probe sets should not exhibit fluorescence growth curves that cross the threshold line. If any of the NTC reactions exhibit a growth curve that crosses the cycle threshold, sample contamination may have occurred. Invalidate the run and repeat the assay with stricter adherence to the

12

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guidelines.

d) Detection limit:

Analytical sensitivity was demonstrated for three influenza strains by determining the limit of detection (LoD) of each primer and probe set in the CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel. The following strains were characterized and used for the LoD study:

A/California/07/09, 2009 H1N1 virus, $10^{8.4}$ EID$_{50}$/mL stock concentration
A/New York/18/09, 2009 H1N1 virus, $10^{7.8}$ EID$_{50}$/mL stock concentration
A/Brisbane/59/07, seasonal H1N1 virus, $10^{8.4}$ EID$_{50}$/mL stock concentration

Five-fold serial dilutions of virus isolated from characterized embryonated chicken egg (ECE)-grown influenza viruses were tested five times (N=5) to identify an end-point for detection with each influenza primer and probe set. RNA was extracted from each virus using the Roche MagNA Pure Compact RNA kit. Once the end point or range was established, the end-point dilution and one dilution above and below the end point was tested and repeated twenty times for each virus and primer and probe set. The limit of detection for each primer and probe set was calculated to indicate the range of lowest detectable concentration of influenza virus (EID$_{50}$/mL) with a 95.0% or greater positivity. The limit of detection for each primer and probe set was determined using the results of the higher virus concentration of the two, type and subtype markers.

Limit of Detection of the CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel

| | Limit of Detection (EID_{50}/mL) | | | |
| --- | --- | --- | --- | --- |
| 2009 H1N1 Virus | InfA | pdm InfA | pdm H1 | Final LoD |
| A/CA/07/2009 | 10^{1.6} | 10^{1.6} | 10^{1.6} | 10^{1.6} |
| A/NY/18/2009 | 10^{1.3} | 10^{0.6} | 10^{1.3} | 10^{1.3} |

e) Analytical specificity:

Inclusivity

To demonstrate the specificity of the primer and probe sets to detect a diverse population of 2009 H1N1 influenza viruses, ten 2009 H1N1 influenza viruses propagated in either Madin-Darby canine kidney (MDCK) cells in culture or in ECE as described previously in this section, were grown to high titer, and harvested. Each virus was subsequently serially diluted to approximately $10^{2}$ ID$_{50}$/mL, near the limit of detection of the assay. The diluted 2009 H1N1 influenza viruses were extracted using Roche MagNA Pure Compact RNA kit and were tested in triplicate using the CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel.

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2009 H1N1 Influenza Viruses Tested with the CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel

| 2009 A/H1N1 Virus | Stock Virus Titer | | ID50/mL Tested | Average InfA Ct Value (N=3) | Average pdm InfA Ct Value (N=3) | Average pdm H1 Ct Value (N=3) |
| --- | --- | --- | --- | --- | --- | --- |
| | TCID50/mL | EID50/mL | | | | |
| A/MEXICO/4108/2009 | - | 10^{8.5} | 10^{2.5} | 33.46 | 36.20 | 32.86 |
| A/CALIFORNIA/8/2009 | - | 10^{9.2} | 10^{2.2} | 29.75 | 31.57 | 28.80 |
| A/CALIFORNIA/7/2009 | - | 10^{8.4} | 10^{2.4} | 34.16 | 35.28 | 33.78 |
| A/CALIFORNIA/04/2009 | 10^{7.9} | - | 10^{1.9} | 32.26 | 33.54 | 32.13 |
| A/TEXAS/48/2009 | 10^{8.0} | - | 10^{2.0} | 32.20 | 33.14 | 32.06 |
| A/WASHINGTON/29/2009 | 10^{7.5} | - | 10^{2.5} | 30.58 | 32.35 | 30.10 |
| A/SOUTH CAROLINA/18/2009 | 10^{6.6} | - | 10^{2.6} | 25.44 | 24.55 | 26.90 |
| A/NEW YORK/18/2009 | - | 10^{7.8} | 10^{2.7} | 27.95 | 26.42 | 28.23 |
| A/ENGLAND/195/2009 | 10^{7.0} | - | 10^{2.0} | 26.92 | 27.03 | 29.36 |
| A/NORTH CAROLINA/39/2009 | 10^{7.7} | - | 10^{2.7} | 27.23 | 26.47 | 27.96 |

Exclusivity with Seasonal Influenza Viruses

Characterized seasonal influenza specimens representing various geographical locations (12-A/H1, 12-A/H3, 12-Influenza B) were tested with the CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel to demonstrate the specificity of the primer and probe sets. Viruses were propagated in ECE or in MDCK cells in culture, grown to high titer, and harvested. RNA was extracted using the automated Roche MagNA Pure Compact RNA kit. The purified RNA was tested once with each primer and probe set in the panel following the CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel protocol.

Seasonal Influenza Viruses Tested with the CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel

| Type | Virus | Titer (log TCID50/mL) | InfA | pdm InfA | pdm H1 |
| --- | --- | --- | --- | --- | --- |
| H1N1 | A/JIANGXI/160/2005 | 5.6 | + | - | - |
| | A/SOLOMON ISLANDS/03/2006 | 6.2 | + | - | - |
| | A/TAIWAN/42/2006 | 4.7 | + | - | - |
| | A/FUKUSHIMA/141/2006 | 5.7 | + | - | - |
| | A/MEXICO/1744/2007 | 5.3 | + | - | - |
| | A/MEXICO/1729/2007 | 4.8 | + | - | - |

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| | A/MEXICO/1677/2007 | 5.7 | + | - | - |
| --- | --- | --- | --- | --- | --- |
| | A/MEXICO/949/2007 | 5.1 | + | - | - |
| | A/BANGLADESH/7286/2007 | 6.1 | + | - | - |
| | A/MEXICO/2010/2007 | 5.1 | + | - | - |
| | A/BRISBANE/59/2007 | 8.4 (EID50/mL) | + | - | - |
| | A/PARAGUAY/61/2009 | 7.2 | + | - | - |
| H3N2 | A/HAWAII/08/2006 | 7.8 | + | - | - |
| | A/WISCONSIN/03/2007 | 8.2 | + | - | - |
| | A/HENAN/JINSHUI/147/2007 | 8.1 | + | - | - |
| | A/BRISBANE/10/2007 | 6.8 | + | - | - |
| | A/MEXICO/922/2007 | 7.8 | + | - | - |
| | A/AFGHANISTAN/2903/2008 | 5.0 | + | - | - |
| | A/MEXICO/1938/2007 | 5.1 | + | - | - |
| | A/MEXICO/1995/2007 | 4.3 | + | - | - |
| | A/ANHUI/1239/2005 | 8.1 | + | - | - |
| | A/MEXICO/1842/2007 | 6.1 | + | - | - |
| | A/PERTH/16/2009 | 8.2 (EID50/mL) | + | - | - |
| | A/WISCONSIN/15/2009 | 8.1 (EID50/mL) | + | - | - |
| Inf B | B/FLORIDA/04/2006 | 5.7 | - | - | - |
| | B/CHONGQING/YONGCHUAN18/2007 | 7.7 | - | - | - |
| | B/FLORIDA/02/2006 | 6.0 | - | - | - |
| | B/PENNSYLVANIA/05/2007 | 6.6 | - | - | - |
| | B/BANGLADESH/5972/2007 | 4.7 | - | - | - |
| | B/BANGLADESH/3461/2007 | 3.7 | - | - | - |
| | B/BANGLADESH/7110/2007 | 4.2 | - | - | - |
| | A/MEXICO/1819/2007 | 5.1 | - | - | - |
| | B/TEXAS/17/2007 | 5.7 | - | - | - |
| | B/TEXAS/03/2007 | 5.6 | - | - | - |
| | B/BRISBANE/60/2008 | 8.5 (EID50/mL) | - | - | - |
| | B/TEXAS/26/2008 | 8.2 (EID50/mL) | - | - | - |

Exclusivity with Influenza Viruses with Pandemic Potential

To demonstrate the ability of the influenza A primer and probe sets to detect influenza A/H5 viruses with pandemic potential, the reactivity of the CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel was tested with ten avian A/H5 influenza viruses that have been shown to infect humans. The avian A/H5 influenza viruses were grown and harvested from allantoic/amniotic fluid from infected ECE and titered. Each virus was extracted using the Roche MagNA Pure Compact RNA Isolation kit, and the purified RNA was tested in triplicate with each marker following the CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel protocol.

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16

Avian A/H5 Influenza Viruses Tested with the CDC rRT-PCR 2009
A(H1N1)pdm Flu Panel

| Type | Virus | Clade | Titer (log EID_{50}/mL) | InfA | pdm InfA | pdm H1 |
| --- | --- | --- | --- | --- | --- | --- |
| H5N1 | A/Japanese white eye/Hong Kong/1038/2006 | 2.3.4 | 9.2 | + (3/3) | - | - |
| | A/Duck/Hunan/795/2002 | 2.1 | 9.9 | + (3/3) | - | - |
| | A/Chicken/Yunnan/1251/2003 | 1 | 9.3 | + (3/3) | - | - |
| | A/Common magpie/Hong Kong/645/2006 | 2.3.2 | 9.2 | + (3/3) | - | - |
| | A/Vietnam/1194/2004 | 1 | 9.3 | + (3/3) | - | - |
| | A/Egypt/321/2007 | 2.2 | 9.2 | + (3/3) | - | - |
| | A/Anhui/1/2005 | 2.3.4 | 9.3 | + (3/3) | - | - |
| | A/Chicken/India/NIV3487/2006 | 2.2 | 9.3 | + (3/3) | - | - |
| | A/Chicken/Vietnam/NCVD-016/2008 | 7 | 9.1 | + (3/3) | - | - |
| | A/Cambodia/R040505/2007 | 1 | 8.5 | + (3/3) | - | - |

To demonstrate the ability of the influenza A primer and probe sets to detect potential pandemic influenza A viruses, the reactivity of the CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel was also tested with fifteen non-human influenza viruses that have been shown to infect humans. The non-human influenza viruses were propagated in ECE, grown to high titer, and harvested as described previously. Each virus was extracted using Roche MagNA Pure Compact RNA kit and the purified RNA was tested in triplicate with each marker following the CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel protocol.

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Animal Influenza Viruses Tested with the CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel

| Species | Virus | Type | Titer (log EID_{50}/mL) | InfA | pdm InfA | pdm H1 |
| --- | --- | --- | --- | --- | --- | --- |
| EQUINE | A/equine/Ohio/01/2003 | EQ-H3N8 | 7.5 | + (3/3) | - | - |
| SWINE | A/Maryland/12/1991 | SW-H1N1 | 9.0 | + (3/3) | - | + (3/3) |
| SWINE | A/Iowa/1/2006 | SW-H1N1 | 9.0 | + (3/3) | + (3/3) | + (3/3) |
| AVIAN | A/chicken/Pennsylvania/298101-4/2004 | H2N2 | 9.5 | + (3/3) | - | - |
| CANINE | A/canine/Florida/43/2004 | H3N8 | 7.2 | + (3/3) | - | - |
| AVIAN | A/chicken/Alabama/1975 | H4N8 | 10.0 | + (3/3) | - | - |
| AVIAN | A/chucker/Minnesota/14591-7/1998 | H5N2 | 7.2 | + (3/3) | - | - |
| AVIAN | A/duck/Singapore-Q/F119-3/1997 | H5N3 | 8.5 | + (3/3) | - | - |
| AVIAN | A/duck/Pennsylvania/1969 | H6N1 | 9.2 | + (3/3) | - | - |
| AVIAN | A/chicken/California/32213-1/2000 | H6N2 | 9.1 | + (3/3) | - | - |
| AVIAN | A/chicken/New York/13237-6/1998 | H6N8 | 10.0 | + (3/3) | - | - |
| AVIAN | A/mallard/NL/12/2000 IB-CDC-1 | H7N7 | 9.5 | + (3/3) | - | - |
| AVIAN | A/turkey/Wisconsin/66 | H9N2 | 9.7 | + (3/3) | - | - |
| AVIAN | A/chicken/New Jersey/15906-9/1996 | H11N1 | 6.5 | + (3/3) | - | - |
| SWINE | A/swine/Wisconsin/125/1997 | SW-H1N1 | 7.9 | + (3/3) | - | - |

Cross reactivity with Common Non-Influenza Human Respiratory Pathogens

Analytical specificity was evaluated by testing each primer/probe set within the device panel with nucleic acids extracted from 34 organisms (15 viruses, 18 bacteria, and 1 yeast) representing common respiratory pathogens or flora commonly present in specimens collected from the nasopharynx region. Each sample (bacteria and virus) was extracted using either the Roche MagNA Pure Compact Nucleic Acid Isolation Kit I or the Roche MagNA Pure Compact RNA kit. Each nucleic acid sample was tested once following the testing procedure previously described for the CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel.

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Common Respiratory Bacteria and Yeast Tested with the CDC rRT-PCR 2009
A(H1N1)pdm Flu Panel

| Strain | Bacteria | Titer (log CFU/mL) | InfA | pdm InfA | pdm H1 |
| --- | --- | --- | --- | --- | --- |
| A639 | Bordetella pertussis | 8.3 | - | - | - |
| 2001-21-196 | Candida albicans (yeast) | 8.8 | - | - | - |
| TW183 | Chlamydia pneumoniae | 40 IFU/mL | - | - | - |
| - | Corynebacterium diphtheriae | 10.0 | - | - | - |
| K12 | Escherichia coli | 9.6 | - | - | - |
| 7790-06 | Streptococcus pyogenes | 7.5 | - | - | - |
| M15709 | Haemophilus influenzae | 6.4 | - | - | - |
| - | Lactobacillus plantarum | 8.8 | - | - | - |
| - | Legionella pneumophila | 10.3 | - | - | - |
| M15757 | Moraxella catarrhalis | 9.5 | - | - | - |
| H37Rv | Mycobacterium tuberculosis | 95 ng/μl L | - | - | - |
| M129 | Mycoplasma pneumoniae | 7.7 | - | - | - |
| - | Neisseria elongata | 8.6 | - | - | - |
| M2578 | Neisseria meningitidis | 7.9 | - | - | - |
| - | Pseudomonas aeruginosa | 10.5 | - | - | - |
| - | Staphylococcus aureus | 10.7 | - | - | - |
| - | Staphylococcus epidermidis | 10.5 | - | - | - |
| 249-06 | Streptococcus pneumoniae | 6.6 | - | - | - |
| SS1672 | Streptococcus salivarius | 8.4 | - | - | - |

Common Respiratory Viruses Tested with the CDC rRT-PCR 2009
A(H1N1)pdm Flu Panel

| Strain | Virus | Titer (log TCID_{50}/mL) | InfA | pdm InfA | pdm H1 |
| --- | --- | --- | --- | --- | --- |
| Echo 6 | Enterovirus | 6.9 | - | - | - |
| Ad. 71 | Human Adenovirus, type 1 | 9.2 | - | - | - |
| S-1058 | Human Adenovirus, type 7a | 7.1 | - | - | - |
| OC43 | Human Coronavirus | 50.4 ng/μl L | - | - | - |
| 229E | Human Coronavirus | 31.6 ng/μl L | - | - | - |
| 1A | Human Rhinovirus A | 5.8 | - | - | - |
| | Human Parainfluenza 1 virus | 3.0 ng/μl L | | | |
| Greer | Human Parainfluenza 2 virus | 3.1 | - | - | - |
| C-243 | Human Parainfluenza 3 virus | 7.9 | - | - | - |
| CH93-18b | Respiratory Syncytial Virus (RSV) | 6.8 | - | - | - |
| KOS | Herpes Simplex Virus | 8.4 | - | - | - |
| AV92-3 | Varicella-zoster Virus | 4.4 | - | - | - |
| B95-8 | Epstein Barr Virus | 1.7 ng/μl L | - | - | - |
| Edmonston | Measles Virus | 5.2 | - | - | - |

18

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No cross reactivity was detected for InfA, pdm InfA, or pdm H1.

f) Assay cut-off:

To assess the CDC Influenza 2009 A(H1N1)pdm Real-Time RT-PCR Panel assay cut-off, the data from the limit of detection study was analyzed. Data were plotted as virus concentration (log $\mathrm{EID}_{50} / \mathrm{mL}$ ) versus cycle threshold (Ct) value at or below the limits of detection for each marker as shown in Figures 1 through 3. The average Ct value at the lower end of the range of the limit of detection plus two standards of deviation afforded the Ct value cut-off for each of the markers in the CDC Influenza 2009 A(H1N1)pdm Real-Time RT-PCR Panel as shown by the red line (InfA = 39; pdm InfA = 37; pdm H1 = 40).

Figure 1: Assay Cut-Off Plot using Limit of Detection Data for the InfA Primer and Probe Set

Figure 2: Assay Cut-Off Plot using Limit of Detection Data for the pdm InfA Primer and Probe Set

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Figure 3: Assay Cut-Off Plot using Limit of Detection Data for the pdm H1 Primer and Probe Set

Based on this analysis, the analytical cutoff value defined for the CDC Influenza 2009 A(H1N1)pdm Real-Time RT-PCR Panel was less than 38. These data indicate that the InfA and pdm H1 primer and probe sets may be more sensitive. However, setting the cutoff Ct value too high could result in false positive results. The clinical study also supports this cutoff value.

# 2. Comparison studies:

a. Method comparison with gold standard/reference method:

See clinical studies

b. Matrix Comparison:

Not Applicable

c. Performance in Fresh vs. Frozen Clinical Specimens:

To determine the effect of a freeze/thaw cycle of a specimen on the CDC Influenza 2009 A(H1N1)pdm Real-Time RT-PCR Panel result, a comparison study was performed utilizing a subset of specimens collected prospectively for the 2009-2010 clinical evaluation of the CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel. Based upon the initial CDC Influenza 2009 A(H1N1)pdm Real-Time RT-PCR Panel and virus culture results, fifty-one positive and twenty negative specimens were chosen among three different U.S. Public Health Laboratories. Frozen aliquots of these specimens were

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thawed, extracted using the bioMérieux NucliSENS® easyMAG® (California), the manual QIAamp® Viral RNA Mini Kit (Massachuesettes), or the Roche MagNA Pure LC 2.0 with Total Nucleic Acid (TNA) Kit (Wisconsin), and retested using the CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel. The study showed $100\%$ concordance (qualitative) between the initial CDC Influenza 2009 A(H1N1)pdm Real-Time RT-PCR Panel result and the result after one freeze/thaw cycle.

# The scatter plots of Ct values of four markers (Inf A, pdm Inf A, pdm H1, RP)





Deming regression was performed to account for the measurement errors for fresh and frozen specimens. Scatter plot demonstrates the overall relationship between fresh and frozen specimens.

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# The bias plots for $\Delta \mathrm{Ct}$ (Fresh Ct - Frozen Ct) of four markers (Inf A, pdm Inf A, pdm H1, RP)



Bias plot displays the difference between fresh and frozen specimens versus their average.


# 3. Clinical studies:

Performance characteristics of the CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel were established during a prospective study at 8 U.S. public health laboratories and a Department of Defense (DoD) laboratory during the 2009-2010 respiratory virus season (February-April). Samples used for this study were collected for routine influenza testing at each site from individuals who were symptomatic with influenza-like illness (ILI) and included both upper and lower respiratory specimen types. Because of the low prevalence of influenza, retrospective specimens were used to supplement prospectively collected specimens.

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The reference methods utilized in this study were virus culture with Immunofluorescent Antibody (IFA) or Direct Fluorescent Antibody (DFA) for identification of influenza type and bi-directional sequencing for confirmation of 2009 H1N1 influenza subtype. InfA analyte results from the CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel were compared to virus culture results in the analysis. Sequencing was performed only with specimens that were first identified as positive for influenza A by virus culture. Pdm InfA and pdm H1 analyte results from the CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel were compared to bi-directional sequencing results in the analysis.

A total of 1901 patient specimens were evaluated at the nine clinical testing sites: 1191 nasopharyngeal swabs (NPS) and nasal swabs (NS), 50 throat swabs, 519 nasal washes and nasal aspirates, 99 dual NPS/TS, and 42 lower respiratory specimens.

## Specimens Received for the Performance Evaluation of the CDC Influenza 2009 A(H1N1)pdm Real-Time RT-PCR Panel

| Age Group (years) | Total # Tested per Age Group and Specimen Type | | | | | | | Total # Tested per Age Group |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | NPS | NS | TS | NW | NA | Lower Respiratory | NPS/TS | |
| 0-16 | 358 | 78 | 25 | 159 | 32 | 2 | 31 | 685 |
| 17-54 | 389 | 44 | 12 | 246 | 10 | 23 | 55 | 779 |
| ≥55 | 240 | 46 | 12 | 55 | 15 | 17 | 13 | 398 |
| Age Not Reported | 34 | 2 | 1 | 2 | 0 | 0 | 0 | 39 |
| Total # of Each Specimen Type | 1021 | 170 | 50 | 462 | 57 | 42 | 99 | 1901 |

## Prospective Study

The U.S. World Health Organization (WHO) and National Respiratory and Enteric Virus Surveillance System (NREVSS) reported an average of positive influenza specimens at approximately 4% during the prospective clinical evaluation of the CDC Influenza 2009 A(H1N1)pdm Real-Time RT-PCR Panel. A total of 1323 specimens were collected and tested with the CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel. Individual specimen types included in the analysis were 302 nasal aspirates and nasal washes, 877 nasopharyngeal swabs and nasal swabs, 65 dual-collected nasopharyngeal swabs/throat swabs, and 30 lower respiratory specimens.

The prospective study results are stratified by specimen type and are presented in the tables below.

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Prospective Results from Nasal Aspirate (NA) and Nasal Wash (NW) specimens for Universal Influenza A (InfA) and 2009 H1N1 (pdm InfA & pdm H1)

| InfA Analyte | Virus Culture | | | | |
| --- | --- | --- | --- | --- | --- |
| | | Influenza A Positive | Influenza A Negative | Total | Performance |
| CDC H1N1 rRT-PCR | Influenza A Positive | 19 | 2 | 21 | 100 % Sensitivity 83.2-100 (95% CI) |
| | Influenza A Negative | 0 | 281 | 281 | 99.3 % Specificity 97.5-99.8 (95% CI) |
| | Total | 19 | 283 | 302 | |
| pdm A and pdm H1 Analyte | Comparator Method | | | | |
| --- | --- | --- | --- | --- | --- |
| | | 2009 A/H1 Influenza Positive | 2009 A/H1 Influenza Negative | Total | Performance |
| CDC H1N1 rRT-PCR | 2009 A/H1 Influenza Positive | 16 | 4 | 20 | 100 % Sensitivity 80.6-100 (95% CI) |
| | 2009 A/H1 Influenza Negative | 0 | 282 | 282 | 98.6 % Specificity 96.5-99.5 (95% CI) |
| | Total | 16 | 286 | 302 | |

Prospective Results from Nasopharyngeal swab (NPS) and Nasal Swab (NS) specimens for Universal Influenza A (InfA) and 2009 H1N1 (pdm InfA & pdm H1)

| InfA Analyte | Virus Culture | | | | |
| --- | --- | --- | --- | --- | --- |
| | | Influenza A Positive | Influenza A Negative | Total | Performance |
| CDC H1N1 rRT-PCR | Influenza A Positive | 61 | 31 | 92 | 96.8 % Sensitivity 89.1-99.1 (95% CI) |
| | Influenza A Negative | 2 | 783 | 785 | 96.2 % Specificity 94.6-97.3 (95% CI) |
| | Total | 63 | 814 | 877 | |
| pdm A and pdm H1 Analyte | Comparator Method | | | | |
| --- | --- | --- | --- | --- | --- |
| | | 2009 A/H1 Influenza Positive | 2009 A/H1 Influenza Negative | Total | Performance |
| CDC H1N1 rRT-PCR | 2009 A/H1 Influenza Positive | 57 | 27 | 84 | 100 % Sensitivity 93.7-100 (95% CI) |
| | 2009 A/H1 Influenza Negative | 0 | 793 | 793 | 96.7 % Specificity 95.3-97.7 (95% CI) |
| | Total | 57 | 820 | 877 | |

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Prospective Results from Nasopharyngeal/Throat Swab (NP/TS) Dual-collected Specimens for Universal Influenza A (InfA) and 2009 H1N1 (pdm InfA & pdm H1)

| InfA Analyte | Virus Culture | | | | |
| --- | --- | --- | --- | --- | --- |
| | | Influenza A Positive | Influenza A Negative | Total | Performance |
| CDC H1N1 rRT-PCR | Influenza A Positive | 6 | 0 | 6 | 100 % Sensitivity 61.0-100 (95% CI) |
| | Influenza A Negative | 0 | 59 | 59 | 100 % Specificity 93.9-100 (95% CI) |
| | Total | 6 | 59 | 65 | |
| pdm A and pdm H1 Analyte | Comparator Method | | | | |
| --- | --- | --- | --- | --- | --- |
| | | 2009 A/H1 Influenza Positive | 2009 A/H1 Influenza Negative | Total | Performance |
| CDC H1N1 rRT-PCR | 2009 A/H1 Influenza Positive | 5 | 1 | 6 | 100 % Sensitivity 56.6-100 (95% CI) |
| | 2009 A/H1 Influenza Negative | 0 | 59 | 59 | 98.3 % Specificity 91.1-99.7 (95% CI) |
| | Total | 5 | 60 | 65 | |

Prospective Results from Lower Respiratory Tract Specimens (BAL, BW, and TA) specimens for Universal Influenza A (InfA) and 2009 H1N1 (pdm InfA & pdm H1)

| InfA Analyte | Virus Culture | | | | |
| --- | --- | --- | --- | --- | --- |
| | | Influenza A Positive | Influenza A Negative | Total | Performance |
| CDC H1N1 rRT-PCR | Influenza A Positive | 5 | 4 | 9 | 83.3 % Sensitivity 43.6-97.0 (95% CI) |
| | Influenza A Negative | 1 | 20 | 21 | 83.3 % Specificity 64.1-93.3 (95% CI) |
| | Total | 6 | 24 | 30 | |
| pdm A and pdm H1 Analyte | Comparator Method | | | | |
| --- | --- | --- | --- | --- | --- |
| | | 2009 A/H1 Influenza Positive | 2009 A/H1 Influenza Negative | Total | Performance |
| CDC H1N1 rRT-PCR | 2009 A/H1 Influenza Positive | 5 | 4 | 9 | 100 % Sensitivity 56.6-100 (95% CI) |
| | 2009 A/H1 Influenza Negative | 0 | 21 | 21 | 84.0 % Specificity 65.3-93.6 (95% CI) |
| | Total | 5 | 25 | 30 | |

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A total of 46 prospective and two retrospective throat swabs were tested. A total of eight (8) specimens were correctly identified as 2009 H1N1 influenza positive. Performance characteristics for 2009 H1N1 influenza virus using throat swab (TS) specimens were established testing a limited number of specimens. Users may wish to establish the sensitivity of this test for throat swab specimens by testing additional samples

## Retrospective Study

The prevalence of influenza decreased significantly during the prospective clinical evaluation of the CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel. This decrease prompted the supplementation of prospective specimens with archived specimens from earlier in the 2009-2010 influenza season. The archived specimens were selected for incorporation into the study if they met the same inclusion criteria as the prospective specimens and were also tested by virus culture at the time they were first received at each site. Individual specimen types included in the retrospective study were 214 nasal aspirates and nasal washes, 312 nasopharyngeal swabs and nasal swabs, 31 dual-collected nasopharyngeal swabs/throat swabs, and 10 lower respiratory specimens.

The retrospective study results are stratified by specimen type and are presented in the tables below

Retrospective Results from Nasal Aspirate (NA) and Nasal Wash (NW) specimens for Universal Influenza A (InfA) and 2009 H1N1 (pdm InfA & pdm H1)

| | InfA Analyte | Virus Culture | | | |
| --- | --- | --- | --- | --- | --- |
| | | Influenza A Positive | Influenza A Negative | Total | Performance |
| CDC H1N1 rRT-PCR | Influenza A Positive | 209 | 0 | 209 | 97.7 % Percent Positive Agreement 94.6-99.0 (95% CI) |
| | Influenza A Negative | 5 | 0 | 5 | NA |
| | Total | 214 | 0 | 214 | |
| | pdm A and pdm H1 Analyte | Comparator Method | | | |
| | | 2009 A/H1 Influenza Positive | 2009 A/H1 Influenza Negative | Total | Performance |
| CDC H1N1 rRT-PCR | 2009 A/H1 Influenza Positive | 203 | 5 | 208 | 99.0 % Percent Positive Agreement 96.5-99.7 (95% CI) |
| | 2009 A/H1 Influenza Negative | 2 | 4 | 6 | 44.4% Percent Negative Agreement 18.9-73.3 (95% CI) |
| | Total | 205 | 9 | 214 | |

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Retrospective Results from Nasopharyngeal swab (NPS) and Nasal Swab (NS) specimens for Universal Influenza A (InfA) and 2009 H1N1 (pdm InfA & pdm H1)

| | InfA Analyte | Virus Culture | | | |
| --- | --- | --- | --- | --- | --- |
| | | Influenza A Positive | Influenza A Negative | Total | Performance |
| CDC H1N1 rRT-PCR | Influenza A Positive | 310 | 0 | 310 | 99.4 % Percent Positive Agreement 97.7-99.8 (95% CI) |
| | Influenza A Negative | 2 | 0 | 2 | NA |
| | Total | 312 | 0 | 312 | |
| | pdm A and pdm H1 Analyte | Comparator Method | | | |
| --- | --- | --- | --- | --- | --- |
| | | 2009 A/H1 Influenza Positive | 2009 A/H1 Influenza Negative | Total | Performance |
| CDC H1N1 rRT-PCR | 2009 A/H1 Influenza Positive | 301 | 6 | 307 | 99.7 % Percent Positive Agreement 98.1-99.9 (95% CI) |
| | 2009 A/H1 Influenza Negative | 1 | 4 | 5 | 40.0 % Percent Negative Agreement 16.8-68.7 (95% CI) |
| | Total | 302 | 10 | 312 | |

Retrospective Results from Nasopharyngeal/Throat Swab (NP/TS) Dual-collected Specimens for Universal Influenza A (InfA) and 2009 H1N1 (pdm InfA & pdm H1)

| | InfA Analyte | Virus Culture | | | |
| --- | --- | --- | --- | --- | --- |
| | | Influenza A Positive | Influenza A Negative | Total | Performance |
| CDC H1N1 rRT-PCR | Influenza A Positive | 31 | 0 | 31 | 100 % Percent Positive Agreement 89.0-100 (95% CI) |
| | Influenza A Negative | 0 | 0 | 0 | NA % |
| | Total | 31 | 0 | 31 | |
| | pdm A and pdm H1 Analyte | Comparator Method | | | |
| --- | --- | --- | --- | --- | --- |
| | | 2009 A/H1 Influenza Positive | 2009 A/H1 Influenza Negative | Total | Performance |
| CDC H1N1 rRT-PCR | 2009 A/H1 Influenza Positive | 29 | 2 | 31 | 100 % Percent Positive Agreement 88.3-100 (95% CI) |
| | 2009 A/H1 Influenza Negative | 0 | 0 | 0 | NA |
| | Total | 29 | 2 | 31 | |

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Retrospective Results from Lower Respiratory Tract Specimens (BAL, BW, and TA) specimens for Universal Influenza A (InfA) and 2009 H1N1 (pdm InfA & pdm H1)

| | InfA Analyte | Virus Culture | | | |
| --- | --- | --- | --- | --- | --- |
| | | Influenza A Positive | Influenza A Negative | Total | Performance |
| CDC H1N1 rRT-PCR | Influenza A Positive | 10 | 0 | 10 | 100 % Percent Positive Agreement 72.3-100 (95% CI) |
| | Influenza A Negative | 0 | 0 | 0 | NA |
| | Total | 10 | 0 | 10 | |
| | pdm A and pdm H1 Analyte | Comparator Method | | | |
| --- | --- | --- | --- | --- | --- |
| | | 2009 A/H1 Influenza Positive | 2009 A/H1 Influenza Negative | Total | Performance |
| CDC H1N1 rRT-PCR | 2009 A/H1 Influenza Positive | 10 | 0 | 10 | 100 % Percent Positive Agreement 72.3-100 (95% CI) |
| | 2009 A/H1 Influenza Negative | 0 | 0 | 0 | NA |
| | Total | 10 | 0 | 10 | |

The CDC 2009 A(H1N1)pdm rRT-PCR Panel Clinical Sensitivity, Specificity, and Percent Agreement Summary

| | InfA | | | 2009 H1N1 | | |
| --- | --- | --- | --- | --- | --- | --- |
| Specimen Type | Sensitivity % (95% CI) | Specificity % (95% CI) | Positive Percent Agreement Retrospective | Sensitivity % (95% CI) | Specificity % (95% CI) | Positive Percent Agreement Retrospective |
| NPS/NS | 96.8 (89.1-99.1) | 96.2 (94.6-97.3) | 99.4 (97.7-99.8) | 100 (93.7-100) | 96.7 (95.3-97.7) | 99.7 (98.1-99.9) |
| NA/NW | 100 (83.2-100) | 99.3 (97.5-99.8) | 97.7 (94.6-99.0) | 100 (80.6-100) | 98.6 (96.5-99.5) | 99.0 (96.5-99.7) |
| NPS/TS | 100 (61.0-100) | 100 (93.9-100) | 100 (89.0-100) | 100 (56.6-100) | 98.3 (91.1-99.7) | 100 (88.3-100) |
| BAL, TA, BW | 83.3 (43.6-97.0) | 83.3 (64.1-93.3) | 100 (72.3-100) | 100 (56.6-100) | 84.0 (65.3-93.6) | 100 (72.3-100) |

{28}

The overall call rate for the CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel in the clinical evaluation at nine different sites was greater than 99%.

The clinical sensitivity of the CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel for all markers tested using upper respiratory specimens was greater than 96% and the clinical specificity for all markers was greater than 96%. The positive percent agreement was greater than 97% for all markers tested.

The clinical sensitivity and specificity of the CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel was also evaluated with lower respiratory specimens. The clinical sensitivity and specificity for all markers in the panel using these specimens were greater than 83% when compared to culture. The positive percent agreement of the CDC rRT-PCR 2009 A(H1N1)pdm Flu Panel was 100%.

4. Clinical cut-off:
Not applicable

5. Expected values/Reference range:

N. Instrument Name:
Applied Biosystems (ABI) 7500 Fast Dx Real-Time PCR (K082562)

O. System Descriptions:
1. Modes of Operation:
The Applied Biosystems 7500 Fast Dx Real-Time PCR instrument integrates a thermal cycler, a fluorimeter and application specific software. The instrument houses the thermal cycler and the fluorimeter, while the application software is run on a PC that is attached to the instrument. Samples are placed in a tube strip or 96-well low-head space plate that is moved to a Peltier-based thermal block and positioned relative to the optics using a tray loading mechanism.

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

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

29

{29}

Yes ☐ X ☐ or No ☐

3. Specimen Identification:
Sample ID is manually entered by user.

4. Specimen Sampling and Handling:
Not applicable

5. Calibration:
Not applicable

6. Quality Control:
Quality control is addressed for each specific assay to be run on the instrument (separately cleared).

P. Other Supportive Instrument Performance Characteristics Data Not Covered In the "Performance Characteristics" Section above:
Not applicable

Q. Proposed Labeling:
The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

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

30