The Influenza A/H5 (Asian Iineage) Virus Real-time RT-PCR Primer and Probe Set is intended for the in vitro qualitative detection of Influenza A/H5 (Asian lineage) virus RNA either directly in patient respiratory specimens or in viral cultures for the presumptive laboratory identification of Influenza A/H5 (Asian lineage) virus.

Testing with the Influenza A/H5 (Asian lineage) Virus Real-time RT-PCR Primer and Probe Set should be used in conjunction with other laboratory testing and clinical observations for the following indications:

Providing epidemiological information for the surveillance of human infection with Influenza A/H5 (Asian lineage) virus
Identifying patients who may be infected with Influenza A/H5 (Asian lineage) virus based on clinical and epidemiological risk factors
The definitive identification of influenza A/H5 (Asian lineage) either directly from patient specimens or from viral cultures requires additional laboratory testing, along with clinical and epidemiological assessment in consultation with national influenza surveillance experts.

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

Use of this assay is limited to Laboratory Response Network (LRN) designated laboratories.

Device Description

The Influenza A/H5 (Asian lineage) Virus Real-time RT-PCR Primer and Probe Set is composed of 2 primer pairs (4 unlabeled oligonucleotides) and 3 labeled probes, along with an inactivated virus control. These reagents are for use in single-tube realtime RT-PCR testing to directly detect novel Influenza A virus specific RNA in human respiratory specimens, or in viral cultures. The real-time PCR process simultaneously amplifies and detects nucleic acid targets in the same reaction.

The primer and probe sets (FluA2 and FluA3) target two distinct RNA regions that are both present in the influenza A/H5 (Asian lineage) hemagglutinin (HA) gene of highly pathogenic H5N1 viruses from the Asian lineage. These target regions were chosen to allow specific detection of Asian lineage influenza A/H5 viruses without detection of other influenza virus subtypes, including the North American lineage influenza A/H5 viruses (e.g., avian H5N2 strains).

Note: There are two lineages of avian influenza A/H5 viruses: the Eurasian (Asian) and North American (American) lineages. Viruses from these two lineages are genetically different. All known human influenza H5 infections have been caused by highly pathogenic viruses of the Asian lineage.

Primers/Probes: The 2 primer and probe sets (FluA2 and FluA3), each target a different region within the HA gene. The FluA2 target is in the HA2 region of the HA gene and the FluA3 target spans the cleavage site of the HA gene. These sets were selected from multiple candidates based on preliminary assessment of reaction efficiency and primer-dimer effects. FluA2 contains an equal mixture of two probes, to ensure optimum homology with viruses within both clades. The probes are labeled with FAM (6-carboxyfluorescein) and Blackhole Quencher™ 1 (BHQTM1). The BHQ chemistry is designed to minimize non-specific fluorescence. Experimental efficiency values of 100% +/- 5% are considered optimal. Tagman reaction efficiencies of FluA2 and FluA3 sets were demonstrated to be 100.6% (R2=0.996) and 100.3% (R2=0.996).
Influenza A/H5N1 positive control (500 µL of virus suspended in 0.01 M PBS): a reverse-engineered vaccine candidate virus that may safely be handled in BSL-2. Inactivated with beta- propiolactone (0.05%). The reassortant virus is noninfectious in chickens and USDA has removed it from the select agent list, classifying it as a BSL2 infectious agent. Additionally, the inactivated virus preparation is non-infectious in embryonated chicken eggs.

Other reagents or accessories required to perform testing with the device are:

Qiagen QuantiTect™ Probe RT-PCR Kit (Qiagen), a master-mix for reversetranscription and cDNA amplification.
Materials for extraction and purification of specimens (three commercial extraction kits are suggested for use in isolating RNA in the test procedure based on specifications meeting the requirements for quality of RNA to be used in the FluA2 and FluA3 reactions). These extraction procedures were used during development and testing of the Influenza A/H5 (Asian lineage) Virus Real-time RT-PCR Primer and Probe Set.
An RNase P (RP) Real-time PCR Primer and probe set that targets the human ribonuclease P (RP) sequence. Extracted clinical specimens should contain human RNA. The RP assay thus serves as a control to ensure RNA resulted from the extraction of clinical specimens.

AI/ML Overview

1. A table of acceptance criteria and the reported device performance

The document does not explicitly state pre-defined acceptance criteria for device performance. However, based on the reproducibility study results, the implied criteria for positive detection (presumptive positive) appear to relate to the virus concentration and agreement with expected results. For negative samples, the acceptance criterion is a 100% agreement with expected results or near 100% given the one equivocal observation.

Performance Metric	Acceptance Criteria (Implied)	Reported Device Performance
Reproducibility (Positive Samples)	High agreement with expected positive results at various concentrations, especially at higher concentrations. Specific thresholds are not explicitly stated, but generally, high accuracy is expected.	Overall (across all instruments): - 400 TCID50/mL: All 36 samples detected (100%) - 40 TCID50/mL: 47/55 (85.5%) positive; 52/55 (94.5%) positive or equivocal - 4 TCID50/mL: 32/36 (88.9%) positive; 35/36 (97.2%) positive or equivocal Specific Instrument Performance (Agreement with Expected Results for Positive Samples): - ABI 7000: 100% at 400, 40, and 4 TCID50/mL (Swab, low 0.4 TCID50/mL: 10% positive, 30% equivocal, 70% negative) - ABI 7700: 100% at 400, 40, and 4 TCID50/mL (Swab, low 0.4 TCID50/mL: 0% positive, 28.5% equivocal, 71.5% negative) - LightCycler®: 100% at 400 TCID50/mL, 78% at 40 TCID50/mL, 89% at 40 TCID50/mL (liquid), 67% at 4 TCID50/mL - SmartCycler®: 100% at 400 TCID50/mL, 67% at 40 TCID50/mL, 100% at 40 TCID50/mL (liquid), 90% at 4 TCID50/mL
Reproducibility (Negative Samples)	High agreement with expected negative results (e.g., 100% negative).	Overall (across all instruments): 71 negative control samples had 1 equivocal result, yielding 98.6% agreement with expected results. Specific Instrument Performance (Agreement with Expected Results for Negative Samples): - ABI 7000: 100% for both liquid and swab negative samples - ABI 7700: 100% for both liquid and swab negative samples - LightCycler®: 100% for liquid negative, 89% for swab negative (due to 1 equivocal) - SmartCycler®: 100% for both liquid and swab negative samples
Detection Limit	Not explicitly stated as a criterion, but values are given as a performance characteristic.	- ABI 7000 and ABI 7700: 4 TCID50/mL for each target - LightCycler® and SmartCycler®: 40 TCID50/mL for each target
Analytical Specificity (Cross-reactivity)	No cross-reactivity with non-H5 Asian HA types, non-influenza respiratory viruses, or bacteria.	No cross-reactivity observed with the tested viral strains or bacterial species (17 stock strains of non-H5 influenza, 8 non-influenza respiratory viruses, 11 bacterial species).
Purity of Virus Control	Non-infectious	The inactivated virus control is non-infectious in chickens and embryonated chicken eggs.
Clinical Sensitivity	Not estimable due to rarity of Asian H5 virus infections.	Not estimated.
Clinical Specificity	Not estimable due to rarity of Asian H5 virus infections.	Not estimated.
Agreement with Lab-Confirmed Cases (H5N1)	High agreement with confirmed patient diagnosis.	10/22 (45%) specimens were presumptive positive, and another 9 were equivocal. Thus, 19/22 (86%, 65.1-97.1% CI) specimens showed A/H5 (Asian lineage) virus detection in one or both probe-set reactions, agreeing with the patient case diagnosis (from 10 patients). All lab-confirmed cases had at least one positive test when multiple specimens were tested.
Agreement with Banked Respiratory Specimens (Negative)	High agreement with expected negative results.	Overall agreement was 98.5% (201/204, 95.8-99.7% CI). One sample was positive (confirmed H5N1), and two were equivocal (contained A/H3).
Agreement with Banked Tissue Culture Samples (Negative)	High agreement with expected negative results.	Agreement was 95.2% (20/21, 76.2-99.9% CI). One sample was equivocal (contained A/H3).

2. Sample size used for the test set and the data provenance (e.g. country of origin of the data, retrospective or prospective)

Reproducibility Study (Simulated Specimens):
- Test Set Size: 6 simulated respiratory specimens (3 swabs, 3 liquid) at various virus concentrations (including negative), tested in 10 laboratories (ABI 7000: 60 tests), 7 laboratories (ABI 7700: 42 tests), 9 laboratories (LightCycler®: 54 tests), and 10 laboratories (SmartCycler®: 60 tests).
- Total Samples Tested: 216 tests distributed across the instruments. Each simulated specimen type was likely tested multiple times within each lab or across labs.
- Data Provenance: The simulated specimens were "spiked with both human cellular material and titered amounts of the virus control material." The control virus itself is a "reverse-engineered vaccine candidate virus." This indicates contrived samples, likely prepared in a laboratory setting (e.g., CDC). This is a prospective study design for performance evaluation.
Clinical Study (Laboratory-confirmed patient cases):
- Test Set Size: 27 specimens from 10 laboratory-confirmed H5N1 patients and 2 specimens from 1 contact patient (not confirmed for H5N1).
- Data Provenance: Retrospective. Specimens were from "laboratory-confirmed patient cases (from Indonesia) of influenza H5N1 virus."
Clinical Study (Banked human respiratory specimens):
- Test Set Size: 204 banked human respiratory specimens.
- Data Provenance: Retrospective. Specimens were received by CDC Influenza Branch Laboratory from Vietnam (168), United States (21), Thailand (11), Kuwait (4).
Clinical Study (Banked tissue culture samples):
- Test Set Size: 21 banked tissue culture samples.
- Data Provenance: Retrospective. Received by CDC Influenza Branch Laboratory from international locations (18) and the United States (3).

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts

Reproducibility Study: No human experts were involved in establishing ground truth for the simulated test set. The ground truth was based on the known concentration of the spiked virus and the expected negative status of non-spiked samples.
Clinical Study (Laboratory-confirmed patient cases):
- Number of Experts: Not explicitly stated, but "Confirmation by independent (WHO or Indonesian) laboratory or sequence analysis" indicates multiple experts and methodologies were involved. "Multiple WHO methods (culture, sequencing, RT-PCR testing for multiple HA targets) were used to characterize H5N1 in these patients."
- Qualifications: "National influenza surveillance experts" and staff in "WHO-recognized RT-PCR assays" and the "WHO Collaborating Center for Surveillance, Epidemiology and Control of Influenza" are implied. These would be highly qualified virologists, microbiologists, and molecular biologists.
Clinical Study (Banked human respiratory specimens):
- Number of Experts: Unspecified, but confirmed by "Influenza Branch4 Lab Real-time RT-PCR Panel Result." This refers to the CDC's Influenza Branch Laboratory, a WHO Collaborating Center, implying a team of experts.
- Qualifications: Experts at a "WHO Collaborating Center for Surveillance, Epidemiology and Control of Influenza" and a "Bioterrorism Rapid Response and Advanced Technology Laboratory" would be highly qualified specialists in virology, molecular diagnostics, and public health.
Clinical Study (Banked tissue culture samples):
- Number of Experts: Unspecified, but "Influenza Branch3 Lab Real-time RT-PCR Panel Result" implies experts from the CDC Influenza Branch Laboratory.
- Qualifications: Similar to the above, highly qualified specialists in virology and molecular diagnostics.

4. Adjudication method (e.g. 2+1, 3+1, none) for the test set

Reproducibility Study: No adjudication method was necessary for the simulated test set, as the ground truth was based on engineered virus concentrations.
Clinical Studies (Laboratory-confirmed patient cases, Banked human respiratory specimens, Banked tissue culture samples):
- The term "adjudication" in the sense of resolving discrepancies between multiple readers is not directly applicable. For patient cases, confirmation was achieved through "independent (WHO or Indonesian) laboratory or sequence analysis" and "Multiple WHO methods (culture, sequencing, RT-PCR testing for multiple HA targets)." This suggests a consensus-based approach or reconciliation of results by reference methods rather than an explicit "X+Y" adjudication.
- For banked specimens, the "Influenza Branch Lab Real-time RT-PCR Panel Result" serves as the reference standard, implying a thorough process by a specialized laboratory.

5. If a multi reader multi case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance

There was no MRMC comparative effectiveness study done.
This device is a diagnostic kit (RT-PCR primer and probe set), not an AI or imaging device that would typically involve human "readers" in the sense of interpreting outputs that AI could aid. The output is a quantitative (CT value) and qualitative (positive/equivocal/negative) result based on amplification.

6. If a standalone (i.e. algorithm only without human-in-the-loop performance) was done

Yes, the performance studies primarily assess the standalone performance of the diagnostic kit. The "reproducibility study" evaluates the kit's performance across different laboratories and instruments, and the "clinical studies" evaluate its ability to detect the virus in patient and banked samples, independent of human interpretation improvements.
While laboratory technicians operate the equipment, the interpretation of the RT-PCR results (based on crossing thresholds and specific target detection) follows established protocols without a human "in-the-loop" decision-making process in the same way an AI-assisted diagnostic device might be evaluated. The result is directly derived from the reaction.

7. The type of ground truth used (expert consensus, pathology, outcomes data, etc)

Reproducibility Study: Engineered/Known Concentration. The ground truth was based on the known, spiked concentrations of the Influenza A/H5N1 virus into simulated respiratory specimens.
Clinical Study (Laboratory-confirmed patient cases): Multi-method Laboratory Confirmation / Consensus. The ground truth was established by "independent (WHO or Indonesian) laboratory or sequence analysis" and "Multiple WHO methods (culture, sequencing, RT-PCR testing for multiple HA targets)." This represents a form of expert-driven, multi-modal laboratory confirmation, akin to an "expert consensus" based on advanced diagnostic techniques.
Clinical Studies (Banked human respiratory specimens and tissue culture samples): Reference Laboratory Panel Result. The ground truth was established by the "Influenza Branch Lab Real-time RT-PCR Panel Result" at CDC (a WHO Collaborating Center). This acts as a highly reliable reference standard, representing the most definitive laboratory diagnosis available.

8. The sample size for the training set

The document describes a diagnostic kit (reagents), not an AI or machine learning model. Therefore, there is no specific "training set" in the conventional sense of data used to train an algorithm.
The "development" of the primer and probe sets involved selecting them from "multiple candidates based on preliminary assessment of reaction efficiency and primer-dimer effects." This involved testing, but it's part of the assay design and optimization, not machine learning model training.

9. How the ground truth for the training set was established

As stated above, there is no "training set" for an algorithm in this context. The development process for the primer and probe sets involved analytical testing with known viral strains and concentrations to ensure specificity and efficiency, which inherently relies on established knowledge of viral genetics and laboratory standards (e.g., known positive controls, established culture methods).

Summary

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510(k) DENOVO DECISION SUMMARY

A. 510(k) Number: K060159

B. Purpose for Submission: Marketing authorization of new device
C. Measurand: Novel influenza A/H5 ribonucleic acid (RNA)
D. Type of Test: Qualitative real-time RT-PCR
E. Applicant: Centers for Disease Control and Prevention

F. Proprietary and Established Names:

Influenza A/H5 (Asian lineage) Virus Real-time RT-PCR Primer and Probe Set

Reagents for Detection of Specific Novel Influenza A Virus Reagents

G. Regulatory Information:

1. Regulation section: 21 CFR 866.3332 Reagents for Detection of Specific Novel Influenza A viruses
1. Classification: Class II (de novo)
1. Product code: NXD, (nucleic acid amplification, novel influenza A virus, A/H5 RNA)
1. Panel: 83 (Microbiology)

H. Intended Use:

1. Intended use(s):
  The Influenza A/H5 (Asian Iineage) Virus Real-time RT-PCR Primer and Probe Set is intended for the in vitro qualitative detection of Influenza A/H5 (Asian lineage) virus RNA either directly in patient respiratory specimens or in viral cultures for the presumptive laboratory identification of Influenza A/H5 (Asian lineage) virus.

Providing epidemiological information for the surveillance of human infection with 1. Influenza A/H5 (Asian lineage) virus

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Identifying patients who may be infected with Influenza A/H5 (Asian lineage) virus 2. based on clinical and epidemiological risk factors
The definitive identification of influenza A/H5 (Asian lineage) either directly from patient specimens or from viral cultures requires additional laboratory testing, along with clinical and epidemiological assessment in consultation with national influenza surveillance experts.

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

Use of this assay is limited to Laboratory Response Network (LRN) designated laboratories.

1. Indication(s) for use: Same as intended use.
1. Special conditions for use statement(s): Restricted Device
1. Special instrument requirements:

Real-time PCR instrumentation (Roche LightCycler®, Cepheid SmartCycler® Applied Biosystems ABI 7000 Sequence Detection System or ABI Prism® 7700 Sequence Detection) along with accessory supplies and lab equipment recommended for each instrument system.

I. Device Description:

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highly pathogenic viruses of the Asian lineage.

. Primers/Probes: The 2 primer and probe sets (FluA2 and FluA3), each target a different region within the HA gene. The FluA2 target is in the HA2 region of the HA gene and the FluA3 target spans the cleavage site of the HA gene. These sets were selected from multiple candidates based on preliminary assessment of reaction efficiency and primer-dimer effects. FluA2 contains an equal mixture of two probes, to ensure optimum homology with viruses within both clades. The probes are labeled with FAM (6-carboxyfluorescein) and Blackhole Quencher™ 1 (BHQTM1). The BHQ chemistry is designed to minimize non-specific fluorescence. Experimental efficiency values of 100% +/- 5% are considered optimal. Tagman reaction efficiencies of FluA2 and FluA3 sets were demonstrated to be 100.6% (R2=0.996) and 100.3% (R2=0.996).
Influenza A/H5N1 positive control (500 µL of virus suspended in 0.01 M PBS): . a reverse-engineered vaccine candidate virus that may safely be handled in BSL-2. Inactivated with beta- propiolactone (0.05%). The reassortant virus is noninfectious in chickens and USDA has removed it from the select agent list, classifying it as a BSL2 infectious agent. Additionally, the inactivated virus preparation is non-infectious in embryonated chicken eggs.

Other reagents or accessories required to perform testing with the device are:

. Qiagen QuantiTect™ Probe RT-PCR Kit (Qiagen), a master-mix for reversetranscription and cDNA amplification.
Materials for extraction and purification of specimens (three commercial . extraction kits are suggested for use in isolating RNA in the test procedure based on specifications meeting the requirements for quality of RNA to be used in the FluA2 and FluA3 reactions). These extraction procedures were used during development and testing of the Influenza A/H5 (Asian lineage) Virus Real-time RT-PCR Primer and Probe Set.
An RNase P (RP) Real-time PCR Primer and probe set that targets the human . ribonuclease P (RP) sequence. Extracted clinical specimens should contain human RNA. The RP assay thus serves as a control to ensure RNA resulted from the extraction of clinical specimens.

J. Substantial Equivalence Information:

1. Predicate device name(s): None.
1. Predicate 510(k) number(s): None.
1. Comparison with predicate: Not applicable.

There are other devices that generally detect Influenza A viruses, however, there is no identifiable predicate that detects specific novel Influenza A viruses. There are other IVD devices that use probe/primer sets for the detection of other specific pathogenic

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microorganisms, and that rely on real-time PCR methods for their principles of operation.

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

A special controls guidance document will be promulgated.
CDRH Draft Guidance for Industry and FDA Staff titled: Nucleic Acid Based In Vitro Diagnostic Devices for Detection of Microbial Pathogens, December 8, 2005.

L. Test Principle:

Human respiratory specimens (nasopharyngeal swabs or aspirates, throat swabs, nasal swabs, or liquefied lower respiratory samples) from symptomatic patients, and viral cultures must be processed initially to extract and purify RNA from the cellular specimen matrix, using general purpose reagents.

Each extracted RNA sample is reacted separately with each primer and probe set in a single-tube real-time RT-PCR reaction. After initial reverse transcription of RNA into cDNA, amplification cycles proceed during which the fluorogenic probes, (each duallabeled with a reporter dye (FAM) attached to the 5' end and a quencher dye attached at or near the 3' end), anneal to specific target sequences located between the forward and reverse primers. During the extension phase of the PCR cycle, the 5' nuclease activity of Taq polymerase degrades the probe, causing the reporter dye to separate from the quencher dye, generating a fluorescent signal. With each cycle, additional reporter dye molecules are cleaved from their respective probes, increasing fluorescence intensity. Fluorescence intensity is monitored at each PCR cycle by fluorimeter modules within specified real-time instruments.

If both of the H5 HA targets (FluA2 and FluA3) are detected, testing is presumptive positive for Influenza A/H5 (Asian lineage) virus. When only one or the other of the targets is detected, results are reported as equivocal for Influenza A/H5 (Asian lineage) virus. Testing is negative if neither target is detected. A positive RP target test assures that human cellular material was extracted and that the PCR chemistry is unaffected by inhibition. All presumptive positive and equivocal results are subjected to additional testing procedures to definitively identify an influenza A/H5 (Asian lineage) virus.

M. Performance Characteristics (if/when applicable):

1. Analytical performance:

a. Precision/Reproducibility:

At two different times, 6 simulated respiratory specimens (3 swabs and 3 liquid) that were spiked with both human cellular material and titered amounts of the virus control material were distributed to multiple laboratories. These samples were tested in 10 laboratories using the ABI 7000, 7 laboratories using ABI 7700, 9

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laboratories using LightCycler®, and 10 laboratories using SmartCycler® Real-time PCR instrument systems.

With these contrived samples that contained levels of virus at the lower end of the levels that have been recovered from respiratory specimens from human cases (10 -10° TCIDs(mL), all 36 samples (tested in different laboratories, using the 4 different real-time PCR instruments) containing 400 TCID50/mL, and the majority of samples containing 40 TCID50/mL were detected (47/55 or 85.5% of these samples were positive; 52/55 or 94.5% were positive or equivocal, with 3 negative for both targets). At the 4 TCID50 level, 32/36 or 88.9% were positive (both targets), while 35/36, or 97.2% were positive in at least one target (positive or equivocal). Of the 72 negative control samples, 1 was equivocal for an agreement with expected results of 98.6%. Breakouts for each instrument by sample level are below.

Combined laboratory results for the different instruments are shown below:

Summary of Reproducibility Study by 10 LRN Laboratories using the ABI7000
Simulatedspecimentype	VirusconcentrationTCID50/mL¹	Influenza A/H5 (Asian lineage) Virus Real-time RT-PCRPrimer and Probe Set Result			Agreement withexpected results
		Presumptivepositive	Equivocal	Negative
Liquid, mod	400	10	-	-	100% (10/10)
Swab, mod	40	10	-	-	100% (10/10)
Liquid, low	4	10	-	-	100% (10/10)
Swab, low	0.4	1	2	7	10% (1/10)
Liquid, neg	none	-	-	10	100% (10/10)
Swab, neg	none	-	-	10	100% (10/10)
Overall agreement with expected result 85% (51/60)

Virus concentration (TCIDs(mL) was extrapolated for each simulated specimen by comparing RT-PCR crossing threshold (Ct) values to a standard curve prepared by testing serial dilutions of an Influenza A/H5N1 virus of a known concentration (TCID50/mL). TCID30/mL was determined using Madin-Darby canine kidney (MDCK) cells.

Summary of Reproducibility Study by 7 LRN Laboratories using the ABI7700
Simulatedspecimentype	VirusconcentrationTCID50/mL1	Influenza A/H5 (Asian lineage) Virus Real-time RT-PCRPrimer and Probe Set Result			Agreement withexpected results
		Presumptivepositive	Equivocal	Negative
Liquid, mod	400	7	-	-	100% (7/7)
Swab, mod	40	7	-	-	100% (7/7)
Liquid, low	4	7	-	-	100% (7/7)
Swab, low	0.4	-	2	5	0% (0/7)
Liquid, neg	none	-	-	7	100% (7/7)
Swab, neg	none	-	-	7	100% (7/7)
		Overall agreement with expected result			83% (35/42)

Virus concentration (TCIDs(mL) was extrapolated for each simulated specimen by comparing RT-PCR crossing threshold (Ct) values to a standard curve prepared by testing serial dilutions of an Influenza A/H5N1 virus of a known concentration (TCID50/mL). TCID50/mL was determined using Madin-Darby canine kidney (MDCK) cells.

1 Ng EKO, Cheng PKC, Ng AYY, Hoang TL, Lim WWL,Jnfluenza A H5N1 DetectionEmerg Infect Dis. 2005 Aug 25,2005

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Summary of Multicenter Reproducibility Study by 9 LRN Laboratoriesusing the LightCycler®
Simulatedspecimentype	VirusconcentrationTCID50/mL1	Influenza A/H5 (Asian lineage) Virus Real-time RT-PCRPrimer and Probe Set Result			Agreement withexpected results
		Presumptivepositive	Equivocal	Negative
Liquid, mod	400	9	-	-	100% (9/9)
Swab, mod	40	7	1	1	78% (7/9)
Liquid, low	40	8	-	1	89% (8/9)
Swab, low	4	6	2	1	67% (6/9)
Liquid, neg	none	-	-	9	100% (9/9)
Swab, neg	none	-	1	8	89% (8/9)

Overall agreement with expected result | 87% (47/54)

1 Virus concentration (TCID30 mL) was extrapolated for each simulated specimen by comparing RT-PCR crossing threshold (Ct) values to a standard curve prepared by testing serial dilutions of an Influenza A/H5N1 virus of a known concentration (TCLDs/mL). TCID50/mL was determined using Madin-Darby canine kidney (MDCK) cells

Summary of Multicenter Reproducibility Study by 10 LRN Laboratories using the SmartCycler®

using the SmartCycler
Simulatedspecimentype	VirusconcentrationTCID50/mL1	Influenza A/H5 (Asian lineage) Virus Real-time RT-PCRPrimer and Probe Set Result			Agreement withexpected results
		Presumptivepositive	Equivocal	Negative
Liquid, mod	400	10	-	-	100% (10/10)
Swab, mod	40	6	3	1	67% (6/10)
Liquid, low	40	9	1	-	100% (9/10)
Swab, low	4	9	1	-	90% (9/10)
Liquid, neg	None	-	-	10	100% (10/10)
Swab, neg	None	-	-	10	100% (10/10)
Overall agreement with expected result					90% (54/60)

Virus concentration (TCID39/ml) was extrapolated for each simulated specimen by comparing RT-PCR crossing threshold (Ct) values to a standard curve prepared by testing serial dilutions of an Influenza A/H5N1 virus of a known concentration (TCIDs) mL).

TCID50/mL was determined using Madin-Darby canine kidney (MDCK) cells

TCID36/mL was determined using Madin-Darby canine kidney (MDCK) cells
2 RP reaction was not performed on one of the ten samples. Probe reactions for this sample were negative

b. Linearity/assay reportable range: NA

c. Traceability, Stability, Expected values (controls, calibrators, or methods):
The Influenza A/H5N1 (Asian lineage) Virus Real-time RT-PCR Positive Control is expected to yield a crossing threshold within 25-35 cycles.
d. Detection limit:
The detection level using the ABI 7000 and ABI 7700 instrument system is approximated to be 4 TCID50/mL in MDCK cells for each target (with 10-fold dilutions and up to 5 replicates). The detection level using the LightCycler®, or SmartCycler® instrument system is approximated to be 40 TCID50/mL in MDCK cells for each target.

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e. Analytical specificity:
Cross-reactivity with non-H5 Asian HA types (17 stock strains from freshly grown culture lysates including representative non-H5s, two H7s and one H9). All subtypes that are known to infect humans, either sporadically or commonly are represented except for H7N3. Non-reactivity with H7N3 is presumed because of similarity of this H7 with H7N7 that was tested.

Note: high viral titers would be expected with confluent tissue cultures (>10E7 TCID50 per the WHO manual).

Additional cross-reactivity with non-influenza respiratory viruses and bacteria (8 viruses tested are broadly representative of other known respiratory viruses; 11 broadly representative bacterial species). Sequence mining including updated BLAST searches reduces the potential crossreactivity for known bacteria and viruses.

No cross-reactivity with the tested viral strains or bacterial species was observed.

f. Assay cut-off:
PCR reactions are run for 45 cycles. Growth curves with crossing thresholds within 45 cycles with either the FluA2 or FluA3 targets (when controls are acceptable) are considered reactive. The FluA2 and FluA3 reactions should both have crossing thresholds when Asian H5 (Asian lineage) hemagglutinin (HA) viral RNA is present. An equivocal result (only one of the two target reactions positive) may be due to contamination, failure of one primer/probe set to react, partial or incomplete inhibition, or non-specific reactivity. Both presumptive positive and equivocal results are subject to additional testing for definitive identification. The approach with 2 targets optimizes detection capability for influenza A/H5 (Asian lineage) viruses.
1. Comparison studies: NA
1. Clinical studies:
- a. Clinical Sensitivity: Sensitivity could not be estimated due to rarity of Asian H5 (Asian lineage) virus infections.
- Clinical specificity: Specificity could not be estimated due to rarity of Asian b. H5 (Asian lineage) virus infections.
- c. Other clinical supportive data (when a. and b. are not applicable):

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Testing with the device on respiratory specimens from laboratory-confirmed patient cases (from Indonesia) of influenza H5N1 virus is shown below.

		Laboratory-confirmed cases1(n= 10 patients2; 27 specimens3)	Contact patient(Not laboratory confirmed)(1 patient; 2 specimens)
SpecimenType	Influenza A/H5 (Asian lineage)Primer/probe Result			Influenza A/H5 (Asianlineage)Primer/probe Result
	PresumptivePositive(FluA2 andFluA3)	Equivocal(FluA2 orFluA3)	Negative	PresumptivePositive orEquivocal	Negative
Throat Swab	7	1	0	0	1
Nasal Swab	1	5	2	0	1
Other4	2	3	1	0	0
Total	10	9	3	0	2

Clinical Case Results

1 Confirmation by independent (WHO or Indonesian) laboratory or sequence analysis

2 Patient ages: ≤ 4 years (2); 5-18 years (2); 19-40 years (7)

3 Lung biopsy (1), pleural fluid (2), rectal swab (1) and serum (1) specimen results excluded

4 Includes broncho suctions (3), lung aspirate (1), pharyngeal aspirate (1) and endotracheal wash (1)

No influenza A/H5 (Asian lineage) virus was detected in the samples from the one contact case by a WHO laboratory or by additional testing done at CDC.

For the 10 laboratory-confirmed H5N1 patient cases: 10/22 (45%, 24.4-67.8%) specimens were presumptive positive for the virus, while another 9 were equivocal. Thus 19/22 specimens (86%, 65.1-97.1%) had A/H5 (Asian lineage) virus detected in one or both of the probe-set reactions, agreeing with the patient case diagnosis. All lab-confirmed cases had at least one positive test when multiple specimens were tested (all patients contributed at least 2 samples, usually at least a throat and a nasal swab). Multiple WHO methods (culture, sequencing, RT-PCR testing for multiple HA targets) were used to characterize H5N1 in these patients.

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Testing with the device on banked respiratory specimens from individuals with influenza-like illness is shown below.

BRRAT3 LabInfluenza A/H5Primer/ ProbeResult	Influenza Branch4 LabReal-time RT-PCR Panel Result
	InfluenzaA/H1Positive	InfluenzaA/H3Positive	InfluenzaA/H5Positive	No Influenzadetected
PresumptivePositive	0	0	1	0
Equivocal	0	2	0	0
Negative	5	75	0	121

Banked1 Human Respiratory2 Specimen Results

Specimens received by CDC Influenza Branch Laboratory from Vietnam (168), United States (21), Thailand (11), Kuwait (4)

² Throat swab (171), nasopharyngeal swab (26), pharyngeal swab (2), oropharyngeal aspirate (2), nasal wash (1), and endotracheal aspirate (1)

3 Bioterrorism Rapid Response and Advanced Technology Laboratory at CDC

4 Influenza Branch Laboratory at CDC serves as the World Health Organization (WHO) Collaborating Center for Surveillance, Epidemiology and Control of Influenza and is one of four regional WHO collaborating centers and reference laboratories involved in surveillance and characterization of circulating influenza viruses around the world.

Overall agreement with an expected negative result was 98.5% (201/204), 95.8-99.7% (95% CI). One sample positive in both FluA2 and FluA3 was characterized by a panel of WHO-recognized RT-PCR assays as H5N1. The other non-negative samples were equivocal (reactive in only one of the primer/probe assays). Additional testing showed these samples contained A/H3 virus. Testing with the device on banked respiratory specimens from tissue culture samples is shown below.

Banked1 Tissue Culture Sample Results

BRRAT2 LabInfluenza A/H5Primer/ ProbeResult	Influenza Branch3 LabReal-time RT-PCR Panel Result
	InfluenzaA/H3Positive	InfluenzaA/H5Positive	Influenza BPositive	No Influenzadetected
PresumptivePositive	0	0	0	0
Equivocal	1	0	0	0
Negative	7	0	1	12

Tissue cultures received by CDC Influenza Branch Laboratory from international locations (18), and United States (3)

2 Bioterrorism Rapid Response and Advanced Technology Laboratory at CDC

3 Influenza Branch Laboratory at CDC serves as the World Health Organization (WHO) Collaborating Center for Surveillance, Epidemiology and Control of Influenza

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Agreement with the expected negative result was 95.2% (20/21), 76.2-99.9% (95% CI).

1. Clinical cut-off: NA
1. Expected values/Reference range:

Currently, influenza A/H5 (Asian lineage) virus has not been detected in poultry or humans in the United States. False-positive results are more likely to occur when disease prevalence in the community is low. Testing of patient specimens is indicated for symptomatic patients meeting the clinical criteria and epidemiologic risk for infection with influenza A/H5 (Asian lineage) virus .

N. Proposed Labeling:

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

O. Conclusion:

The petition for Evaluation of Automatic Class III Designation for this device is accepted. 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.

Regulation Number and Section

§ 866.3332 Reagents for detection of specific novel influenza A viruses.

(a)
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.(b)
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.