K Number

Device Name

CDC DENV-1-4 REAL TIME RT-PCT ASSAY

Manufacturer

CENTERS FOR DISEASE CONTROL AND PREVENTION

Date Cleared

2012-05-24

(73 days)

Product Code

OZB

Regulation Number

866.3946

Intended Use

The CDC DENV-1-4 Real-Time RT-PCR Assay is intended for use on an Applied Biosystems (ABI) 7500 Fast Dx Real-Time PCR Instrument: - For the diagnosis of dengue in serum or plasma collected from patients with signs and symptoms consistent with dengue (mild or severe) during the acute phase: - For the identification of dengue virus serotypes 1, 2, 3 or 4 from viral RNA in serum or plasma (sodium citrate) collected from human patients with dengue during the acute phase; - To provide epidemiologic information for surveillance of circulating dengue viruses.

Device Description

The CDC DENV-1-4 Real-Time RT-PCR Assay is a panel of oligonucleotide primers and dual-labeled hydrolysis (TaqMan®) probes designed to be utilized in real-time RT-PCR assays using the ABI 7500 Fast Dx Real-Time PCR instrument for detection of DENV-1, -2, -3 and -4 viral RNA in serum or plasma from human patients with dengue. The CDC DENV-1-4 Real-Time RT-PCR Assay includes control materials as described below: - . Positive control materials, which consist of heat-inactivated DENV-1 Haw, DENV-2 NGC, DENV-3 H87, and DENV-4 H241 viruses. - A Human Specimen Control (HSC), consisting of noninfectious (beta ● propiolactone inactivated) cultured human cell material that provides a positive signal in the assay and demonstrates successful recovery of RNA as well as the integrity of the RNA extraction reagents. - . 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. RNA extractions can be done using the Qiagen OIAamp® DSP Viral RNA Mini Kit either manually or in combination with the QIAcube Instrument (small centrifuge) and/or Roche MagNA Pure LC total nucleic acid isolation kit and Magna Pure Instrument. The RT-PCR assay is run on the Applied Biosystems® 7500 Fast Dx Real-Time PCR instrument using Invitrogen SuperScript™ III Platinum® One-Step Quantitative RT-PCR System. The assay can be run in singleplex (each DENV serotype detected in a separate reaction) or in multiplex (four DENV serotypes are run in the same reaction). The two formats provide equal sensitivity.

More Information

Contact

Type

Center

Panel

Date Received

Product Code

Subsequent Product Codes

Applicant Name (Manufacturer)

Device Name

Decision

Street 1

Street 2

City

State

Country Code

ZIP

Postal Code

Class Advise Committee

SSP Indicator

Third Party Reviewed

Expedited Review

Predicate Devices

Not applicable

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The device description and performance studies focus on standard molecular diagnostic techniques (RT-PCR) and do not mention any AI or ML components for data analysis or interpretation.

Therapeutic

This device is intended for the diagnosis of dengue and identification of dengue virus serotypes, which are diagnostic purposes, not therapeutic.

Diagnostic

Yes

Explanation: The "Intended Use / Indications for Use" section explicitly states that the assay is "For the diagnosis of dengue in serum or plasma collected from patients".

SaMD (Software as a Medical Device)

The device is a panel of oligonucleotide primers and probes, control materials, and relies on specific hardware (ABI 7500 Fast Dx Real-Time PCR Instrument, Qiagen QIAamp DSP Viral RNA Mini Kit, QIAcube Instrument, Roche MagNA Pure LC total nucleic acid isolation kit, Magna Pure Instrument, Invitrogen SuperScript III Platinum One-Step Quantitative RT-PCR System) for its function. It is a diagnostic assay kit, not a software-only device.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

Here's why:

Intended Use: The intended use explicitly states it's for the "diagnosis of dengue in serum or plasma collected from patients" and for the "identification of dengue virus serotypes... from viral RNA in serum or plasma... collected from human patients." This clearly indicates it's used to examine specimens derived from the human body to provide information for diagnostic purposes.
Device Description: The device is a panel of primers and probes designed to detect viral RNA in human serum or plasma using a real-time RT-PCR assay. This is a common method used in IVD tests for detecting pathogens.
Specimen Type: The device is intended for use with serum or plasma, which are human specimens.
Performance Studies: The document includes detailed performance studies (Reproducibility, LoD, Analytical Reactivity, Cross Reactivity, Interference, Carry-Over/Cross Contamination, Fresh vs. Frozen Specimens, Prospective Clinical Study, Retrospective Clinical Study) which are typical for the validation of an IVD device.
Key Metrics: The document provides key metrics like Positive Percent Agreement and Negative Percent Agreement, which are standard performance indicators for diagnostic tests.

Based on the definition of an In Vitro Diagnostic device (a device intended for use in the collection, preparation, and examination of specimens taken from the human body for the purpose of diagnosing disease or other conditions, including a determination of the state of health, in order to cure, mitigate, treat, or prevent disease or its sequelae), this device fits the description.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The CDC DENV-1-4 Real-Time RT-PCR Assay is intended for use on an Applied Biosystems (ABI) 7500 Fast Dx Real-Time PCR Instrument:

For the diagnosis of dengue in serum or plasma collected from patients . with signs and symptoms consistent with dengue (mild or severe) during the acute phase:
For the identification of dengue virus serotypes 1, 2, 3 or 4 from viral . RNA in serum or plasma (sodium citrate) collected from human patients with dengue during the acute phase;
. To provide epidemiologic information for surveillance of circulating dengue viruses.

Product codes

OZB, NSU

Device Description

The CDC DENV-1-4 Real-Time RT-PCR Assay includes control materials as described below:

. Positive control materials, which consist of heat-inactivated DENV-1 Haw, DENV-2 NGC, DENV-3 H87, and DENV-4 H241 viruses.
A Human Specimen Control (HSC), consisting of noninfectious (beta ● propiolactone inactivated) cultured human cell material that provides a positive signal in the assay and demonstrates successful recovery of RNA as well as the integrity of the RNA extraction reagents.
. 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.

RNA extractions can be done using the Qiagen OIAamp® DSP Viral RNA Mini Kit either manually or in combination with the QIAcube Instrument (small centrifuge) and/or Roche MagNA Pure LC total nucleic acid isolation kit and Magna Pure Instrument.

The RT-PCR assay is run on the Applied Biosystems® 7500 Fast Dx Real-Time PCR instrument using Invitrogen SuperScript™ III Platinum® One-Step Quantitative RT-PCR System. The assay can be run in singleplex (each DENV serotype detected in a separate reaction) or in multiplex (four DENV serotypes are run in the same reaction). The two formats provide equal sensitivity.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Not Found

Indicated Patient Age Range

Not Found

Intended User / Care Setting

Not Found

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

Not Found

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Precision/Reproducibility:
Study Type: Reproducibility study
Sample Size: Not explicitly stated for total, but for each DENV serotype and sample type (moderate positive, low positive, high negative, negative), 60 tests were performed across three sites (20 tests per site).
Key Results:

DENV-1: Moderate Positive 100% agreement, Low Positive 96% agreement, High Negative 21% agreement, Negative 100% agreement.
DENV-2: Moderate Positive 100% agreement, Low Positive 95% agreement, High Negative 23% agreement, Negative 100% agreement.
DENV-3: Moderate Positive 100% agreement, Low Positive 100% agreement, High Negative 25% agreement, Negative 100% agreement.
DENV-4: Moderate Positive 100% agreement, Low Positive 95% agreement, High Negative 23% agreement, Negative 100% agreement.

Limit of Detection (LoD):
Study Type: Analytical performance study
Sample Size: 5 replicas per dilution for each virus strain (DENV-1, -2, -3, -4) in serum and plasma.
Key Results: The observed LoD for both Singleplex and Multiplex assays in serum and plasma was 1 x 10 pfu/mL.

Analytical Reactivity:
Study Type: Analytical performance study
Sample Size: Triplicate samples for each of 29 Dengue-1-4 isolates at 10^3 and 10^2 pfu/mL dilutions.
Key Results: The observed LoD of the assay was similar in cultured isolates obtained from patients from different countries.

Analytical Specificity (Cross Reactivity):
Study Type: Analytical performance study
Sample Size: Triplicate samples for 12 organisms (viruses and bacteria).
Key Results: No cross reactivity was observed with any panel member tested at clinically significant concentrations. All negative results were obtained for all triplicate samples.

Interference studies:
Study Type: Analytical performance study
Sample Size: Three tests per viral dilution (1:10 higher than LoD, equal to LoD, 1:10 below LoD) for each potential interfering substance (bilirubin, cholesterol, hemoglobin, triglycerides, genomic DNA) in normal human serum.
Key Results: No significant interference was observed at the tested levels.

Carry-Over/Cross Contamination:
Study Type: Analytical performance study
Sample Size: 8 replica sets of DENV-1, -2, -3, -4 (8 high-positive and 8 high-negative replicas) were tested in an alternating series.
Key Results: All results were as expected (32/32 negative samples tested negative, 32/32 positive samples tested positive).

Fresh vs. Frozen Specimens:
Study Type: Comparison study
Sample Size: Triplicate sets of moderate and low positive dilutions of spiked human serum samples, subject to 5 freeze/thaw cycles.
Key Results: 100% qualitative agreement between initial results and results after each freeze/thaw cycle.

Clinical Performance (Prospective Studies):
Study Type: Prospective clinical study
Sample Size: 86 serum samples.
Key Results:

Positive Percent Agreement: 97.92% (47/48) with 95% CI (89.10% – 99.63%) compared to CDC molecular detection method followed by sequencing.
Negative Percent Agreement: 100% (38/38) with 95% CI (90.82% – 100%) compared to CDC molecular detection method followed by sequencing.
One sample negative by CDC DENV-1-4 Real-Time RT-PCR Assay was positive for DENV-3 by bi-directional sequencing.

Clinical Performance (Retrospective Studies):
Study Type: Retrospective clinical study
Sample Size: 371 serum samples.
Key Results:

Positive Percent Agreement: 98.04% (100/102) with 95% CI (93.13% - 99.46%) compared to IgM anti-DENV seroconversion.
Negative Percent Agreement: 98.51% (265/269) with 95% CI (96.24% - 99.42%) compared to IgM anti-DENV seroconversion.
Two samples negative by the CDC-DENV-1-4 Real-Time RT-PCR Assay were confirmed positive by sequencing.
Four samples showed positive RT-PCR results but were not confirmed by IgM anti-DENV seroconversion; these were confirmed by E gene bi-directional sequencing.

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

Prospective Studies:
Positive Percent Agreement: 97.92% (47/48)
Negative Percent Agreement: 100% (38/38)

Retrospective Studies:
Positive Percent Agreement: 98.04% (100/102)
Negative Percent Agreement: 98.51% (265/269)

Predicate Device(s)

Not applicable

Reference Device(s)

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information

Not Found

Regulation Number and Section

§ 866.3946 Dengue virus nucleic acid amplification test reagents.

(a)
Identification. Dengue virus nucleic acid amplification test reagents are devices that consist of primers, probes, enzymes, and controls for the amplification and detection of dengue virus serotypes 1, 2, 3, or 4 from viral ribonucleic acid (RNA) in human serum and plasma from individuals who have signs and symptoms consistent with dengue (mild or severe). The identification of dengue virus serotypes 1, 2, 3, or 4 in human serum and plasma (sodium citrate) collected from human patients with dengue provides epidemiologic information for surveillance of circulating dengue viruses.(b)
Classification. Class II (special controls). The special control is FDA's guideline entitled “Class II Special Controls Guideline: Dengue Virus Nucleic Acid Amplification Test Reagents.” For availability of the guideline document, see § 866.1(e).

Summary

510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

A. 510(k) Number: K113336
B. Purpose for Submission: New device
C. Measurand: Dengue Virus (DENV) RNA target sequence
D. Type of Test: An in vitro molecular diagnostic test that consists of a panel of oligonucleotide primers and dual-labeled hydrolysis (TaqMan®) probes for the qualitative detection of dengue virus target sequences in serum and plasma using nucleic acid isolation, amplification, and detection on the ABI 7500 Fast Dx Real-Time PCR instrument.
E. Applicant: Centers for Disease Control and Prevention (CDC)
F. Proprietary and Established Names: CDC DENV-1-4 Real-Time RT-PCR Assay. Common Name: Dengue Real-Time RT-PCR

G. Regulatory Information:

| 1. Regulation section: | 21 CFR 866.3946: Dengue Virus Nucleic Acid
Amplification Test Reagents |
|------------------------|---------------------------------------------------------------------------|
| 2. Classification: | Class II (de novo) |
| 3. Product code: | OZB, NSU |
| 4. Panel: | Microbiology (83) |

H. Intended Use:

1. Intended use(s):
  The CDC DENV-1-4 Real-Time RT-PCR Assay is intended for use on an Applied Biosystems (ABI) 7500 Fast Dx Real-Time PCR Instrument:
For the diagnosis of dengue in serum or plasma collected from patients . with signs and symptoms consistent with dengue (mild or severe) during the acute phase:
For the identification of dengue virus serotypes 1, 2, 3 or 4 from viral . RNA in serum or plasma (sodium citrate) collected from human patients with dengue during the acute phase;
. To provide epidemiologic information for surveillance of circulating dengue viruses.

Testing of clinical blood specimens (serum or plasma) with the CDC DENV-1-4 Real-Time RT-PCR Assay should not be performed unless the patient clinical and/or epidemiologic criteria for testing suspect dengue cases.

The CDC DENV-1-4 Real-Time RT-PCR Assay is not FDA cleared or approved for the screening of blood or plasma donors.

Negative results obtained with this test do not preclude the diagnosis of dengue and should not be used as the sole basis for treatment or other patient management decisions.

This device is for distribution to laboratories with personnel who have training and experience in standardized molecular diagnostic testing procedures and viral diagnosis, and appropriate biosafety equipment and containment

1. Indication(s) for use:
  Same as Intended Use
1. Special conditions for use statement(s):
  For in vitro diagnostic use
1. Special instrument requirements:

Extraction:

Roche MagNA Pure LC Total NA Kit and Magna Pure Instrument. a.
Qiagen QIAamp® DSP Viral RNA Mini Kit (Manually) and/or in b. combination with the QIAcube Instrument (small centrifuge)

PCR: Applied Biosystems® 7500 Fast Dx Real-Time PCR Assay instrument with System Sequence Detection 1.4 Software provided by manufacturer.

I. Device Description:

The CDC DENV-1-4 Real-Time RT-PCR Assay includes control materials as described below:

. Positive control materials, which consist of heat-inactivated DENV-1 Haw, DENV-2 NGC, DENV-3 H87, and DENV-4 H241 viruses.
A Human Specimen Control (HSC), consisting of noninfectious (beta ● propiolactone inactivated) cultured human cell material that provides a positive signal in the assay and demonstrates successful recovery of RNA as well as the integrity of the RNA extraction reagents.
. 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.

Device Components (Provided)

Box 1: Detection Kit (Primer and Probe Sets) and Package Insert/Instructions for Use Box 2: Positive Control Kit (A mix of heat inactivated DENV-1, -2, -3 and -4

standards)

Box 3: Human Specimen Extraction Control (HSC)

Ancillary Reagents (Not provided): The following is a list of ancillary reagents that are not supplied with the CDC DENV-1-4 Real-Time RT-PCR Assay. The Invitrogen and Roche products are included in CDC's reagent qualification program.

	Reagents	Quantity	Catalog No
rRT-PCR
Enzyme
Mastermix
Options	Invitrogen SuperScript™ III
Platinum® reactions One-Step
Quantitative RT-PCR System
(without Rox)*	100
reactions
500
reactions	11732-020

*The CDC DENV-1-4 Real-Time RT-PCR Assay test performance requires that only qualified ancillary reagent lots be used with the device. Any lots not specifically qualified by the CDC-Dengue Branch for use with the CDC DENV-1-4 Real-Time RT-PCR Assay are not valid for use with this device, and may affect device performance.

**Qiagen QIAamp® DSP Viral RNA Mini Kit and Qiagen QIAcube are produced under Good Manufacturing Practices (GMP).

Lots which are qualified under this process are communicated through a Qualified Reagent Lot List. This list is maintained by CDC and made available to the end users of the CDC DENV-1-4 Real-Time RT-PCR by including it with the Product Insert as well as through the Product Support mechanism identified in the Product Insert.

J. Substantial Equivalence Information:

1. Predicate device name(s): Not applicable
1. Predicate Numbers (s): Not applicable
1. Comparison with predicate: Not applicable

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

1. CLSI EP5: Evaluation of Precision Performance of Clinical Chemistry Devices- Second Edition, Villanova PA
1. CLSI EP7-A2: Interference Testing in Clinical Chemistry; Approved Guideline, 2nd Ed.
1. CLSI EP17-A: Protocols for Determination of Limits of Detection (2004).

Note: A Special Controls Guidance Document will be promulgated.

L. Test Principle:

The CDC DENV-1-4 Real-Time RT-PCR Assay is used in rRT-PCR on an ABI 7500 Fast Dx Real-Time PCR Instrument. The CDC DENV-1-4 Real-Time RT-PCR assay includes a set of oligonucleotide primers and dual-labeled hydrolysis (Taqman®) probes for in vitro qualitative detection of dengue virus serotypes 1, 2, 3 or 4 from serum or plasma collected from human patients with signs and symptoms consistent with dengue (mild or severe). The targeted regions of viral RNA are transcribed into complementary 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.

A positive control virus mix is also included, which consists of heatinactivated DENV-1 Haw, DENV-2 NGC, DENV-3 H87, and DENV-4 H241 viruses. A Human Specimen Control (HSC) is a noninfectious cultured human cell material that provides a positive signal in the assay and demonstrates successful recovery of RNA as well as the integrity of the RNA extraction reagent. The human RNase P RNA (RP) is present in cultured cell material and in most clinical samples and detectable by RT-PCR using the primers and probes provided. The CDC DENV-1-4 Real-Time RT-PCR Assay can be run in singleplex (each DENV serotype detected in a separate reaction) or in multiplex (four DENV serotypes are run in the same reaction). These two formats provide equal sensitivity.

Summary of Dengue Testing Process

Image /page/5/Figure/1 description: This image is a flowchart describing the steps of the CDC DENV-1-4 RT-PCR Assay. The first steps, upon receipt of the assay, are to resuspend primers and probes, aliquot and store, extract DENV-1-4 RNA, and dilute DENV-1-4 RNA 1:10. Upon sample receipt, the steps are to extract samples RNA and HSC RNA, prepare master mix (20 uL), prepare RT-PCR plate (5 uL RNA), run CDC RT-PCR assay on ABI 7500Fast Dx, analyze data, and report results.

Recording and interpretation of the assay results:

CDC DENV-1-4 Real-Time RT-PCR Assay Users Guide for Interpretation of Results - Quick Reference and Reporting

DENV-1	DENV-2	DENV-3	DENV-4	RP Target	Report
+				는	Positive DENV-1 detection*
	+			ー	Positive DENV-2 detection*
		+		ਜ	Positive DENV-3 detection*
			+	ਜ	Positive DENV-4 detection*
				+	Negative for DENV, result
does not preclude infection
					Inconclusive test, likely poor
extraction or sample quality**

If sample is positive for 2 serotypes, repeat the test. If sample is repetitively reactive for both serotypes the result is indicative of a dual infection and should be confirmed by the CDC-Dengue Branch.

** When an inconclusive result is obtained, re-extract the specimen and test the newly extracted RNA (recommended) or re-test the extracted RNA. If the re-tested sample is negative for all markers and all controls exhibit the expected performance, the result is "Inconclusive" and a new specimen should be collected if possible.

RNase P (RP)

All clinical samples should exhibit fluorescence amplification curves in the RNase P reaction that cross the threshold line within 37 cycles (DENV-1 Strains | | | | | | | | | | |
| 2003 | Brazil | African/American | 31.99 | 1.74 | 3/3 | 1.89 x $10^4$ | NA | NA | 0/3 | N/A |
| 2007 | Mexico | African/American | 34.45 | 0.47 | 2/3 | 9.87 x $10^4$ | NA | NA | 0/3 | N/A |
| 2007 | Venezuela | African/American | 32.32 | 1.08 | 3/3 | 1.58 x $10^4$ | NA | NA | 0/3 | N/A |
| 1994 | Sri Lanka | Asian | 32.88 | 0.18 | 3/3 | 8.99 x $10^3$ | NA | NA | 0/3 | N/A |
| 2004 | Philippines | South Pacific | 33.29 | 0.71 | 3/3 | 7.23 x $10^4$ | NA | NA | 0/3 | N/A |
| 2004 | Bangkok | Asian | 36.21 | 1.42 | 2/3 | 2.75 x $10^4$ | NA | NA | 0/3 | N/A |
| DENV-2 Strains | | | | | | | | | | |
| 2003 | Brazil | SE Asian/American | 32.39 | 0.32 | 3/3 | 8.11 x $10^4$ | NA | NA | 0/3 | N/A |
| 2005 | Colombia | SE Asian/American | 31.69 | 0.34 | 3/3 | 7.66 x $10^4$ | NA | NA | 0/3 | N/A |
| 2007 | Ivory Cost | SE Asian | 31.67 | 0.34 | 3/3 | 2.46 x $10^4$ | 36.6 | 1.37 | 2/3 | 7.22 x $10^3$ |
| 2003 | Viet Nam | Asian I | 32.18 | 0.40 | 3/3 | 2.17 x $10^4$ | 36.11 | NA | 1/3 | 6.89 x $10^1$ |
| 2006 | Thailand | SE Asian | 32.14 | 0.87 | 3/3 | 9.56 x $10^3$ | NA | NA | 0/3 | N/A |
| 2007 | Dominican R. | SE Asian/American | 32.78 | 0.32 | 3/3 | 7.24 x $10^4$ | 35.87 | NA | 1/3 | 3.44 x $10^3$ |
| 2001 | Costa Rica | SE Asian/American | 32.04 | 0.14 | 3/3 | 3.15 x $10^4$ | NA | NA | 0/3 | N/A |
| 2003 | Peru | SE Asian/American | 32.39 | 0.32 | 3/3 | 5.99 x $10^2$ | 37.12 | 1.26 | 1/3 | 1.88 x $10^3$ |
| 2006 | Burkina Faso | Cosmopolitan | 32.86 | 1.05 | 3/3 | 2.12 x $10^4$ | 37.44 | 0.86 | 1/3 | 7.98 x $10^3$ |
| DENV-3 Strains | | | | | | | | | | |
| 2006 | Puerto Rico | Indian Subcont. | 32.04 | 0.42 | 3/3 | 6.99 x $10^4$ | NA | NA | 0/3 | N/A |
| 2003 | Brazil | Indian Subcont. | 31.99 | 1.74 | 3/3 | 1.39 x $10^4$ | 36.99 | 1.27 | 1/3 | 8.9 x $10^3$ |
| 1995 | Samoa | Indian Subcont. | 36.69 | 1.47 | 2/3 | 5.12 x $10^4$ | NA | NA | 0/3 | NA |
| 2003 | Bangkok | Indian Subcont. | 32.16 | 0.81 | 3/3 | 2.10 x $10^4$ | NA | NA | 0/3 | N/A |
| 2000 | Ecuador | Indian Subcont. | 32.88 | 0.18 | 3/3 | 1.87 x $10^4$ | 36.41 | 0.91 | 1/3 | 6.34 x $10^3$ |
| 1991 | Cook Island | SE Asian/S. Pacific | 33.29 | 0.71 | 3/3 | 2.11 x $10^4$ | NA | NA | 0/3 | N/A |
| DENV-4 Strains | | | | | | | | | | |
| 2006 | Colombia | Indonesian | 36.60 | 1.30 | 2/3 | 2.23 x $10^4$ | NA | NA | 0/3 | N/A |
| 2006 | Mexico | Indonesian | 32.39 | 0.32 | 3/3 | 9.15 x $10^4$ | NA | NA | 0/3 | N/A |
| 1992 | Sri Lanka | SE Asian | 31.70 | 0.33 | 3/3 | 2.44 x $10^4$ | 36.59 | 0.76 | 1/3 | 3.65 x $10^3$ |
| 2006 | Thailand | Indonesian | 35.78 | 0.40 | 2/3 | 1.45 x $10^4$ | NA | NA | 0/3 | N/A |
| 1994 | St. Croix | Indonesian | 32.18 | 0.40 | 3/3 | 2.89 x $10^4$ | NA | NA | 0/3 | N/A |
| 1999 | Ecuador | Indonesian | 32.14 | 0.87 | 3/3 | 5.35 x $10^4$ | 36.56 | NA | 1/3 | 7.98 x $10^3$ |
| 1995 | Micronesia | Asian | 32.78 | 0.32 | 3/3 | 2.13 x $10^4$ | NA | NA | 0/3 | 2.77 x $10^3$ |

DENV isolates tested with the CDC DENV-1-4 Real-Time RT-PCR Assay (Multiplex)

GCE/mL- genome copy equivalent/mL

e. Analytical specificity:

Cross Reactivity: The analytical specificity of the CDC DENV-1-4 Real-Time RT-PCR Assay was evaluated by testing the device with nucleic acids extracted from 12 organisms representing common pathogens present in blood, serum or plasma samples of patients with febrile illness, included in the differential diagnosis of dengue. These pathogens were obtained from CDC repositories. Ten of these pathogens were used to spike human serum (confirmed negative for dengue virus) at the clinically significant concentrations. These 10 organisms included four RNA arboviruses (West Nile virus [WNV], Yellow Fever virus [YFV], Saint Louis Encephalitis virus [SLEV], and Chikungunya virus [CHIKV]). WNV, YFV and SLEV are

flaviviruses related to DENV. Herpes simplex virus 1 and 2 (HSV-1 and -2), cytomegalovirus (CMV), and varicella zoster virus (VZV) are DNA viruses selected for this study. Two bacterial organisms, Leptospira and Borrellia, were also spiked in serum for cross reactivity studies.

The CDC DENV-1-4 Real Time RT-PCR Assay was performed on all 12 samples in triplicate. RNA was extracted using the Qiagen QIAamp® DSP Viral RNA Mini Kit and was tested with the CDC DENV-1-4 Real-Time RT-PCR Assay using the Invitrogen SuperScript®III Platinum OneStep Quantitative RT-PCR System on the Applied Biosystems® 7500 Fast Dx thermocycler. Negative results were obtained with the CDC DENV-1-4 Real Time RT-PCR Assay in all triplicate samples for all 12 tested organisms. No cross reactivity was observed with any panel member tested at clinically significant concentrations.

Cross Reactivity Panel

| Pathogen | Sample type | Concentration
pfu/ml | DENV RT-
PCR
Rate positive |
|-------------------------|----------------|-------------------------|----------------------------------|
| Virus | | | |
| WNV | spiked serum | 6.9x107 | 0/3 |
| YFV | spiked serum | 3.7x106 | 0/3 |
| SLEV | spiked serum | 3.7x106 | 0/3 |
| CHIKV | spiked serum | 4.0x106 | 0/3 |
| HCV | clinical serum | unknown* | 0/3 |
| HAV | clinical serum | unknown* | 0/3 |
| HSV-1 | spiked serum | 1.0x105 | 0/3 |
| HSV-2 | spiked serum | 1.0x105 | 0/3 |
| CMV | spiked serum | 1.0x105 | 0/3 |
| VZV | spiked serum | 1.0x105 | 0/3 |
| Bacteria | | bacteria/ml | |
| Leptospira | spiked serum | 2.5 x105 | 0/3 |
| Borrelia
burgdorferi | spiked serum | 1.0x106 | 0/3 |

*Presence of these pathogens in clinical samples was established by non-quantitative RT-PCR assays (concentration of pathogen is unknown)

f. Interference studies:

Performance of the CDC DENV-1-4 Real-Time RT-PCR Assay was evaluated in the presence of potential interfering substances which could reasonably be expected to be present in serum and plasma specimens. All interference studies were carried out in the presence of cultured, quantified (pfu/mL) stocks of whole virus (strains DENV-1 Haw, DENV-2 NGC, DENV-3 H87, DENV-4 H241) diluted to concentrations 1:10 higher than the LoD dilution, equal to the LoD dilution, and the 1:10 dilution below the LoD. Each viral dilution was tested three times in normal human serum (NHS) or in NHS containing bilirubin (342 µmol/L), cholesterol (13 mmol/L), hemoglobin (2 g/L), triglycerides (37 mmol/L) and genomic DNA (400 ug/100 mL). The levels tested for each endogenous substance were based on the Clinical

Laboratories Institute (CLSI) Standard, EP7-A2 (2005).

Viral RNA from every sample was extracted using the Qiagen QIAamp® DSP Viral RNA Mini Kit. Extracted viral RNAs were tested following the procedure described for the CDC DENV-1-4 Real-Time RT-PCR Assay using the Invitrogen SuperScript®III Platinum OneStep Quantitative RT-PCR System on the Applied Biosystems® 7500 Fast Dx thermocycler.

No significant interference was observed in the presence of the described potential interfering substances at the tested levels.

g. Carry-Over/Cross Contamination:

To assess possible cross contamination of samples in the CDC DENV-1-4 Real-Time RT-PCR Assay, 8 replica sets of DENV-1 Haw, DENV-2 NGC, DENV-3 H87, DENV-4 H241 were tested in an alternating series. Cultured, quantified (pfu/mL) stocks of DENV-1. -2. -3 and 4 were diluted to highpositive (10' pfu/ml) and high-negative (5x102 pfu/ml) concentrations. Eight high-positive and 8 high-negative replicas were tested in an alternating series by the CDC DENV-1-4 Real-Time RT-PCR Assay. Viral RNA from all replica samples was extracted using the Oiagen OIAamp® DSP Viral RNA Mini Kit. Each viral RNA elution was tested following the procedure described for the CDC DENV-1-4 Real-Time RT-PCR Assay using the Invitrogen SuperScript®III Platinum OneStep Quantitative RT-PCR System on the Applied Biosystems® 7500 Fast Dx thermocycler. All results were as expected. All negative samples tested negative (32/32) and all positive samples tested positive (32/32).

h. Fresh vs. Frozen Specimens:

To determine the effect of freezing and the effect of freeze/thaw cycles on the CDC DENV-1-4 Real-Time RT-PCR Assay results, a comparison study was performed using spiked human serum samples. Based upon the initial CDC DENV-1-4 Real-Time RT-PCR Assay results obtained with serially diluted virus in serum, CDC chose moderate and low positive dilutions and prepared them in triplicate sets. These sets were frozen at -80°C for 24 hours and subject to five consecutive freeze/thaw cycles. Frozen aliquots (-80 ℃ for 24h.) of these specimens were thawed, extracted using the Qiagen OIAamp® DSP Viral RNA Mini Kit (cat# 61904) following manufacturer's protocol, and compared to the original fresh sample test results. Each nucleic acid sample was tested following the procedure described for the CDC DENV-1-4 Real-Time RT-PCR Assay using the Invitrogen SuperScript®III Platinum OneStep Quantitative RT-PCR System (cat# 11732-088) on the Applied Biosystems® 7500 Fast Dx thermocvcler. The study showed 100% agreement (qualitative) between the initial CDC DENV-1-4 Real-Time RT-PCR Assay (0) result and the result after each freeze/thaw cycle (1, 2, 3, 4 and 5).

2. Comparison studies:

a. Method comparison with predicate device:

There is no predicate device for this assay. However, the clinical performance of the CDC DENV-1-4 Real-Time RT-PCR Assay was evaluated against two laboratory developed assays: 1) CDC IgM anti-DENV Capture Enzyme Linked Immunosorbent Assay (MAC-ELISA) and 2) CDC molecular detection method (run at CDC) followed by bi-directional sequencing of the DENV E gene (1485 bp). Positive and negative percent agreement for method comparison studies were calculated in comparison to these two different methods.

For additional details please see section 3 Clinical studies subsection c.

b. Matrix Comparison:
Matrix comparison was not required because the LoD of the CDC DENV-1-4 Real-Time RT-PCR Assay in serum and plasma showed no difference.
1. Clinical studies:
a. Clinical Sensitivity: N/A
b. Clinical specificity: N/A
c. Other clinical supportive data (when a. and b. are not applicable):

Clinical Performance:

Prospective Studies: Performance characteristics of the CDC DENV-1-4 RT-PCR Assay were established during a prospective study at 3 public health laboratories (2009-2011). A total of 86 serum samples were prospectively collected from dengue-suspected, febrile patients during the first 5 days of symptoms and were tested at the three sites. There were twenty five (25) and thirty six (36) serum samples tested at Site 1 and 3 respectively using the Qiagen QIAamp® DSP Viral RNA Mini Kit (cat# 61904) extraction procedure following the manufacturer's protocol. Twenty five (25) serum samples were tested at Site 2 using the Roche MagNA Pure LC Total Nucleic Acid Isolation Kit (03 038 505 001). The eluted viral RNA was tested using the Invitrogen SuperScript®III Platinum OneStep Quantitative RT-PCR System (cat# 11732-088) on the Applied Biosystems® 7500 Fast Dx thermocycler. All 86 samples subsequently underwent bi-directional sequencing of the DENV E gene (1485 bp). The prospectively collected samples were not accompanied by a second, convalescent specimen. Percent agreement was determined in

comparison to the CDC molecular detection method followed by sequencing. Overall prospective comparison results are presented in the table below.

Multiplex CDC DENV-1-4 Real-Time RT-PCR Assay Comparison Results
	Reference Method (Sequencing)
		Positive	Negative	Total
CDC DENV-1-4
Real-Time RT-
PCR Assay	Positive	47	0	47
	Negative	1*	38	39
	Total	48	38	86

		Value	95% Confidence Interval
Positive Percent Agreement		97.92% (47/48)	89.10% – 99.63%
Negative Percent Agreement		100% (38/38)	90.82% – 100%

Overall Prospective Comparison Results

"One sample was negative on the CDC DENV-1-4 Real-Time RT-PCR Assay which was positive for DENV-3 by bi-directional sequencing. All other negative RT-PCR samples were also negative by E gene bi-directional sequencing.

Retrospective Studies: Performance characteristics of the CDC DENV-1-4 Real-Time RT-PCR Assay were established during a retrospective study at the CDC Dengue Branch. Three hundred seventy one serum samples were obtained from the archived CDC routine dengue surveillance specimens collected in pairs during 2007-2011. The first sample (acute) was collected during the first five days of illness, and the second sample (convalescent) was obtained at least 6 days after the onset of symptoms. These samples were tested with the IgM anti-DENV Capture Enzyme Linked Immunosorbent Assay (CDC MAC-ELISA - validated in-house) in order to establish seroconversion. The results of the CDC DENV-1-4 Real-Time RT-PCR Assay (Multiplex) obtained for the acute samples were compared to the IgM anti-DENV seroconversion results in the paired samples.

Nucleic acid from all acute samples was extracted using the Qiagen QIAamp® DSP Viral RNA Mini Kit (cat# 61904) following manufacturer's protocol. Each nucleic acid sample was tested following the procedure described for the CDC DENV-1-4 Real-Time RT-PCR Assay (Multiplex) using the Invitrogen SuperScript®III Platinum OneStep Quantitative RT-PCR System (cat# 11732-088) on the Applied Biosystems® 7500 Fast Dx thermocycler. The percent agreement is calculated for the number of samples that received positive or negative results in the CDC DENV-1-4 RT-PCR assay and using IgM anti-DENV seroconversion as a comparator. In addition, bi-directional sequencing of the DENV E gene (1485 bp) was obtained. and corroborated the results of, the CDC

DENV-1-4 Real-Time RT-PCR Assay on all positive samples except for one. The following table summarizes the results obtained on the 371 specimens that originated from the following sources: non-United States dengue cases (n=39); Puerto Rico dengue cases (n=82); 250 negative cases from Puerto Rico (no IgM anti-DENV conversion). Overall retrospective comparison results are presented in the table below.

Multiplex CDC DENV-1-4 Real-Time RT-PCR Assay Comparison Results
		Reference Method
(IgM anti-DENV Conversion)
		Positive	Negative	Total
CDC DENV-1-4
Real-Time RT-
PCR Assay	Positive	100*	4***	104
	Negative	2**	265	267
	Total	102	269	371

		Value	95% Confidence Interval
Positive Percent Agreement		98.04% (100/102)	93.13% - 99.46%
Negative Percent Agreement		98.51% (265/269)	96.24% - 99.42%

Overall Retrospective Comparison Results

*One DENV-1 case was positive on the CDC DENV-1-4 Real-Time RT-PCR Assay (multiplex) and confirmed positive by IgM anti-DENV conversion, but not sequenced effectively enough to produce an interpretable result. **Two samples were negative by the CDC-DENV-1-4 Real-Time RT-PCR Assay (multiplex). One of these samples was DENV-3 positive by the CDC-DENV-1-4 Real-Time RT-PCR Assay (singleplex) and was further confirmed DENV-3 positive by sequencing. The other sample was confirmed DENV-3 positive by sequencing. *** Four samples showed positive RT-PCR results and were not confirmed by IgM anti-DENV seroconversion. Two of these samples were positive results for DENV-3.and the other two samples were DENV-4 positive. These results were confirmed by E gene bi-directional sequencing.

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

The percent of positive cases identified by the CDC DENV-1-4 Real-Time RT-PCR Assay will vary depending on the nature of the surveillance process, given that an unknown proportion of febrile cases reported may not be dengue. It is expected that the proportion of serotypes identified will vary with time, location and patient population. In dengue endemic areas, usually one serotype may be more prevalent than the others. A serotype may not be detected for long periods and then re-emerge and become the predominant serotype. Different countries may have different serotype predominance; therefore travelers returning to the United States

from dengue-endemic countries may have been infected with different serotypes. Puerto Rico has had the four DENV serotypes circulating at different times. During the 2007 epidemic, DENV-2, and DENV-3 were the predominant serotypes and during the 2010 epidemic, DENV-1 and DENV-4 were predominant. Florida has experienced autochthonous DENV-1 transmission in 2009 and 2010; and travel-associated cases (Florida residents travelling internationally) have been identified with DENV-1, DENV-2 and DENV-4.

In serologically confirmed cases, the CDC DENV-1-4 Real-Time RT-PCR Assay positively identified 93% of cases. A 97% agreement between positive results on the CDC DENV-1-4 Real-Time RT-PCR Assay and sequencing was obtained.

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 866.3946 with special controls. The special controls guidance document "Class II Special Controls Guidance Document: Dengue virus Nucleic Acid Amplification Test Reagents" will shortly be available.