(73 days)
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.
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.
Acceptance Criteria and Device Performance for CDC DENV-1-4 Real-Time RT-PCR Assay
1. Table of Acceptance Criteria and Reported Device Performance
For the CDC DENV-1-4 Real-Time RT-PCR Assay, the acceptance criteria are implicitly derived from the performance goals demonstrated in the clinical studies. These studies evaluate the assay's ability to accurately detect and serotype Dengue Virus RNA in human serum samples.
| Performance Characteristic | Acceptance Criteria (Implied from Study Results) | Reported Device Performance |
|---|---|---|
| Reproducibility | High positive samples (2-3x LoD) should show 100% agreement with expected results across sites and operators. | DENV-1: 100% (60/60) agreement for Moderate Positive samples. DENV-2: 100% (60/60) agreement for Moderate Positive samples. DENV-3: 100% (60/60) agreement for Moderate Positive samples. DENV-4: 100% (60/60) agreement for Moderate Positive samples. |
| Low positive samples (equal to LoD) should show high agreement with expected results across sites and operators. | DENV-1: 96% (58/60) agreement for Low Positive samples. DENV-2: 95% (57/60) agreement for Low Positive samples. DENV-3: 100% (60/60) agreement for Low Positive samples. DENV-4: 95% (57/60) agreement for Low Positive samples. | |
| Negative samples should consistently show 100% agreement with expected results (negative). | DENV-1, DENV-2, DENV-3, DENV-4: 100% (60/60) agreement for Negative samples. | |
| Limit of Detection (LoD) | Consistent LoD across serotypes in serum and plasma, regardless of extraction method. | Singleplex & Multiplex (all extraction methods) in Serum & Plasma: 1 x 10³ pfu/mL for all DENV serotypes. |
| Analytical Specificity (Cross-Reactivity) | No cross-reactivity with common pathogens present in blood, serum, or plasma of patients with febrile illness (differential diagnosis for dengue). | No cross-reactivity observed with any of the 12 tested organisms (including WNV, YFV, SLEV, CHIKV, HSV-1/2, CMV, VZV, Leptospira, Borrelia) at clinically significant concentrations. (0/3 positive for DENV RT-PCR for all cross-reactants) |
| Interference | No significant interference from endogenous substances (e.g., bilirubin, cholesterol, hemoglobin, triglycerides, genomic DNA) at specified concentrations. | No significant interference observed for DENV-1, -2, -3, and -4 at tested levels. |
| Carry-Over/Cross Contamination | No evidence of carry-over or cross-contamination between high-positive and negative samples. | 100% agreement for negative samples (32/32) and positive samples (32/32) in alternating series testing. |
| Specimen Stability (Freeze/Thaw) | No qualitative difference in results after multiple freeze/thaw cycles. | 100% qualitative agreement between initial results and results after 1, 2, 3, 4, and 5 freeze/thaw cycles. |
| Clinical Performance (Positive Percent Agreement to Comparator) | High positive percent agreement with established molecular detection methods (sequencing and MAC-ELISA) for confirmed dengue cases. | Prospective Study (vs. Sequencing): 97.92% (47/48) Positive Percent Agreement. Retrospective Study (vs. IgM anti-DENV Conversion): 98.04% (100/102) Positive Percent Agreement. |
| Clinical Performance (Negative Percent Agreement to Comparator) | High negative percent agreement with established molecular detection methods (sequencing and MAC-ELISA) for non-dengue cases. | Prospective Study (vs. Sequencing): 100% (38/38) Negative Percent Agreement. Retrospective Study (vs. IgM anti-DENV Conversion): 98.51% (265/269) Negative Percent Agreement. |
2. Sample Sizes Used for the Test Set and Data Provenance
-
Reproducibility Study:
- For each DENV serotype (DENV-1, -2, -3, -4) and control type (moderate positive, low positive, high negative, negative), 20 samples were tested per site (total of 60 samples per condition across 3 sites).
- Data Provenance: Cultured, quantified (pfu/mL) stocks of whole virus (strains DENV-1 Haw, DENV-2 NGC, DENV-3 H87, and DENV-4 H241) spiked into dengue-negative human serum. The study was performed at three testing sites, including the CDC Dengue Branch Lab and two external independent sites. This appears to be a prospective experimental study.
-
Limit of Detection (LoD) Study:
- For each DENV serotype, dilution, and matrix (serum/plasma), 5 replicas were tested.
- Data Provenance: panels prepared from cultured, quantified (pfu/mL) stocks of whole virus (strains DENV-1 Haw, DENV-2 NGC, DENV-3 H87, DENV-4 H241) serially diluted into dengue-negative human serum or plasma. This is a retrospective/controlled laboratory study using spiked samples.
-
Analytical Reactivity Study:
- For each of the 29 DENV-1-4 isolates, triplicate samples were tested at 10³ pfu/mL and 10² pfu/mL dilutions.
- Data Provenance: 29 DENV-1 - 4 isolates obtained from patients from different countries, cultured and quantified. This is a retrospective/controlled laboratory study using cultured clinical isolates.
-
Cross-Reactivity Study:
- Triplicate samples for each of the 12 pathogens were tested.
- Data Provenance: Nucleic acids extracted from 12 organisms (including WNV, YFV, SLEV, CHIKV, HSV-1/2, CMV, VZV, Leptospira, Borrelia, HCV, HAV). Ten of these were spiked into human serum (confirmed negative for dengue virus); HCV and HAV were clinical serum samples with unknown concentration. This is a retrospective/controlled laboratory study using spiked samples and some clinical samples.
-
Interference Study:
- For each DENV serotype, viral dilution (1:10 above LoD, at LoD, 1:10 below LoD) and interfering substance, three replicate samples were tested.
- Data Provenance: Cultured, quantified (pfu/mL) stocks of whole virus (strains DENV-1 Haw, DENV-2 NGC, DENV-3 H87, DENV-4 H241) diluted into normal human serum (NHS) or NHS containing specific interfering substances. This is a retrospective/controlled laboratory study using spiked samples.
-
Carry-Over/Cross Contamination Study:
- 8 high-positive and 8 high-negative replicas were tested in an alternating series for each DENV serotype. Total of 32 negative and 32 positive samples tested.
- Data Provenance: Cultured, quantified (pfu/mL) stocks of DENV-1, -2, -3 and -4 diluted to high-positive and high-negative concentrations. This is a retrospective/controlled laboratory study using spiked samples.
-
Fresh vs. Frozen Specimens Study:
- Moderate and low positive dilutions prepared in triplicate sets, subjected to 5 freeze/thaw cycles.
- Data Provenance: Spiked human serum samples. This is a retrospective/controlled laboratory study using spiked samples.
-
Clinical Performance - Prospective Study:
- Sample Size: 86 serum samples.
- Data Provenance: Prospectively collected from dengue-suspected, febrile patients during the first 5 days of symptoms at 3 public health laboratories (2009-2011). Country of Origin: Not explicitly stated, but likely US (given public health labs) or US territories (e.g., Puerto Rico). This is a prospective study.
-
Clinical Performance - Retrospective Study:
- Sample Size: 371 serum samples.
- Data Provenance: Archived CDC routine dengue surveillance specimens collected in pairs (acute/convalescent) during 2007-2011. Country of Origin: Non-United States dengue cases (n=39); Puerto Rico dengue cases (n=82); 250 negative cases from Puerto Rico. This is a retrospective study.
3. Number of Experts Used to Establish the Ground Truth for the Test Set and Their Qualifications
-
For Analytical Studies (Reproducibility, LoD, Analytical Reactivity, Cross-Reactivity, Interference, Carry-Over, Fresh vs. Frozen): The ground truth was established by preparing samples with known concentrations of specific DENV serotypes or other pathogens, often quantified by plaque-forming units (pfu/mL). Therefore, no human experts were explicitly used or required to establish the ground truth for these in vitro analytical studies. The accuracy relies on the lab's ability to precisely prepare and quantify these samples.
-
For Clinical Performance - Prospective Study:
- Ground Truth Method: Bi-directional sequencing of the DENV E gene (1485 bp). Genetic sequencing is considered a highly definitive molecular method for pathogen identification and sub-typing.
- Number of Experts/Qualifications: The document does not specify the number or qualifications of experts involved in performing or interpreting the sequencing results. However, molecular sequencing is typically performed and interpreted by trained laboratory professionals, often with expertise in molecular diagnostics, virology, and bioinformatics.
-
For Clinical Performance - Retrospective Study:
- Ground Truth Method: IgM anti-DENV Capture Enzyme Linked Immunosorbent Assay (MAC-ELISA) for seroconversion, validated in-house by CDC, combined with bi-directional sequencing of the DENV E gene (1485 bp) for confirmation of RT-PCR positive samples. IgM seroconversion is a standard diagnostic method for recent dengue infection.
- Number of Experts/Qualifications: The document does not specify the number or qualifications of experts involved in performing or interpreting the MAC-ELISA or sequencing results. Similar to the prospective study, skilled laboratory professionals would perform these tests.
4. Adjudication Method for the Test Set
The document does not describe an explicit "adjudication method" in the sense of a committee of experts reconciling conflicting results for the test sets.
- For the prospective clinical study, the CDC DENV-1-4 RT-PCR Assay results were directly compared to bi-directional sequencing results. Any discrepancies were noted (e.g., one sample negative by RT-PCR but positive by sequencing).
- For the retrospective clinical study, RT-PCR results were compared to IgM anti-DENV seroconversion. For discrepant or positive RT-PCR results, bi-directional sequencing was performed for corroboration. The document highlights specific discrepancies and their resolution by sequencing.
This approach suggests a direct comparison to a reference standard (sequencing or seroconversion), with sequencing serving as a higher-level confirmation for discrepancies rather than an adjudicated consensus amongst multiple independent readers.
5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done, What was the Effect Size of How Much Human Readers Improve with AI vs. Without AI Assistance
This section is Not Applicable (N/A). The CDC DENV-1-4 Real-Time RT-PCR Assay is an in vitro molecular diagnostic test designed for automated or semi-automated analysis and detection of DENV RNA. It is not an imaging or diagnostic device that involves "human readers" interpreting results in the way an AI-assisted diagnostic tool might, for which an MRMC study would be relevant. The output (Ct values) is typically interpreted against predefined thresholds by trained laboratory personnel, rather than a subjective interpretation by a "reader." Therefore, no MRMC study or AI assistance effect size is reported.
6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done
Yes, a standalone (algorithm only) performance was effectively performed. The CDC DENV-1-4 Real-Time RT-PCR Assay, as a molecular diagnostic test, operates autonomously once the sample preparation (RNA extraction) is complete and the reagents are loaded into the ABI 7500 Fast Dx Real-Time PCR instrument. The instrument's software detects fluorescence amplification and determines Ct values without human intervention during the detection phase. The "algorithm" here is the chemical reaction itself driven by the primers and probes, and the instrument's software for signal detection and thresholding. The results (presence/absence of DENV RNA, and serotype) are derived directly from the instrument's output. Human involvement comes in interpreting these results according to the provided Users Guide (Section M. Performance Characteristics and the Clinical Studies sections predominantly represent this standalone performance, demonstrating the assay’s inherent accuracy in detecting DENV RNA and distinguishing serotypes).
7. The Type of Ground Truth Used
- For Analytical Studies: The ground truth was primarily based on spiked samples with known concentrations of cultured, quantified viruses (pfu/mL) and other pathogens.
- For Clinical Performance - Prospective Study: Bi-directional sequencing of the DENV E gene (1485 bp) was used as the ground truth.
- For Clinical Performance - Retrospective Study: A combination of IgM anti-DENV seroconversion (using CDC MAC-ELISA) and bi-directional sequencing of the DENV E gene was used. Sequencing served to corroborate RT-PCR positive results and to resolve discrepancies from seroconversion.
8. The Sample Size for the Training Set
The document does not explicitly describe a "training set" in the context of an machine learning algorithm. This is a molecular diagnostic assay that uses defined reagents (primers and probes) rather than a machine learning model that would require a dedicated training set. The assay's parameters (e.g., thermal cycling conditions, detection thresholds within the instrument software) would have been optimized during its development and validation phase, which might involve iterative testing on various samples, but these are not explicitly termed a "training set" in the context used for AI/ML.
9. How the Ground Truth for the Training Set Was Established
Since there is no explicitly defined "training set" for an AI/ML model for this molecular diagnostic assay, this point is Not Applicable (N/A). The assay's design (primers, probes) and operational parameters were established through standard molecular biology and assay development procedures, which inherently ensure that the chemical reactions are specific and sensitive to the target RNA.
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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 AcidAmplification Test Reagents |
|---|---|
| 2. Classification: | Class II (de novo) |
| 3. Product code: | OZB, NSU |
| 4. Panel: | Microbiology (83) |
H. Intended Use:
-
- 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:
- Intended use(s):
-
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.
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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
-
- Indication(s) for use:
Same as Intended Use
- Indication(s) for use:
-
- Special conditions for use statement(s):
For in vitro diagnostic use
- Special conditions for use statement(s):
-
- 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 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.
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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.
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.
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| Reagents | Quantity | Catalog No | |
|---|---|---|---|
| rRT-PCREnzymeMastermixOptions | Invitrogen SuperScript™ IIIPlatinum® reactions One-StepQuantitative RT-PCR System(without Rox)* | 100reactions500reactions | 11732-02011732-088 |
| NucleicAcidPurificationKit Options | Qiagen QIAamp® DSP ViralRNA Mini Kit and/or QiagenQIAcube ** | 50Extractions | 61904 or9001292 |
| MagNA Pure LC total NucleicAcid Isolation Kit on theMagNA Pure LC 2.0instrument* | 192Extractions | 03 038 505001 |
*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:
-
- Predicate device name(s): Not applicable
-
- Predicate Numbers (s): Not applicable
-
- Comparison with predicate: Not applicable
K. Standard/Guidance Documents Referenced (if applicable):
-
- CLSI EP5: Evaluation of Precision Performance of Clinical Chemistry Devices- Second Edition, Villanova PA
-
- CLSI EP7-A2: Interference Testing in Clinical Chemistry; Approved Guideline, 2nd Ed.
-
- CLSI EP17-A: Protocols for Determination of Limits of Detection (2004).
Note: A Special Controls Guidance Document will be promulgated.
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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.
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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.
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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, resultdoes not preclude infection | ||||
| Inconclusive test, likely poorextraction 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 (< 37 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:
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- If the DENV-1, -2, -3 and -4 are positive, even in the presence of a ● negative RP, the dengue result should be considered valid. It is possible, that some samples may fail to exhibit RP amplification curves due to low levels in the original clinical sample. A negative RP signal does not preclude the presence of dengue virus in a clinical specimen.
- If all dengue markers and RP are 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 retest, 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.
M. Performance Characteristics (if/when applicable):
-
- Analytical performance:
- a. Precision/Reproducibility:
The reproducibility of the CDC DENV-1-4 Real-Time RT-PCR Assay was evaluated at three testing sites (CDC Dengue Branch Lab and two external independent sites). Operator to operator, run to run and site to site reproducibility were evaluated using four test panels which included negative (unspiked sample), high negative (1:10 dilution below the LoD), low positive (equal to the LoD) and moderate positive (2-3x higher than the LoD ) samples. In order to develop these panels, cultured, quantified (pfu/mL) stocks of whole virus (strains DENV-1 Haw, DENV-2 NGC, DENV-3 H87 and DENV-4 H241) were used to spike into dengue negative human serum. Each sample of the panel was tested twice a day for at least 5 days by two operators with each operator running the panel members two times a day. Test operators were blinded with respect to test panel members for each run and device lot to ensure that panel results could not be predicted by the operator.
Two RNA extraction methods validated for the CDC DENV-1-4 Real-Time RT-PCR were used in the reproducibility study. The CDC Dengue Branch Lab and one external site used the Qiagen QIAamp® DSP Viral RNA Mini Kit and second external site used the MagNA Pure LC total Nucleic Acid Isolation Kit. The manufacturer's instructions for use provided in the package insert were followed. Results generated for each of the extraction methods are summarized in the Table below.
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Reproducibility Studies for the CDC DENV-1-4 Real-Time RT-PCR Assay on the ABI 7500 Fast Dx Real-Time PCR Instrument and SSD version 1.4 Software at three sites
| Site 1 | Site 2 | Site 3 | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| AVGCT | %GV | AgreementwithExpectedResult | AVGCT | %CV | AgreementwithExpectedResult | AVGCT | %GV | AgreementwithExpectedResult | ||
| Moderate | ||||||||||
| Positive | 24.23 | 5.24 | 20/20 | 25.83 | 2.40 | 20/20 | 17.91 | 3.96 | 20/20 | |
| LowPositive | 30.45 | 3.68 | 19/20 | 31.15 | 1.32 | 19/20 | 21.28 | 3.34 | 20/20 | |
| DENV-1 | High | |||||||||
| Negative | 38.75 | 3.51 | 16//20 | 38.70 | 4.16 | 15/20 | 19.41 | 5.87 | 16/20 | |
| Negative | NA | NA | 20/20 | NA | NA | 20/20 | 38.54* | NA | 19/20 | |
| Moderate | ||||||||||
| PositiveLow | 26.89 | 5.09 | 20/20 | 24.97 | 2.76 | 20/20 | 18.65 | 5.52 | 20/20 | |
| Positive | 32.02 | 3.94 | 18/20 | 30.80 | 3.21 | 19/20 | 22.27 | 4.63 | 20/20 | |
| DENV-2 | HighNegative | 40.24 | 2.91 | 14/20 | 38.08 | 2.42 | 17/20 | 19.72 | 8.01 | 15/10 |
| Negative | NA | NA | 20/20 | 39.22 | NA | 20/20 | 41.72* | NA | 19/20 | |
| Moderate | 4.97 | |||||||||
| PositiveLow | 25.53 | 20/20 | 25.25 | 4.32 | 20/20 | 18:51 | 4.16 | 20/20 | ||
| Positive | 30.62 | 3.66 | 20/20 | 30.88 | 2.33 | 20/20 | 21.56 | 3.57 | 20/20 | |
| DENV-3 | HighNegative | 38.91 | 3.16 | 15/20 | 38.49 | 4.65 | 16/20 | 21.10 | 5.73 | 16/20 |
| Negative | NA | NA | 20/20 | 39.71 | NA | 20/20 | 38.63* | NA | 19/20 | |
| ModeratePositive | 24.83 | 4.27 | 20/20 | 25.46 | 3.61 | 20/20 | 17.78 | 6.13 | 20/20 | |
| DENV-4 | LowPositive | 30.10 | 2.82 | 19/20 | 30.40 | 6.09 | 19/20 | 21.30 | 3.62 | 19/20 |
| HighNegative | 39.14 | 3.68 | 14/20 | 38.02 | 3.10 | 15/20 | 26.90 | 8.18 | 17/20 | |
| Negative | NA | NA | 20/20 | NA | NA | 20/20 | NA | NA | 20/20 | |
| NegativeControl | NA | NA | 20/20 | NA | NA | 20/20 | NA | NA | 20/20 |
NA: Not applicable (samples did not obtain a CT value).
*Only one specimen obtained a CT value.
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Reproducibility Study Summary for the CDC DENV-1-4 Real-Time RT-PCR Assay on the ABI 7500 Fast Dx Real-Time PCR Instrument and SSD version 1.4 Software
| AVG CT | % CV | Agreement with expected Result | 95% CI | ||
|---|---|---|---|---|---|
| DENV-1 | Moderate Positive | 25.52 | 3.41 | 60/60 (100 %) | 94.0 - 100 |
| Low Positive | 31.03 | 2.42 | 58/60 (96 %) | 88.6 - 99.11 | |
| High Negative | 38.60 | 3.55 | 47/60 (21 %) | 15.9 - 39.6 | |
| Negative | 38.54* | NA | 60/60 (100 %) | NA | |
| DENV-2 | Moderate Positive | 26.32 | 3.91 | 60/60 (100 %) | 94.0 - 100 |
| Low Positive | 31.77 | 3.43 | 57/60 (95 %) | 86.3 - 98.29 | |
| High Negative | 38.91 | 3.14 | 46/60 (23%) | 14.4 - 35.4 | |
| Negative | 40.32* | NA | 60/60 (100 %) | NA | |
| DENV-3 | Moderate Positive | 26.01 | 5.96 | 60/60 (100 %) | 94.0 - 100 |
| Low Positive | 31.10 | 4.57 | 60/60 (100 %) | 94.0 - 100 | |
| High Negative | 39.25 | 3.59 | 45/60 (25%) | 15.8 - 37.23 | |
| Negative | 39.17* | NA | 60/60 (100 %) | NA | |
| DENV-4 | Moderate Positive | 25.47 | 4.00 | 60/60 (100 %) | 94.0 - 100 |
| Low Positive | 30.62 | 3.89 | 57/60 (95 %) | 86.3 - 98.29 | |
| High Negative | 38.62 | 4.17 | 46/60 (23%) | 14.4 - 35.4 | |
| Negative | NA | NA | 60/60 (100 %) | NA | |
| NegativeControl | NA | NA | 10/10 (100 %) | NA |
*AVG CT value is based on one or two samples.
b. Linearity/assay reportable range: Not applicable, qualitative assay
- c. Traceability, Stability, Expected values (controls, calibrators, or methods):
Before analyzing the samples prepared using the CDC DENV-1-4 Real-Time RT-PCR Assay 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 DENV-1-4 Real-Time RT-PCR Assay and the ABI 7500 Fast Dx Real-Time PCR instrument User Manual.
Assay Quality Control
Quality control in laboratories performing the CDC DENV-1-4 Real Time RT-PCR Assay must be performed in conformance with local, state, and federal regulations or accreditation requirements and the user's laboratory's standard quality control procedures. It is recommended that the user refer to
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CLSI document C24-A2 - Statistical Quality Control for Quantitative Measurements.
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 amplification curves that cross the threshold line. If any of the NTC reactions exhibit an amplification curve that crosses the cycle threshold, sample contamination may have occurred. Invalidate the run and repeat the assay with strict adherence to the guidelines.
DENV-1- 4 Positive Controls
The positive control consists of a mix of DENV-1, -2, -3, and -4 dengue virus from C636 cell culture supernatant. Purified RNA from the positive control will yield a positive result with the primer and probe sets designed for DENV-1, DENV-2, DENV-3 and DENV-4.
Extraction Control - Human Specimen Control (HSC)
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 dengue specific markers.
A negative amplification/detection control (NTC) and a positive control should be included in each run. An HSC extraction control must proceed through nucleic acid isolation for each batch of specimens to be tested.
d. Limit of Detection (LoD):
i) LoD of CDC DENV-1-4 Real-Time RT-PCR Assay (Singleplex) using a manual RNA extraction method (Qiagen QIAamp® DSP Viral RNA Mini Kit):
The LoD of the CDC DENV-1-4 Real-Time RT-PCR Assay (Singleplex) was determined using a 5-replica panel of viruses serially diluted into dengue negative human serum or plasma at 1:10 dilutions. The panels were prepared from cultured, quantified (pfu/mL) stocks of whole virus (strains DENV-1 Haw, DENV-2 NGC, DENV-3 H87, DENV-4 H241). Viral RNA from all replica samples was extracted using the Qiagen QIAamp® DSP Viral RNA Mini Kit (cat# 61904). 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 One Step Quantitative RT-PCR System (cat# 11732-088) on the Applied Biosystems® 7500 Fast Dx thermocycler. The table below shows the Average CT value for the 5 replicas
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of each dilution and the corresponding standard deviation, number of positive samples over the total number of replicas and the positive rate.
| Serum | Plasma | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Viruses | VirusConcent.(pfu/ml) | Avg CT | STD | Positive/Total | PositivityRate | Avg CT | STD | Positive/Total | PositivityRate |
| 10' | 21.67 | 0.29 | ર્સ્ડ | 100% | 22.46 | 0.32 | ર્ટાર | 100% | |
| 106 | 26.15 | 0.23 | ર્સ્ડ | 100% | 26.82 | 0.23 | રાર | 100% | |
| 105 | 30.90 | 0.73 | ર્સ્ટ | 100% | 31.47 | 0.76 | ર્ટાર | 100% | |
| DENV-1 | 104 | 34.65 | 2.14 | ર્સ્ટ | 100% | 35.40 | 0.89 | ર્ટાર | 100% |
| 103 | 36.36 | 1.73 | ર્સ્ટ | 100% | 35.82 | 3.20 | ર્સ્ટ | 100% | |
| 102 | N/A | N/A | 0/2 | 0 | N/A | N/A | 0/5 | 0 | |
| 10 | N/A | N/A | 0/5 | 0 | N/A | N/A | 0/5 | 0 | |
| 100 | N/A | N/A | 0/5 | 0 | N/A | N/A | 0/2 | 0 | |
| 10' | 21.65 | 0.23 | ર્સ્ટ | 100% | 21.12 | 0.26 | ર્ટાર | 100% | |
| 106 | 24.74 | 0.31 | ર્સ્ટ | 100% | 24.36 | 0.28 | ર્ટાર | 100% | |
| 105 | 28.83 | 0.27 | ર્સ્ડ | 100% | 27.15 | 0.26 | ર્ટાર | 100% | |
| DENV-2 | 104 | 31.75 | 0.52 | ર્સ્ટ | 100% | 32.39 | 0.84 | ર્ટાર | 100% |
| 103 | 35.78 | 0.23 | ર્સ્ટ | 100% | 36.56 | 0.67 | ર્સ્ટ | 100% | |
| 10- | 39.67 | N/A | 0/2 | 0 | 39.6 | N/A | 0/5 | 0 | |
| 10 | 41.9 | N/A | 0/2 | 0 | 42.5 | N/A | 0/5 | 0 | |
| 10% | N/A | N/A | 0/2 | 0 | N/A | N/A | 0/5 | 0 | |
| 10' | 20.67 | 0.25 | ર્સ્ડ | 100% | 22.21 | 0.38 | ર્ટાર | 100% | |
| 106 | 24.18 | 0.77 | ર્સ્ટ | 100% | 25.34 | 0.27 | ર્ટાર | 100% | |
| 103 | 28.48 | 0.55 | ર્સ્ટ | 100% | 29.39 | 0.14 | રાર | 100% | |
| 104 | 32.27 | 0.73 | ર્સ્ડ | 100% | 32.85 | 0.18 | ર્ટાર | 100% | |
| 103 | 36.58 | 1.32 | 4/5 | 80% | 35.36 | 1.03 | ર્ટાર | 100% | |
| DENV-3 | 102 | 39.71 | N/A | 0/2 | 0 | 38.36 | 0.06 | 3/5 | 60% |
| 10- | 42.01 | N/A | 0/5 | 0 | 41.87 | N/A | 0/5 | 0 | |
| 100 | N/A | N/A | 0/5 | 0 | N/A | N/A | 0/2 | 0 | |
| 10' | 22.46 | 0.13 | ર્સ્ટ | 100% | 24.75 | 0.18 | ર્ટાર | 100% | |
| 10€ | 26.33 | 0.48 | ર્સ્ટ | 100% | 28.20 | 0.12 | ર્ટ રિ | 100% | |
| 105 | 29.64 | 0.83 | ર્સ્ટ | 100% | 31.29 | 0.40 | ર્સ્ટ | 100% | |
| DENV-4 | 104 | 32.61 | 0.49 | ર્સ્ટ | 100% | 34.05 | 1.33 | રાર | 100% |
| 103 | 36.68 | 0.46 | ર્સ્ટ | 100% | 36.72 | 1.18 | ર્સ્ટ | 100% | |
| 10- | 39.55 | N/A | 0/2 | 0 | 38.73 | 2.34 | ા (ર | 20% | |
| 10- | N/A | N/A | 0/2 | 0 | N/A | N/A | 0/5 | 0 | |
| 100 | N/A | N/A | 0/5 | 0 | N/A | N/A | 0/5 | 0 |
Limit of Detection of the CDC DENV-1-4 Real-Time RT-PCR Assay (Singleplex) in Serum and Plasma
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The observed LoD of the CDC DENV-1-4 Real-Time RT-PCR Assay (Singleplex) in serum and plasma was 1 x 10 pfu/mL.
The LoD of the CDC DENV-1-4 Real-Time RT-PCR Assay (Multiplex) was also determined using a 5-replica panel of viruses serially diluted into dengue negative human serum or plasma as described for the CDC DENV-1-4 Real-Time RT-PCR Assay (Singleplex). The LoD of the CDC DENV-1-4 Real-Time RT-PCR Assay (Multiplex) in serum and plasma was shown similar (1 x 10 pfu/mL) to the CDC DENV-1-4 Real-Time RT-PCR Assay (Singleplex) using a manual RNA extraction method (Qiagen QIAamp® DSP Viral RNA Mini Kit).
ii) LoD of CDC DENV-1-4 Real-Time RT-PCR Assay (Multiplex) using an automated RNA extraction method (Qiagen QIAamp® DSP Viral RNA Mini Kit and Qiagen QIAcube):
The LoD of the CDC DENV-1-4 Real-Time RT-PCR Assay (Multiplex) was established using a 5-replica panel of viruses sequentially diluted in human serum or plasma at 1:10 dilutions. In order to develop these panels, cultured, quantified (pfu/mL) stocks of whole virus (strains DENV-1 Haw, DENV-2 NGC, DENV-3 H87, DENV-4 H241) were used. Viral RNA from all replica samples was extracted using the Qiagen QIAamp® DSP Viral RNA Mini Kit (cat# 61904) on the Qiagen QIAcube (cat# 9001292) following manufacturer's protocol. 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 Ouantitative RT-PCR System (cat# 11732-088) on the Applied Biosystems® 7500 Fast Dx thermocycler.
The LoD of the CDC DENV-1-4 Real-Time RT-PCR Assay (Multiplex) in serum and plasma was the same (1 x 10 pfu/mL) for both RNA extraction methods, using the automated (Qiagen QIAamp® DSP Viral RNA Mini Kit and Qiagen QIAcube) and the manual procedure (Qiagen QIAamp® DSP Viral RNA Mini Kit).
iii) LoD of CDC DENV-1-4 Real-Time RT-PCR Assay (Multiplex) using an automated RNA extraction method (MagNA Pure LC Total Nucleic Acid Isolation Kit on the MagNA Pure LC 2.0 Instrument):
The LoD of the CDC DENV-1-4 Real-Time RT-PCR Assay (Multiplex) was established using a 5-replica panel of viruses sequentially diluted in human serum or plasma at 1:10 dilutions. In order to develop these panels, cultured, quantified (pfu/mL) stocks of whole virus (strains DENV-1 Haw, DENV-2 NGC, DENV-3 H87, DENV-4 H241) were used. Viral RNA from all replica samples was extracted using Roche MagnaPure LC 2.0 total Nucleic Acid
{13}------------------------------------------------
Isolation Kit (03 038 505.001) on the Roche Magna Pure LC 2.0 instrument following manufacturer's protocol. 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 (cat# 11732-088) on the Applied Biosystems® 7500 Fast Dx thermocycler.
The LoD of the CDC DENV-1-4 Real-Time RT-PCR Assay (Multiplex) in serum and plasma using an automated viral RNA extraction method (MagNA Pure LC total Nucleic Acid Isolation Kit on the MagNA Pure LC 2.0 instrument) was 1 x 10 pfu/mL.
There was no difference in the LoD of the CDC DENV-1-4 Real-Time RT-PCR Assay (Multiplex) using the described three viral extraction methods - an automated MagNA Pure LC total Nucleic Acid Isolation Kit on the MagNA Pure LC 2.0 instrument, an automated Qiagen QIAamp® DSP Viral RNA Mini Kit and Qiagen QIAcube, or a manual procedure (Qiagen QIAamp® DSP Viral RNA Mini Kit).
iv) Analytical Reactivity
In order to assess if the CDC DENV-1-4 Real-Time RT-PCR Assay detects a variety of currently circulating strains, the observed LoD of the CDC DENV-1-4 Real-Time RT-PCR Assay was further confirmed using the additional Dengue-1 - 4 strains. Twenty nine Dengue-1 - 4 isolates obtained from patients from different countries were cultured and quantified. The quantified stocks were serially diluted in human serum at 1:10 dilutions to 102 and 102 pfu/mL and were tested. Triplicate samples of each dilution were tested by the CDC DENV-1-4 Real-Time RT-PCR Assay (Multiplex).
The results showed that the observed LoD of the CDC DENV-1-4 Real-Time RT-PCR Assay was similar in cultured isolates obtained from patients from different countries. Results are shown in the table below.
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| YEAR | Country | Genotype | AVG CT | STD | Pos/Total | $10^3$ pfu/mlGCE/ml | AVG CT | STD | Pos/Total | $10^2$ pfu/mlGCE/ml |
|---|---|---|---|---|---|---|---|---|---|---|
| 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
{15}------------------------------------------------
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.
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Cross Reactivity Panel
| Pathogen | Sample type | Concentrationpfu/ml | DENV RT-PCRRate 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 |
| Borreliaburgdorferi | 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
{17}------------------------------------------------
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).
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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. -
- 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
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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-1-4Real-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
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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-1-4Real-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.
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- Clinical cut-off: N/A
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- 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
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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.
§ 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).