DPP Zika IgM System, DPP Zika IgM System Control Pack, and DPP Micro Reader
Device Facts
| Record ID | K200506 |
|---|---|
| Device Name | DPP Zika IgM System, DPP Zika IgM System Control Pack, and DPP Micro Reader |
| Applicant | Chembio Diagnostic Systems |
| Product Code | QFO · Microbiology |
| Decision Date | Jun 3, 2020 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.3935 |
| Device Class | Class 2 |
Indications for Use
The DPP Zika IgM System is intended for the presumptive qualitative detection of Zika virus IgM antibodies in human serum (plain or separation gel), potassium-EDTA plasma, potassium-EDTA venous whole blood, or fingerstick whole blood specimens, collected from individuals meeting the CDC Zika virus clinical criteria (e.g., a history of clinical signs and symptoms associated with Zika virus infection) and/or CDC Zika virus epidemiological criteria (e.g., history of residence in or travel to a geographic region with active Zika transmission at the time of travel, or other epidemiological criteria for which Zika virus testing may be indicated). Specimens from symptomatic patients or returning travelers from endemic areas should not be collected prior to 8 days after symptom onset or after potential exposure as a sample collected earlier may return a negative result. If testing is needed after day 4 but before day 8 and results are negative, testing must be repeated one week later. Positive results must be confirmed by following the latest CDC guidelines for the diagnosis of Zika virus infection. Results of this test are intended to be used in conjunction with clinical observations, patient history, epidemiological information, and other laboratory results. Zika IgM levels over the course of illness are not well characterized. Zika IgM levels are variable during the course of infection and may be detectable near day 4 post-onset of symptoms and persist up to approximately 12 weeks following initial infection. Negative results may be seen in specimens collected before day four post-onset of symptoms or after the window of detectable IgM closes and therefore do not preclude the possibility of Zika virus infection, past or present. The Chembio DPP Zika IgM System is not indicated for testing blood or plasma donors. The test cannot be visually interpreted by the operator and must be read on the DPP Micro Reader.
Device Story
The DPP Zika IgM System is a lateral cross-flow dual path platform (DPP) immunochromatographic assay for presumptive detection of Zika virus IgM antibodies. Input samples (serum, plasma, or whole blood) are applied to a test device; a sequential flow process separates sample migration from detector nanoparticle release, mimicking ELISA to minimize hook effects. The device uses immobilized recombinant Zika NS-1 antigen in the test area and Protein A in the control area. Results are read exclusively by the DPP Micro Reader, a portable, battery-powered reflectance reader that provides a qualitative (reactive/non-reactive) output on an LCD screen. Used in clinical settings by healthcare providers to aid in diagnosing Zika virus infection in conjunction with patient history and epidemiological data. The system includes an external control pack for quality assurance. The mandatory use of the reader eliminates subjective visual interpretation, reducing operator error and supporting clinical decision-making for patient management.
Clinical Evidence
Clinical performance evaluated via prospective and retrospective studies using serum, plasma, and whole blood. PPA for serum was 95.1% (95% CI 83.9-98.7%) against EUA comparator. Potassium-EDTA plasma PPA was 100% (95% CI 97.8-100.0%) in external sites. Venous whole blood PPA was 92.4% (95% CI 88-95.3%) for days 8-84 post-symptom onset. Fingerstick whole blood PPA was 98.7% (95% CI 96.4-99.6%). NPA was established in endemic and non-endemic populations, ranging from 93.0% to 98.7% depending on matrix and site. Analytical studies included precision, cross-reactivity (Dengue 2%, CMV 5.3%), and interference testing.
Technological Characteristics
Lateral cross-flow dual path platform (DPP) immunochromatographic assay. Materials: membrane strip with immobilized recombinant Zika NS-1 antigen and Protein A. Energy source: battery-powered (CR2032) reflectance reader. Connectivity: standalone. Sterilization: N/A. Software: embedded firmware in DPP Micro Reader for reflectance-based qualitative analysis. Complies with CLSI EP05-A3, EP07, EP12-A2, EP15-A3, EP17-A2, EP25-A, EP37, and IEC 60601-1-2.
Indications for Use
Indicated for presumptive qualitative detection of Zika virus IgM antibodies in human serum, potassium-EDTA plasma, potassium-EDTA venous whole blood, or fingerstick whole blood. For individuals meeting CDC clinical/epidemiological criteria for Zika virus testing. Not for blood or plasma donors.
Regulatory Classification
Identification
Zika virus serological reagents are in vitro diagnostic devices that consist of antigens or antibodies for the detection of Zika virus or Zika antibodies in human specimens from individuals who have signs and symptoms consistent with Zika virus infection and/or epidemiological risk factors. The detection aids in the diagnosis of current or recent Zika virus infection or serological status. Negative results obtained with this test do not preclude the possibility of Zika virus infection, past or present. Positive results should be interpreted with consideration of other clinical information and laboratory findings and should not be used as the sole basis for treatment or other patient management decisions.
Special Controls
The special controls for this device are:
*Classification.* Class II (special controls). The special controls for this device are:(1) The labeling required under § 809.10(b) of this chapter must include: (i) An intended use with a detailed description of what the device detects (Zika IgM antibodies, other Zika antibodies, or Zika antigens), the type of results provided to the user, the specimen type for which testing is indicated ( *e.g.,* serum, whole blood), the clinical indications appropriate for test use, and the specific population(s) for which the test is intended.(ii) Performance characteristics from analytical and clinical studies required under paragraphs (b)(2)(ii) and (iii) of this section. (iii) A detailed explanation of the interpretation of results and criteria for validity of results ( *e.g.,* criteria that internal or external quality controls must meet in order for a test/test run to be valid, minimum signal strength that the sample has to yield to be interpretable as a valid result).(iv) Limiting statements indicating that: (A) Results are not intended to be used as the sole basis for diagnosis, treatment, or other patient management decisions. The test results should be interpreted in conjunction with clinical observations, patient history, epidemiological information, and other laboratory evidence. (B) Device results are intended to be followed up according to the latest professional guidelines ( *e.g.,* recommendations from the Centers for Disease Control and Prevention) for the diagnosis of Zika virus infection.(C) Negative test results do not preclude the possibility of Zika virus infection, past or present. (D) Specimens can result in false negative results on the device if collected outside of the appropriate response window for specific Zika virus antigens or antibodies, as determined by scientific evidence ( *e.g.,* for IgM <7 days post symptom onset (pso) or risk of exposure and if collected past 84 days pso).(v) Detailed instructions for use that minimize the risk of generating a false positive or false negative result ( *e.g.,* co-testing of other matrices).(2) Design verification and validation must include: (i) A detailed device description, including all device parts ( *e.g.,* Zika antigen target, other flavivirus antigen target, capture antibodies), instrument requirements, ancillary reagents required but not provided, and the technological characteristics, including all pre-analytical methods for specimen processing.(ii) Detailed documentation and results from analytical performance studies including: characterization of the cut-off(s), analytical sensitivity to a standardized reference material that FDA has determined is appropriate ( *e.g.,* World Health Organization reference standard or the Centers for Disease Control and Prevention reference standard), class specificity for human antibodies (*e.g.,* IgM or IgG), analytical specificity (cross reactivity including cross reactivity to other flaviviruses), interference, carryover/cross contamination, specimen stability, hook effect (if applicable), matrix equivalency (if applicable), freeze-thaw studies (if applicable), and reproducibility.(iii) Detailed documentation and results from clinical studies, including the clinical study protocol (with a description of the testing algorithm and results interpretation table), detailed clinical study report, including line data of the clinical study results, and other appropriate statistical analysis. The samples used in the clinical study must be collected from subjects representative of the full spectrum of the intended use population ( *e.g.,* endemic and non-endemic regions if both are indicated).
Predicate Devices
- ZIKV Detect 2.0 IgM Capture ELISA (DEN180069)
Related Devices
- K192046 — LIAISON XL Zika Capture IgM II and LIAISON XL Zika Capture IgM II Control Set · DiaSorin, Inc. · Oct 28, 2019
- K191578 — ADVIA Centaur Zika test, ADVIA Centaur Zika Ab (100 tests), ADVIA Centaur Zika IgM (50 tests), ADVIA Centaur Zika Ab Quality Control, ADVIA Centaur Zika IgM Quality Control · Siemens Healthcare Diagnostics, Inc. · Jul 17, 2019
Submission Summary (Full Text)
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Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.gov
# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY
ASSAY AND INSTRUMENT
## I Background Information:
A 510(k) Number
K200506
B Applicant
Chembio Diagnostic Systems
C Proprietary and Established Names
DPP Zika IgM System, DPP Zika IgM System Control Pack, and DPP Micro Reader
## D Regulatory Information
| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| QFO | Class II | 21 CFR 866.3935 - Zika Virus Serological Reagents | MI - Microbiology |
| QCH | Class II | 21 CFR 866.3920 - Assayed quality control material for clinical microbiology assays | MI - Microbiology |
| JJQ | Class I | 21 CFR 862.2300 - Colorimeter, photometer, or spectrophotometer for clinical use | CH - Clinical Chemistry |
## II Submission/Device Overview:
### A Purpose for Submission:
The purpose of this premarket notification is to submit a new device (DPP Zika IgM System) to the FDA for consideration for clearance.
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B Measurand:
Human IgM antibodies against Zika virus.
C Type of Test:
The Chembio DPP Zika IgM System is a qualitative immunochromatographic assay for the presumptive detection of IgM antibodies to Zika virus.
III Intended Use/Indications for Use:
A Intended Use(s):
See Indications for Use below.
B Indication(s) for Use:
DPP Zika IgM System
The DPP Zika IgM System is intended for the presumptive qualitative detection of Zika virus IgM antibodies in human serum (plain or separation gel), potassium-EDTA plasma, potassium-EDTA venous whole blood, or fingerstick whole blood specimens, collected from individuals meeting the CDC Zika virus clinical criteria (e.g., a history of clinical signs and symptoms associated with Zika virus infection) and/or CDC Zika virus epidemiological criteria (e.g., history of residence in or travel to a geographic region with active Zika transmission at the time of travel, or other epidemiological criteria for which Zika virus testing may be indicated). Specimens from symptomatic patients or returning travelers from endemic areas should not be collected prior to 8 days after symptom onset or after potential exposure as a sample collected earlier may return a negative result. If testing is needed after day 4 but before day 8 and results are negative, testing must be repeated one week later. Positive results must be confirmed by following the latest CDC guidelines for the diagnosis of Zika virus infection.
Results of this test are intended to be used in conjunction with clinical observations, patient history, epidemiological information, and other laboratory results. Zika IgM levels over the course of illness are not well characterized. Zika IgM levels are variable during the course of infection and may be detectable near day 4 post-onset of symptoms and persist up to approximately 12 weeks following initial infection.
Negative results may be seen in specimens collected before day four post-onset of symptoms or after the window of detectable IgM closes and therefore do not preclude the possibility of Zika virus infection, past or present.
The Chembio DPP Zika IgM System is not indicated for testing blood or plasma donors.
The test cannot be visually interpreted by the operator and must be read on the DPP Micro Reader.
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DPP Zika IgM System Control Pack
The Chembio DPP Zika IgM System Control Pack is an external quality control kit for use with the DPP Zika IgM System only. The performance characteristics of the DPP Zika IgM System Control Pack have not been established for any other assay or instrument different from the DPP Micro Reader.
DPP MicroReader
The DPP Micro Reader is a reflectance reader used to obtain test results from DPP Zika IgM System. The DPP Micro Reader is necessary to minimize errors from direct visual interpretation; therefore, the results of DPP Zika IgM System cartridges must be read exclusively with the DPP Micro Reader.
## C Special Conditions for Use Statement(s):
Rx - For Prescription Use Only
## D Special Instrument Requirements:
Chembio DPP Micro Reader
## IV Device/System Characteristics:
### A Device Description:
The Chembio DPP Zika IgM System is a qualitative immunochromatographic assay for the presumptive detection of IgM antibodies to Zika virus. The Chembio DPP Zika IgM System includes the DPP Zika Test Device and the DPP Micro Reader.
### MATERIALS PROVIDED
Each kit contains the reagents and tools to perform 20 tests:
20 individually pouched DPP Zika IgM Test Devices, each containing:
- 1 DPP Zika Test Device (membrane strip with immobilized recombinant Zika NS-1 antigen in the TEST (T) area and Protein A in the CONTROL (C) area.
- 1 Desiccant Pouch
20 Disposable 10 µL Microsafe Tubes
20 Sample vials
20 Transfer Pipets (100 µl)
1 DPP Zika IgM Buffer– YELLOW Cap
- 7.5 mL contains sodium phosphate, sodium chloride, EDTA, NP-40, Tween 20, Urea, chicken serum, gentamicin, streptomycin, and sodium azide as preservative.
1 Product Insert for the DPP Zika IgM System
1 Quick Reference Guide for the DPP Zika IgM System
### MATERIALS REQUIRED BUT NOT PROVIDED (SYSTEM RELATED)
- Chembio DPP Micro Reader.
Each kit contains:
- DPP Micro Reader with Zika IgM RFID sticker
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3 Lithium-ion, type CR2032 (3 V/230 mAh), coin cell batteries (installed)
- Custom power adapter cable (USB to 2.0 mm jack)
- Power plug adaptor
- DPP Cartridge Holder
- Microfiber cloth
- User Manual
## ADDITIONAL REQUIRED MATERIALS (ASSAY RELATED)
- Chembio DPP Zika IgM System Control Pack
- 1 DPP Zika Reactive Control (volume of 300 µL; enough to perform 15 tests): undiluted, naturally occurring Zika IgM positive plasma sample.
- 1 DPP Non-Reactive Control (volume of 300 µL; enough to perform 15 tests): undiluted, naturally occurring Zika IgM negative plasma sample.
- 1 DPP Zika Diluent (300 µL; enough to perform 15 tests): undiluted, naturally occurring Zika IgM negative plasma samples.
- 1 Product Insert
All reagents are supplied ready to use.
- Clock, watch, or other timing device
- Pipettor capable of delivering 10-100 µL of sample may be used in lieu of the disposable 10 µL MicroSafe Tube and 100 µL Transfer Pipets supplied with the kit (for serum, potassium-EDTA plasma, and potassium-EDTA venous whole blood specimens or with the Chembio DPP Zika IgM System Control Pack)
- Microcentrifuge Tubes
- Disposable gloves
- Antiseptic wipes
- Biohazard disposal container
- For fingerstick whole blood specimens:
- Sterile gauze
- Sterile Safety Lancets for fingerstick whole blood specimens
- For venous whole blood or serum/plasma specimens:
- Collection devices
For a fingerstick whole blood sample, sample collection uses a 10 µl Microsafe Tube. For human serum (plain or separation gel), potassium-EDTA plasma, potassium-EDTA venous whole blood specimens, 10 µl of sample is collected using a calibrated laboratory pipet. Once the sample and buffer combination are added to the DPP Zika IgM Test Device, results are read by using the Chembio DPP Micro Reader, a portable, maintenance-free, battery-powered instrument that is operated by a single, multi-function button. The results of the DPP Zika IgM Test Device cannot be visually interpreted by the operator. Controls are kit lot specific and must not be interchanged between different DPP Zika IgM System lots.
## B Principle of Operation:
The Chembio DPP Zika IgM System is a qualitative immunochromatographic assay for the presumptive detection of IgM antibodies to Zika virus. The device employs Chembio’s DPP (Dual Path Platform) technology that differs from traditional lateral flow technology by
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delivering the sample directly onto the capture zone, followed by delivery of the colored conjugate. To initiate the test, a $10~\mu \mathrm{L}$ specimen is collected, diluted with kit buffer and applied to the SAMPLE+BUFFER Well#1 of the DPP Zika Test Device. The specimen migrates along the sample path membrane and is delivered to the TEST (T) area of the reagent strip, where Zika NS1 antigens are immobilized. Zika-specific antibodies, if present in the sample, bind to the immobilized NS1 antigens in the T area, while non-specific antibodies bind to the Protein A in the CONTROL (C) area. Successful sample application is indicated by the disappearance of soluble dye lines in the T and C areas. Five minutes after adding the sample, buffer is added into the BUFFER Well #2. The buffer hydrates the dried IgM antibody-binding colored conjugate, which migrates to the T area. Interpretation of reactions is performed by using the Chembio DPP Micro Reader an instrument that uses assay-specific algorithms to verify the presence of the C line and measure color intensity at the T line position.
The DPP Micro Reader interprets the result using assay-specific cut-off values, and reports a REACTIVE, NON-REACTIVE or INVALID result along with a numerical intensity value for the IgM test line after approximately 3 seconds. The results are presented through a 14-segment liquid crystal display (LCD) on the top of the instrument and displays the test results to the operator.
Patient results should be interpreted as follows:
| READER DISPLAY | Result | Interpretation | Follow-Up |
| --- | --- | --- | --- |
| <20 | NON-REACTIVE | Zika IgM antibodies were not detected in the specimen. | Pregnant women and sexually active men not practicing birth control should be re-tested with a later bleed taken at least 7 days from the first specimen. In the case of pregnant women please follow the latest CDC Interim Guidance for Health Care Providers Caring for Pregnant Women with Possible Zika Virus regarding clinical management of negative results (https://www.cdc.gov/zika/hc-providers/index.). |
| ≥20 | REACTIVE | IgM antibodies to Zika virus detected in the specimen. | The result should be confirmed by the latest CDC testing algorithms. For information regarding Zika testing algorithm, please refer to CDC guidance for state and local public health laboratories: https://www.cdc.gov/zika/laboratories/index.. |
| INV | INVALID | The test results cannot be interpreted | It is recommended that the INVALID test be repeated with a new device. |
Note: The magnitude of the reported Index value is not indicative of the amount of Zika virus immunoglobulin present in the patient sample.
Results should be read 15 minutes after addition of the Buffer into Well #2.
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Negative results with specimens collected before 8 days after onset of symptoms must be repeated with a later bleed taken at least 7 days from the first specimen.
The DPP Micro Reader is necessary to minimize errors from direct visual interpretation; therefore, results of DPP Zika IgM System must be read exclusively with the DPP Micro Reader.
The DPP Micro Reader is not configurable by the user. The DPP Micro Reader has been individually adapted for specific use with the DPP Zika IgM System through a product-specific configuration file stored on a dedicated, irremovable, Radio Frequency Identifier (RFID) sticker affixed to the Micro Reader. The RFID must be used to operate the reader. The configuration file on the RFID sticker carries the parameters specific to the detection and quantitation of the "TEST" and "CONTROL" lines and the algorithm used by the reader to qualify and display test line results as reactive, non-reactive or indeterminate. The configuration file is uploaded onto the reader each time a measurement is made by the user.
C Instrument Description Information:
| Modes of Operation | Yes | No |
| --- | --- | --- |
| Does the applicant’s device contain the ability to transmit data to a computer, webserver, or mobile device? | ☐ | ☑ |
| Does the applicant’s device transmit data to a computer, webserver, or mobile device using wireless transmission? | ☐ | ☑ |
| Software | | |
| FDA has reviewed applicant’s Hazard Analysis and software development processes for this line of product types. | ☑ | ☐ |
1. Instrument Name:
DPP Micro Reader
2. Specimen Identification:
Sample identification is manually captured in a paper record.
3. Specimen Sampling and Handling:
Managed by a trained technician.
4. Calibration:
Not applicable
5. Quality Control:
The Chembio DPP Zika IgM System Control Pack is an external quality control kit for use with the DPP Zika IgM System only. The performance characteristics of the DPP Zika IgM
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System Control Pack have not been established for any other assay or instrument different from the DPP Micro Reader.
Chembio DPP Zika IgM System Control Pack Reactive/Non-Reactive Controls are human, plasma-based reagents. The controls are specifically formulated and manufactured to ensure performance of the test and are used to verify the user's ability to properly perform the test and interpret the results. Use of control reagents manufactured by another source may not produce the required results, and therefore, may not meet the requirements for an adequate quality assurance program for the DPP Zika IgM System.
If control values lie outside the expected ranges, the test is invalid and patient results cannot be reported. It is the responsibility of each site using the DPP Zika IgM System to establish an adequate quality assurance program to ensure the performance of the device under its specific locations and conditions of use. Quality control requirements should be followed in conformance with local, state, and federal regulations or accreditation requirements and the site's standard quality control procedures.
V Substantial Equivalence Information:
A Predicate Device Name(s):
ZIKV Detect 2.0 IgM Capture ELISA
B Predicate 510(k) Number(s):
DEN180069
C Comparison with Predicate(s):
| Device & Predicate Device(s): | K200506 | DEN180069 |
| --- | --- | --- |
| Device Trade Name | Chembio DPP Zika IgM System | Inbios ZIKV Detect 2.0 IgM Capture ELISA |
| General Device Characteristic Similarities | | |
| Analyte | Human Zika virus IgM antibodies | Same |
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| Intended Use/Indications For Use | The DPP Zika IgM System is intended for the presumptive qualitative detection of Zika virus IgM antibodies in human serum (plain or separation gel), potassium-EDTA plasma, potassium-EDTA venous whole blood, or fingerstick whole blood specimens, collected from individuals meeting the CDC Zika virus clinical criteria (e.g., a history of clinical signs and symptoms associated with Zika virus infection) and/or CDC Zika virus epidemiological criteria (e.g., history of residence in or travel to a geographic region with active Zika transmission at the time of travel, or other epidemiological criteria for which Zika virus testing may be indicated). Specimens from symptomatic patients or returning travelers from endemic areas should not be collected prior to 8 days after symptom onset or after potential exposure as a sample collected earlier may return a negative result. If testing is needed after day 4 but before day 8 and results are negative, testing must be repeated one week later. Positive results must be confirmed by following the latest CDC guidelines for the diagnosis of Zika virus infection.
Results of this test are intended to be used in conjunction with clinical observations, patient history, epidemiological information, and other laboratory results. Zika IgM levels over the course of illness are not well characterized. Zika IgM levels are variable during the course of infection and may be detectable near day 4 post-onset of symptoms and persist up to approximately 12 weeks following initial infection.
Negative results may be seen in specimens collected before day four post-onset of symptoms or after the window of detectable IgM closes and therefore do not preclude the possibility of Zika virus infection, past or present.
The Chembio DPP Zika IgM System is not indicated for testing blood or plasma donors.
The test cannot be visually interpreted by the operator and must be read on the DPP Micro Reader. | The ZIKV Detect 2.0 IgM Capture ELISA is intended for the qualitative detection of Zika virus IgM antibodies in human sera for the presumptive clinical laboratory diagnosis of Zika virus infection. The assay is intended for use only in patients with clinical signs and symptoms consistent with Zika virus infection, and/or CDC Zika virus epidemiological criteria (e.g., history of residence in or travel to a geographic region with active Zika transmission at the time of travel, or other epidemiological criteria for which Zika virus testing may be indicated). Assay results are for the presumptive detection of IgM antibodies to Zika virus (ZIKV). Positive results must be confirmed by following the latest CDC guidelines for the diagnosis of Zika virus infection.
Results of this test are intended to be used in conjunction with clinical observations, patient history, epidemiological information, and other laboratory evidence to make patient management decisions. Zika IgM levels are variable over the course of the infection and may be detectable near day four post onset of symptoms and persist up to approximately 12 weeks following initial infection.
Negative results may be seen in specimens collected before day four post onset of symptoms or after the window of detectable IgM closes, and therefore do not preclude the possibility of Zika virus infection, past or present.
This assay is not indicated for testing blood or plasma donors. |
| --- | --- | --- |
| General Device Characteristic Differences | | |
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VI Standards/Guidance Documents Referenced:
| Sample Type | Human serum (plain or separation gel), potassium-EDTA plasma, potassium-EDTA venous whole blood, or fingerstick whole blood specimens | Human serum |
| --- | --- | --- |
| Sample Size | Minimum of 10 μL before dilution | Minimum of 4 μL before dilution |
| Antibody target | NS-1 protein | Zika envelope glycoproteins |
| Type of Test | Lateral cross-flow dual path platform (DPP) immunochromatographic assay | IgM Capture enzyme-linked immunosorbent assay (ELISA) |
| Quality Control | Provided Separately | Included |
| Interpretation of Results | <20 NON-REACTIVE
≥20 REACTIVE INVALID
Result is displayed through use of the DPP Microreader. No calculations are required by the user. | Zika Ag OD450 ≥ Threshold Zika Ag OD450 AND Zika ISR value > 1.90: Presumptive Zika Positive
Initial: Zika Ag OD450 ≥ Threshold Zika Ag OD450 AND 1.50 ≤ Zika ISR ≤ 1.90: Retest in duplicate
Retest: Zika Ag OD450 ≥ Threshold Zika Ag OD450 AND Zika ISR value ≥ 1.70
**Presumptive Zika Positive**
Not Presumptive Zika Positive & CCA / NCA ratio ≥ 5.00: Presumptive Other Flavivirus Positive
Not Presumptive Zika Positive & CCA / NCA ratio < 5.00: Negative
Interpretation requires calculations performed by the user prior to reporting the result |
| Time to Result | Time to result is 15 minutes after addition of Buffer into Well#2. | Time to result is approximately 3.5 hrs. |
| Reagent Storage | 2-8°C Refrigerator or up to 23°C | 2-8°C Refrigerator only |
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| Standard Title | Document Number | Publication Date | Recognition Number |
| --- | --- | --- | --- |
| Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline – Third Edition | CLSI EP05-A3 | 2014 | 7-251 |
| Interference Testing in Clinical Chemistry. Third edition | CLSI EP07-3rd Ed. | 2018 | 7-275 |
| User Protocol for Evaluation of Qualitative Test Performance Approved Guideline – Second Edition | CLSI EP12-A2 | 2008 | 7-152 |
| User of Performance for Precision and Trueness, Approved Guideline— Third edition | CLSI EP15-A3 | 2014 | 7-253 |
| Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline - Second Edition | CLSI EP17-A2 | 2012 | 7-233 |
| Evaluation of Stability of In Vitro Diagnostic Reagents; Approved Guideline. | CLSI EP25-A | 2009 | 7-235 |
| Supplemental Tables For Interference Testing In Clinical Chemistry | CLSI EP37 | 2018 | 7-284 |
| EMC-the International Electrotechnical Commission (IEC) | (IEC) 60601-1-2 Edition 4.0 2014-02 | 2014 | 19-8 |
| Quality System Regulation | 21CFR 820 | | N/A |
| Labeling for in vitro diagnostic products | 21CFR 809.10 | | N/A |
| Colorimeter, photometer, or spectrophotometer for clinical use | 21 CFR §862.2300 | | N/A |
| Special controls under, Zika virus serological reagents | 21 CFR §866.3935 | 2019 | N/A |
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VII Performance Characteristics (if/when applicable):
A Analytical Performance:
1. Precision/Reproducibility:
a. DPP Zika IgM System
Multi-site precision of the DPP Zika IgM System was evaluated at three US sites. A four-member panel (negative, high negative, low positive and high positive) plasma samples were measured using three (3) lots of the DPP Zika IgM System. Testing was performed over five days, with two runs per day. Each run was performed by one operator at each site, for a total of 6 operators and instruments (operator is confounded with the run and instrument). Each panel member was tested by only one operator in triplicates for each of the 3 assay lots (5 x 3 x 3 x 2 x 3 design; n = 270 for each sample).
Precision estimates were derived for reproducibility.
Reproducibility Results for the DPP Zika IgM System
| | | | Within-Run | | Between-Operator/Instrument | | Between-Day | | Between-Lot | | Between-Site | | Total* | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample ID | Mean Reader Value | N | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| Moderate Positive | 110.0 | 270 | 13.4 | 12.2 | 2.8 | 2.5 | 0.0 | 0.0 | 6.3 | 5.8 | 0.0 | 0.0 | 15.1 | 13.7 |
| Low Positive | 36.5 | 270 | 5.5 | 15.0 | 2.4 | 6.7 | 0.0 | 0.0 | 3.6 | 1.0 | 0.0 | 0.0 | 7.0 | 19.0 |
| High Negative | 15.1 | 270 | 3.3 | 21.5 | 1.5 | 9.8 | 0.0 | 0.0 | 1.0 | 6.6 | 0.0 | 0.0 | 3.7 | 24.5 |
| Negative | 1.7 | 270 | 1.3 | n/a | 0.0 | n/a | 0.0 | n/a | 0.0 | n/a | 1.3 | n/a | 1.8 | n/a |
%CV coefficient of variation expressed as a percentage; SD standard deviation.
* Total = Within-Run + Between-Operator/Instrument + Between-Day + Between-Lot + Between-Site
b. DPP Zika IgM System Control Pack
The precision of the controls was assayed separately using a single lot of controls and reagents. Testing was performed at a single site, over five days, with two operators per day, two instruments per operator, two runs per instrument, and two replicates per run (5 x 2 x 2 x 2 x 2 design; n = 80 for each sample). The data were analyzed for the Within-Run, Between-Run, Between-Instrument, Between-Operator, Between-Day and Total/Within-Lab.
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5-day Precision Results for DPP Zika IgM System Control Pack
| | | | Within-Run | | Between-Run | | Between-Instrument | | Between-Operator | | Between-Day | | Total* | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample ID | Mean Reader Value | N | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| Moderate Positive Control | 54.1 | 80 | 8.5 | 15.7 | 0.0 | 0.0 | 4.7 | 8.6 | 0.0 | 0.0 | 0.0 | 0.0 | 9.7 | 17.9 |
| Low Positive Control | 31.9 | 80 | 5.6 | 17.5 | 0.9 | 3.0 | 1.8 | 5.6 | 4.2 | 13.0 | 0.0 | 0.0 | 7.2 | 22.7 |
| Negative Control | 3.4 | 80 | 1.3 | n/a | 0.3 | n/a | 0.6 | n/a | 0.0 | n/a | 0.3 | n/a | 1.5 | n/a |
* Total = Within-Run + Between-Run + Between-Instrument + Between-Operator + Between-Day
2. Linearity:
Not applicable
3. Analytical Specificity/Interference:
a. Cross-Reactivity:
The performance of the DPP Zika IgM System was evaluated for the potential interference caused by autoantibodies, and antibodies against other closely related viruses as well as pathogens where infection produces symptoms similar to those observed during Zika virus infection, and potentially naturally occurring cross-reactive samples, previously characterized as positive per the supplier's Certificate of Analysis for various disease states using FDA cleared/ approved devices when possible. Except for specimens with HCV, HBV, Leptospira, ANA, Rheumatoid Factor, HAMA, Malaria, and Yellow Fever Virus Post-Immunization, only specimens confirmed positive for IgM of the respective disease state were included in the analysis.
One Dengue IgM sample (1/48) and two Cytomegalovirus IgM samples (2/38) were cross-reactive using the DPP Zika IgM System. Babesia cross-reactivity was not evaluated.
Summary of DPP Zika IgM System Cross-Reactivity
| | | | DPP Zika IgM System | | |
| --- | --- | --- | --- | --- | --- |
| Organism/Condition | # of Specimens | # Zika Negative | # Zika Positive | % Cross-reactivity | |
| Flavivirus | Anti-Chikungunya virus IgM | 26 | 26 | 0 | 0% |
| | Anti-Dengue Virus IgM | 48 | 47 | 1* | 2% |
| | Anti-West Nile Vile IgM | 28 | 28 | 0 | 0% |
| | Yellow fever virus post-immunization¹ | 32 | 32 | 0 | 0% |
| Other Viruses/diseases | Anti-Malaria/anti-plasmodium falciparum² | 20 | 20 | 0 | 0% |
| | Anti-Borrelia sp. (Lyme Disease) IgM | 10 | 10 | 0 | 0% |
| | Anti-Cytomegalovirus (CMV) IgM | 38 | 36 | 2** | 5.3% |
| | Anti-Epstein Barr Virus (EBV) IgM | 10 | 10 | 0 | 0% |
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| Anti-Hepatitis (B) virus (Total) | 13 | 13 | 0 | 0% |
| --- | --- | --- | --- | --- |
| Anti-Hepatitis (C) virus (Total) | 10 | 10 | 0 | 0% |
| Anti-Herpes simplex virus 1 and 2 (HSV-1, HSV-2) IgM | 23 | 23 | 0 | 0% |
| Anti-Leptospira (Leptospirosis) IgM³ | 16 | 16 | 0 | 0% |
| Anti-nuclear Antibodies (ANA) | 11 | 11 | 0 | 0% |
| Anti-Parvovirus B19 IgM | 7 | 7 | 0 | 0% |
| Anti-Rubella virus IgM | 12 | 12 | 0 | 0% |
| Rheumatoid Factor | 12 | 12 | 0 | 0% |
| Anti-Varicella zoster virus IgM | 3 | 3 | 0 | 0% |
| Human Anti-mouse Antibody (HAMA) | 10 | 10 | 0 | 0% |
| Total | 329 | 326 | 3 | |
* Negative by an FDA cleared Zika Assay; West Nile equivocal.
** Negative by an FDA cleared Zika Assay.
³ IgM levels could not be established.
³ Specimens were confirmed positive for Malaria infection by Giemsa and Microscopy but serological status is not known.
³ Pretreated with rheumatoid factor-absorbent prior to IgM detection.
## b. Endogenous Interference:
The DPP Zika IgM System was evaluated for potential interference with endogenous substances. These interfering substances were spiked into low reactive and normal human plasma samples to evaluate their impact on assay performance. In addition, to assure that the negative plasma itself had no effects on the test result, it was tested alone on the assay giving negative results. To further corroborate that the positive Zika IgM spike was appropriate, it was also tested alone on the assay showing positive results near cut-off. Each sample was tested in triplicate by one operator.
None of the tested endogenous substances cause a change in clinical interpretation for the DPP Zika IgM System.
## Summary DPP Zika IgM System Endogenous Interference
| Interfering substances | Concentration | DPP Zika IgM system | |
| --- | --- | --- | --- |
| | | (#reactive/ #tested) | |
| | | Negative diluent | Zika IgM Spike |
| | | (Non-Reactive) | (Reactive) |
| Hemoglobin | 10 mg/mL | 0/3 | 3/3 |
| Bilirubin, Conjugated | 0.4 mg/mL | 0/3 | 3/3 |
| Bilirubin, Un-Conjugated | 0.4 mg/mL | 0/3 | 3/3 |
| Serum Proteins | 60 mg/mL | 0/3 | 3/3 |
| HAMA | 732 ng/mL | 0/3 | 3/3 |
| Cholesterol | 4 mg/mL | 0/3 | 3/3 |
| Rheumatoid Factor | 2000 IU/mL | 0/3 | 3/3 |
| Triglycerides | 15 mg/mL | 0/3 | 3/3 |
## 4. Assay Reportable Range:
{13}
Not applicable
5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):
a. Traceability/Analytical Sensitivity:
The analytical sensitivity at the cut-off values for the DPP Zika IgM System was established using WHO 1st International standard for anti-Asian lineage Zika virus antibody (human) (NIBSC 16/352). This preparation contains antibodies reactive to Dengue virus. The standard was used to prepare a dilution series. The concentration of the reference reagent that corresponds to average reader values just above the cut-off of 20 (average values of <30 when analyzed by the DPP Micro Reader) and where at least 95% of all replicates tested reactive was 650 IU/mL in serum, 700 IU/mL in potassium-EDTA plasma, and 725 IU/mL in potassium-EDTA venous whole blood.
Results obtained with other NS1 assays suggest the standard has low concentration for the IgM isotype against this analyte, while the response is stronger for envelope-based assays.
b. Stability:
Sample Stability:
The study data supports specimen stability at the following storage temperatures and storage times:
- Potassium-EDTA venous whole blood and fingerstick whole blood immediate use
- Stored at 2–8°C separated serum and potassium-EDTA plasma samples for up to 3 days*
- No more than 2 freeze-thaw cycles for separated serum samples and 5 freeze-thaw cycles for potassium-EDTA plasma
| Summary of Stability Claims for the DPP Zika IgM System Reagents | |
| --- | --- |
| Stability Type | Claim |
| DPP Zika IgM System Reagent Shelf-Life Stability (up to 23 °C) | 6 months |
| DPP Zika IgM System Reagent Shelf-Life Stability (2-8 °C) | 12 months |
| DPP Zika IgM System Reagent In Use (up to 23 °C) | 1 month when used within the expiration date |
The user must not open the pouch until ready to perform a test.
| Summary of Stability Claims for the DPP Zika IgM System Control Pack | |
| --- | --- |
| Stability Type | Claim |
| DPP Zika IgM System Control Shelf-Life Stability (-20 °C) | 6 months |
| DPP Zika IgM System Control In Use Stability (-20 °C) | N/A** |
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| DPP Zika IgM System Control Freeze-Thaw Cycles | 3 |
| --- | --- |
* Based on two instead of three studies.
** It is recommended that the end user divide the controls into smaller aliquots that are then frozen back. The end user dilutes the controls only before use, and immediately discards any remaining material after use. Frozen aliquots are acceptable if they are both within the freeze-thaw cycles and the shelf life claimed above.
6. Detection Limit:
See Section 5.a, Traceability/Analytical Sensitivity.
7. Assay Cut-Off:
The assay cut-off value was determined using the Limit of the Blank (LoB) approach and through the testing of 569 natural serum samples sourced from United States and Mexico, both non-endemic populations; 184 natural plasma samples sourced from a non-endemic population from the United States (n=95) and an endemic population from Peru (n= 89); 215 natural venous whole blood samples sourced from a non-endemic population from the United States; and 102 natural capillary whole blood samples sourced from a non-endemic population from the United States.
The cut-off value was set at 20 when analyzed by the DPP Micro Reader: a reader value of < 20 is considered non-reactive and a value ≥ 20 is considered reactive. The value was corroborated by the clinical studies.
8. Prozone/ Hook Effect:
A high dose hook effect is not expected to occur due to the separation between the capture step and the conjugate addition step in the DPP Zika IgM System. On the primary flow path of DPP devices, the sample migrates towards the immobilized immunoreagents on the horizontal strip, that capture the analyte of interest, if present. Following a brief incubation to maximize binding efficiency, the detector nanoparticles are released from the conjugate pad via the secondary flow path. In addition, the conjugate buffer, devoid of any analyte, "cleans" the test membrane by "pushing" any remaining free analyte along the test strip. This sequential approach resembles the traditional ELISA assay process and minimizes the potential of the prozone (or hook) effect. Formal Hook Effect testing was not performed.
9. Class Specificity:
Antibody class specificity was evaluated on the DPP Zika IgM System. The reducing agent dithiothreitol (DTT) was used to specifically inactivate IgM antibodies. In this study, tests with sample buffer containing 10 mM DTT were run and compared to tests with the standard Zika IgM sample buffer formulation on three clinical Zika IgM positive samples. Sample buffer with 10 mM DTT resulted in non-reactive DPP Zika IgM System results in all the samples, suggesting that the IgM antibody reactivity was abolished. In contrast, standard Zika IgM sample buffer formulation resulted in the expected reactive DPP Zika IgM System results when tested with the same three clinical Zika IgM positive samples.
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DTT Effect on IgM Measurements for the DPP Zika IgM System
| | Specimens | | |
| --- | --- | --- | --- |
| | A
EDTA
PLASMA
IgM+/IgG+ | B (neat)
EDTA
PLASMA
IgM/IgG+ | B (1:64)
EDTA PLASMA
IGM+/ IgG+ |
| | DPP Zika IgM System Results | | |
| Standard Sample buffer | 140 (R) | 320 (R) | 330 (R) |
| Sample buffer with 10 mM DTT | 9 (NR) | 3 (NR) | 1 (NR) |
* This study was performed with an older version of the device that was determined equivalent to the one under clearance. The devices differ in that the cleared device 1) replaces the liquid form of the goat anti-human IgG antibodies and heterophilic antibody (HA) interference blocker for treatment of the specimen with the dehydrated form of the reagents placed in the DPP Zika IgM System device, and 2) replaces the DPP Zika IgM Sample Buffer and DPP Zika IgM Running Buffer vials by a single vial that is labeled DPP Zika IgM Buffer. Class specificity should not be affected by the changes, as the DTT treatment is added to the sample.
The results of these studies demonstrate IgM class specificity for the DPP Zika IgM System.
10. Carry-Over:
Not applicable as a single sample is run per device.
B Comparison Studies:
1. Method Comparison with Predicate Device:
See section "C" below.
2. Matrix Comparison:
The performance of the DPP Zika IgM System was evaluated using different matrices. Potassium-EDTA plasma is considered the reference matrix for the DPP Zika IgM System as it is the matrix presented for the main clinical study involving serial bleeds. Equivalency to potassium-EDTA plasma was evaluated using:
a. 11 positive and negative natural matched frozen potassium-EDTA whole blood, potassium-EDTA plasma, and serum samples
b. 49 plasma-replaced venous whole blood. For plasma replacement, negative whole blood specimens from 49 individuals obtained from a U.S blood bank were centrifuged, the plasma portion was removed, and the pellet was carefully suspended in an equal volume of Zika IgM antibody positive potassium-EDTA plasma from the 49 Zika positive plasma samples.
c. 30 samples for each serum and potassium-EDTA plasma prepared from 5 individual negative sera and plasma sources respectively, spiked with up to a 10% volume of positive potassium-EDTA plasma specimens to obtain high negative, low positive, and 4 values across the dynamic range of the DPP assay and tested in duplicates
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d. 50 matched negative sample sets for serum and potassium-EDTA plasma samples sources and spiked with up to 10% volume of positive potassium-EDTA plasma specimens to obtain high negative, low positive, moderate positive, and high positive specimens tested in duplicates. Un-spiked negative samples were also included.
In conclusion, the results for four studies for the Matrix Equivalency are summarized below.
Summary Table for Matrix Equivalency of the DPP Zika IgM System with Known Positive and Negative Zika Specimens
| | Potassium-EDTA Whole Blood | Serum^ |
| --- | --- | --- |
| Potassium-EDTA Plasma | 100% (60/60) (%CI 94.0 – 100%) | 97.3% (88.5*/91) (%CI 91.5-99.2%) |
^ Average of mean agreement values obtained for each experiment. Some studies have 1 while others 2 replicates per sample. When one replicate agreed and one disagreed, it was counted as 0.5 sample agreement.
* Discordant results were only found in high negative samples prepared by spiking. Above the cut-off of the assay, agreement was 100%.
C Clinical Studies:
1. Positive Predictive Agreement:
Serum
One-hundred and forty-two (142) serum samples were collected from patients residing in flavivirus endemic regions in the Dominican Republic (11), Peru (99), and Brazil (32 samples from 26 individuals bled a first time and 6 repeated a second time). Six out of eleven (6/11) samples from Dominican Republic, all the samples from Peru, and the twenty-six (26) individual donors for the Brazilian samples were positive for Zika virus by rtPCR. One sample from Peru did not have sufficient volume to be tested by the comparator and was excluded. From the 141 samples available for comparator testing, 41 samples were positive by the comparator assay.
A high percentage of false positive results was observed against the comparator, largely contributed to by the source of the samples.
Positive Percent Agreement with the Comparator Assay on Serum from Endemic Areas - Internal site Overall Serum Agreement
| Collection Site | Total (n) | Comparator^{1} Zika IgM Reactive | | | Comparator^{1} Zika IgM Non-Reactive | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | DPP Zika IgM System Reactive (R)^{2} | DPP Zika IgM System Non-Reactive (NR)^{2} | Positive Percent Agreement (95% CI) | DPP Zika IgM System Reactive (R)^{2} | DPP Zika IgM System Non-Reactive (NR)^{2} | Negative Percent Agreement |
| Dominican Republic | 11 | 8 | 2^{3} | 80% (8/10) | 1^{4} | 0 | 0% (0/1) |
| Peru | 98 | 23 | 0 | 100% (23/23)^{5} | 68 | 7 | 9.3% (7/75^{5, 6}) |
| Brazil | 32 | 8^{7} | 0 | 100% (8/8) | 4 | 20 | 83.3% (20/24) |
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| Total | 141 | 39 | 2 | 95.1%
(39/41)^{8}
(83.9-98.7%) | 73 | 27 | 27%
(27/100) |
| --- | --- | --- | --- | --- | --- | --- | --- |
¹ EUA comparator assay reactive samples include Possible and Presumptive Zika Positive Specimens. EUA comparator assay non-reactive samples include Negative and Presumptive Other Flavivirus Positive specimens.
² This study was performed with an older version of the DPP Zika IgM System that was determined equivalent to the one under clearance.
³ Negative for Zika IgM antibody by both the DPP Zika IgM System and a second authorized Zika IgM assay, while positive by an EUA Zika virus rtPCR assay and the EUA test used as a comparator.
⁴ Negative for Zika IgM antibody by both the EUA test used as a comparator and a second authorized Zika IgM assay, while positive by an EUA Zika virus rtPCR assay.
⁵ All samples tested positive by a rtPCR assay for Zika virus.
⁶ All samples were ≤7 days post onset of symptoms. Even when positive by PCR, depending on the sensitivity of the assay, the level of antibodies may be low for detection.
⁷ There were 8 positive samples in total in the 32. Two out of eight (2/8) samples were a single sample picked in both the first and second blood draw; 1/8 was only picked on the first blood draw; 5/8 were picked only on the second blood draw.
⁸ Two of the eight (2/8) Brazilian positive samples were a single sample picked in both the first and second blood draw and counted twice for final performance calculations. Without double counting the specimen, the total PPA would be 95.0% (83.5-98.6%).
# Potassium-EDTA Plasma
## Internal Site
Positive percent agreement (PPA) for potassium-EDTA plasma was evaluated using archived samples consisting of eight serial samples collected from 50 symptomatic subjects from the Dominican Republic. Of these subjects confirmed positive for Zika virus by nucleic acid testing, only forty-eight (48) tested also positive for Zika antibodies in at least one of the serial bleeds by a Zika IgM comparator assay and were included in the analysis, for a total of 299 samples. The 48 subjects included 12 pregnant women. All samples were tested at an internal site and results are presented below.
Positive Percent Agreement with the Comparator Assay on Potassium-EDTA Plasma, Stratified by Number of Days Post-Onset of Symptoms - Internal site
| Days Post Onset of Symptoms | Total (n) | Comparator^{1} Zika IgM Reactive^{2} | | | Comparator^{1} Zika IgM Non-Reactive^{3} | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | DPP Zika IgM System Reactive (R) | DPP Zika IgM System Non-Reactive (NR) | Positive Percent Agreement^{4} (95% CI) | DPP Zika IgM System Reactive (R) | DPP Zika IgM System Non-Reactive (NR) | Negative Percent Agreement |
| 0-7* | 51 | 6 | 5 | 54.6% (6/11) | 11 | 29 | 72.5% (29/40) |
| 8-14 | 39 | 39 | 0 | 100.0% (39/39) | NT | NT | NT |
| 15-28 | 86 | 82 | 0 | 100.0% (82/82) | 4 | 0 | 0% (0/4) |
| 29-42 | 86 | 79 | 0 | 100.0% (79/79) | 7 | 0 | 0% (0/7) |
| 43-56 | 82 | 61 | 0 | 100.0% (61/61) | 21 | 0 | 0% (0/21) |
| 57-70 | 29 | 19 | 0 | 100.0% (19/19) | 9 | 1 | 10% (1/10) |
| 71-84 | 11 | 8 | 0 | 100.0% (8/8) | 3 | 0 | 0% (0/3) |
| Total | 384 | 294 | 5 | 100% (288/288)
(95% CI 98.7-100.0%) | 55 | 30 | 2.2% (1/45) |
* Data from Days 0 – 7 is not included in the calculation of total PPA because obtaining samples prior to 8 days after symptom onset or exposure is not recommended and false negative results are anticipated.
¹ EUA comparator assay
² Comparator reactive samples include Possible and Presumptive Zika Positive Specimens.
³ Comparator non-reactive samples include Negative and Presumptive Other Flavivirus Positive specimens.
⁴ Calculated using the score method.
NT: Not tested.
{18}
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# External Sites
Of the total of 299 positive samples from the Zika IgM endemic region mentioned in the study above, a subset of 171 bleeds from 39 positive individuals, including 11 samples from pregnant women, were divided and distributed among two external clinical sites (82 and 89 samples respectively). Forty-nine (49) prospectively collected plasma samples from asymptomatic pregnant women living in the continental United States known to be negative for Zika IgM antibodies by the comparator were interspersed for blinding (24 and 25 samples per site respectively). The combined data are summarized in the following table:
Positive Percent Agreement with the Comparator Assay on Potassium-EDTA Plasma, Stratified by Number of Days Post-Onset of Symptoms - External Sites
| Days Post Onset of Symptoms | Comparator^{1} Zika IgM Reactive | | Comparator^{2} Zika IgM Non-Reactive |
| --- | --- | --- | --- |
| | Positive Percent Agreement^{3} | 95% Confidence Interval | Negative Percent Agreement^{3} (95% CI) |
| Asymptomatic | N/A | N/A | 98.0% (48^{4}/49) (95% CI 89.3-99.6%) |
| 8-14 | 100.0% (29/29) | 88.3-100.0% | NT |
| 15-28 | 100.0% (48/48) | 92.6-100.0% | NT |
| 29-42 | 100.0% (46/46) | 92.3-100.0% | NT |
| 43-56 | 100.0% (29/29) | 88.3-100.0% | NT |
| 57-70 | 100.0% (12/12) | 75.8-100.0% | NT |
| 71-84 | 100.0% (7/7) | 64.6-100.0% | NT |
| Total | 171/171=100.0% (95%CI 97.8-100.0%) | | |
1 EUA comparator assay reactive samples include Possible and Presumptive Zika Positive Specimens.
2 Cleared comparator assay non-reactive samples include Negative and Presumptive Other Flavivirus Positive (non-Zika) specimens.
3 Calculated using the score method.
4 For one sample, the microreader value indicated a negative result, but the end-user recorded an interpretation of "R" for Reactive.
NT: Not Tested and not included in the calculations.
# Potassium-EDTA Venous Whole blood
The PPA of the DPP Zika IgM System for potassium-EDTA venous whole blood was evaluated by two studies. For the first study, samples were tested at either an internal site or at external sites:
# Internal Site
The positive percent agreement of the assay in potassium-EDTA venous whole blood was evaluated at an internal site using 10 frozen natural potassium-EDTA whole blood from the Dominican Republic that tested positive by the comparator assay, in addition to 42 contrived samples due to the low prevalence of Zika virus during the time of the study.
{19}
The contrived samples were prepared by plasma replacement, using serial bleeds from 6 individuals residing in the Dominican Republic. All subjects from this endemic area confirmed positive for Zika virus by nucleic acid testing were also positive for Zika antibodies in at least one of the serial bleeds by the comparator assay. Potassium-EDTA negative whole blood specimens from 42 individuals obtained from a U.S blood bank were replaced with potassium-EDTA serial plasma from the six Zika immune individuals. Results for all 42 plasma-replaced whole blood samples were reactive for Zika IgM antibodies on the DPP Zika IgM System, with 41 agreeing with the EUA comparator assay.
## External Sites
For the external sites, the same subset of 171 antibody positive plasma specimens plus the 49-antibody negative potassium-EDTA plasma specimen from asymptomatic pregnant women from the US as described in the plasma study above, was used in a plasma replacement study. Results were confirmed by the comparator assay. The one-hundred and seventy-one (171) plasma replaced venous whole blood positive specimens were distributed among two clinical sites (82 and 89 samples per site) combined with 24 or 25 known negative plasma samples respectively.
The summary of the results obtained with potassium-EDTA natural and potassium-EDTA plasma reconstituted venous whole blood across the sites mentioned above is shown below:
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| Positive Percent Agreement with the Comparator Assay on Natural and Potassium-EDTA Plasma Replaced Venous Whole Blood, Stratified by Number of Days Post-Onset of Symptoms Across Internal and Clinical SitesDays Post onset of Symptoms | Comparator¹ Zika IgM Reactive | | Comparator Zika IgM Non- Reactive |
| --- | --- | --- | --- |
| | Positive Percent Agreement² | 95% confidence interval | Negative Percent Agreement² |
| Negative³ | N/A | N/A | 98% (48⁴/49) |
| 0-7* | 83.3% (10/12) | 55.2-95.3% | NT |
| 8-14 | 97.1% (33⁵/34) | 85.1-99.5% | NT |
| 15-28 | 94.7% (54/57) | 85.6-98.2% | NT |
| 29-42 | 96.4% (53/55) | 87.7-99.0% | NT |
| 43-56 | 84.2% (32/38) | 69.6-92.6% | NT |
| 57-70 | 80.0% (12/15) | 54.8-93.0% | 0/1¹,⁶ |
| 71-84 | 90.9% (10/11) | 62.3-98.4% | NT |
| Total | | | 96.0% (48/50) (95% CI 86.5- 98.9%) |
| Total | 92.4% (194/210) (95% CI 88.0- 95.3) | | |
* Data from Days 0 – 7 is not included in the calculation of total PPA because obtaining samples prior to 8 days after symptom onset or exposure is not recommended and false negative results are anticipated.
¹ EUA comparator assay reactive samples include Possible and Presumptive Zika Positive Specimens.
² Calculated using the score method.
³ Cleared comparator assay non-reactive samples include Negative and Presumptive Other Flavivirus Positive (non-Zika) specimens.
⁴ For one sample, the microreader value indicated a negative result, but the end-user recorded an interpretation of “R” for Reactive.
⁵ For one sample, the microreader value indicated a reactive result, but the end-user recorded an interpretation of “NR” for Non-Reactive.
⁶ Negative by a second authorized Zika IgM serology assay.
NT: Not Tested and not included in the calculations.
The second study was conducted at 3 clinics in the US using prospectively-collected potassium-EDTA venous whole blood from subjects enrolled in an “all comers” fashion. One of the 300 subjects was excluded because their naïve whole blood tested reactive in the DPP Zika IgM System without being spiked, leaving 299 subjects for analysis. In order to assess positive agreement, samples were contrived by potassium-EDTA plasma replacement into venous whole blood samples to yield negative, low positive, and moderate positive specimens. The contrived samples were tested in a blinded fashion across three sites. Results were compared against the expected results based on the spiking level for each sample.
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Agreement with Expected Reactivity of Potassium-EDTA Plasma Replaced Venous Whole Blood Across Three Sites
| Zika IgM Target Level | Agreement to Expected Reactivity by Levels¹ | Combined Agreement |
| --- | --- | --- |
| Negative | 100% (120/120)
(95%CI 96.9-100.0%) | NPA
100% (120/120)
(95%CI 96.9-100.0%) |
| Low reactive (<3xLoD) | 99.1% (115/116)
(95%CI 95.3-99.9%) | PPA
99.4% (178/179)
(95%CI 96.9-99.9%) |
| Moderate reactive
(≥3xLoD) | 100.0% (63/63)
(95%CI 94.3-100.0%) | |
¹ Calculated using the score method.
## Capillary Whole Blood
The performance of the assay with capillary whole blood was evaluated in a total of 375 adult subjects across four near-patient sites in the US enrolled on an “all comers” basis. Due to the low prevalence of Zika virus during the time of the study, the evaluation of the performance of the assay with fingerstick was performed using contrived samples. After exclusion of a sample detected as reactive before spiking, the remaining 374 fingerstick samples prospectively collected were immediately placed into pre-labeled samples vials spiked with negative, low or moderate levels of anti-Zika IgM. Presence or absence of Zika IgM antibodies in the spiking material was corroborated by comparator testing. The vials were coded so that study staff and the two trained operators at the clinical laboratory were not aware of the predetermined reactivity of the spiked sample vials. Two samples were later eliminated from the analysis due to deviations in data transcription. In total, there were 372 vials (135 negative, 149 low and 88 moderate levels of Zika antibodies) that were tested with the DPP Zika IgM System. All testing was performed in near-patient settings.
Agreement with Expected Reactivity of Capillary Whole Blood
| Zika IgM Target Level | Agreement to Expected Reactivity by Level¹ | Combined Agreement |
| --- | --- | --- |
| Negative | 97.8% (132/135)
(95%CI 93.7-99.2%) | NPA
97.8% (132/135)
(95%CI 93.7-99.2%) |
| Low reactive
(2.5x - <3x Cut-Off) | 98.0% (146/149)
(95%CI 92.3-99.3%) | PPA
98.7% (234/237)
(95%CI 96.4-99.6%) |
| Moderate reactive
(≥3x - 5x Cut-Off) | 100.0% (88/88)
(95%CI 95.8-100%) | |
¹ Calculated using the score method.
## 2. Negative Predictive Agreement:
Negative percent agreement was evaluated using a total of 500 prospectively collected specimens tested at seven (7) near-patient sites, including four (4) sites in areas endemic for mosquito-borne flaviviruses (250 samples) and three (3) sites in non-endemic areas (250 samples). Potassium-EDTA venous and fingerstick whole blood were tested at near-patient settings. Paired serum, potassium-EDTA plasma and urine were sent to a reference laboratory where they were tested for Zika virus antibodies with the DPP Zika IgM System and the comparator method (data not shown).
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{22}
Samples were expected to be negative for Zika IgM antibodies due to the low prevalence of Zika virus during the study. For those samples found positive, results were expressed as a percentage of positive results in the total population under study. Samples were tested by the DPP Zika IgM System, the cleared comparator assay, and an additional serology EUA test. Results are summarized in the following tables. The different rate of positive results may be partially explained by the differential target of the detected antibodies and an increased false positive rate for each assay in endemic populations due to the low prevalence of the virus at the moment of collection.
Endemic Area
| | NPA# of DPP Zika IgM System by Comparison with Samples Negative By | | | | Percent of Positive Results in Population | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | FDA Cleared Comparator1 | Additional EUA Test2 | Both FDA Cleared and EUA tests | | DPP Zika IgM System3 | FDA Cleared Comparator1 | Additional EUA Test2 |
| Serum | 94.3% (213/226) | 94.8% (218/230) | 95.7% (202/211) | | 8.0% (204/250) | 9.6% (24/250) | 7.6% (19/250) |
| | 95%CI | 95%CI | 95%CI | | | | |
| | 90.4-98.6 | 91.1-97.0 | 92.1-97.7 | | | | |
| Potassium-EDTA Plasma | 93.4% (211/226) | 93.0% (214/230) | 93.8% (198/211) | | 8.8% (226/250) | 9.6% (24/250) | 7.6% (19/250) |
| | 95%CI | 95%CI | 95%CI | | | | |
| | 89.3-95.9 | 89.0-95.7 | 89.8-96.4 | | | | |
| Potassium-EDTA Venous Whole Blood | 93.8% (212/226) | 94.4% (217/230) | 94.8% (200/211) | | 7.6% (194/250) | 9.6% (24/250) | 7.6% (19/250) |
| | 95%CI | 95%CI | 95%CI | | | | |
| | 89.9-96.3 | 90.6-96.7 | 90.9-97.1 | | | | |
| Capillary Whole Blood | 96.0% (217/226) | 96.5% (222/230) | 97.2% (205/211) | | 5.6% (144/250) | 9.6% (24/250) | 7.6% (19/250) |
| | 95%CI | 95%CI | 95%CI | | | | |
| | 92.6-97.9 | 93.3-98.2 | 93.9-98.7 | | | | |
Calculated using the score method.
1 Detects IgM antibodies against envelope
2 Detects IgM and IgG antibodies anti NS-1
3 Detects IgM antibodies anti NS-1.
a 15 agree in 3 or all matrices, the remaining in 2 or less
b 14 agree in 3 or all matrices, the remaining in 2 or less
c 14 agree in 3 or all matrices, the remaining in 2 or less
d 11 agree in 3 or all matrices, the remaining in 2 or less
Non-Endemic Area
| | NPA# of DPP Zika IgM System by Comparison with Samples Negative By | | | | Percent of Positive Results in Population | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | FDA Cleared Comparator1 | Additional EUA Test2 | Both FDA Cleared and EUA tests | | DPP Zika IgM System3 | FDA Cleared Comparator1 | Additional EUA Test2 |
| Serum | 98.0% (239/244) | 98.3% (225/229) | 98.2% (220/224) | | 2.0% (54/250) | 2.4% (6/250) | 8.4% (21/250) |
| | 95%CI | 95%CI | 95%CI | | | | |
| | 95.3-99.1 | 95.6-99.3 | 95.5-99.3 | | | | |
| Potassium-EDTA Plasma | 96.7% (236/244) | 97.8% (224/229) | 97.8% (219/224) | | 3.2% (85/250) | 2.4% (6/250) | 8.4% (21/250) |
| | 95%CI | 95%CI | 95%CI | | | | |
| | 93.7-98.3 | 95.0-99.1 | 94.9-99.0 | | | | |
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| Potassium-EDTA
Venous
Whole
Blood | 97.1%
(237/244)
95%CI
94.2-98.6 | 97.4%
(223/229)
95%CI
94.4-98.8 | 97.3%
(218/224)
95%CI
94.3-98.8 | | 2.8%
(7°/250) | 2.4%
(6/250) | 8.4%
(21/250) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Capillary
Whole
Blood | 98.4%
(239/243)a
95%CI
95.9-99.4%) | 98.7%
(226/229)
95%CI
96.2-99.6 | 98.7%
(221/224)
95%CI
96.1-99.5 | | 1.6%
(4°/250) | 2.4%
(6/250) | 8.4%
(21/250) |
a Calculated using the score method.
b Detects IgM antibodies against envelope;
c Detects IgM and IgG antibodies anti NS-1;
d Detects IgM antibodies anti NS-1.
e FS result from one subject excluded due to protocol deviation (Test read < 15-minutes)
a 4 agree in 3 or all matrices, the remaining in 2 or less;
b 4 agree in 3 or all matrices, the remaining in 2 or less;
c 4 agree in 3 or all matrices, the remaining in 2 or less;
d 3 agree in 3 or all matrices, the remaining in 2 or less
## 3. Performance Against the FDA Plasma Zika Panel:
Performance of the DPP Zika IgM System was evaluated by testing a panel of samples provided to the sponsor by FDA. The panel consists of plasma samples from individuals infected with Zika, West Nile, or Dengue viruses. Samples were collected at different time points from Zika infection documented by PCR; samples taken early or late after infection may be anticipated to be antibody negative. Samples were randomized and blinded for testing. Results were as follows:
| | DPP Zika IgM System | | |
| --- | --- | --- | --- |
| | | Presumptive Zika Positive | Negative |
| Zika IgM
Consensus | Positive (n=24) | 23 | 1 |
| | Negative (n=12) | 1 | 11 |
PPA= 23/24, 95.8%
NPA= 11/12, 91.7%
| | DPP Zika IgM System | | |
| --- | --- | --- | --- |
| | | Presumptive Zika Positive
(False Positives) | Negative |
| Cross-reactivity
Evaluation | West Nile* (n=10) | 1 | 9 |
| | Dengue* (n=10) | 0 | 10 |
* Samples were antibody positive for West Nile or Dengue viruses, and negative for Zika virus.
This evaluation was performed using samples provided by Blood Systems Research Institute (BSRI, now Vitalant Research Institute) from a study supported by Contract No. HHSN268201100001I from the National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health. The panel composition and consensus results are the responsibility of the FDA and do not necessarily represent the official views of BSRI, the NHLBI, or the National Institutes of Health.
## 4. Other Clinical Supportive Data (When 1. and 2. Are Not Applicable):
Not applicable
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D Clinical Cut-Off:
Not applicable
E Expected Values/Reference Range:
Not applicable
F Other Supportive Instrument Performance Characteristics Data:
Not applicable
VIII Proposed Labeling:
The labeling supports the finding of substantial equivalence for this device.
IX Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.
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