HOLOGIC Zika Virus Assay Instruction Manual

: June 1, 2024
: HOLOGIC

HOLOGIC Zika Virus Assay

Product Information

Specifications

Intended Use: For Emergency Use Authorization (EUA) for in vitro diagnostic use only Rx
Method: Transcription-Mediated Amplification (TMA)
Enzymes Used: MMLV reverse transcriptase and T7 RNA polymerase
Specimen Types: Serum, plasma, urine, whole blood
Regulatory Approval: FDA Emergency Use Authorization

Product Usage Instructions

General Information

The Aptima Zika Virus assay is designed for trained clinical laboratory personnel familiar with nucleic acid amplification techniques. Negative results should not be the sole basis for patient management decisions.

Principles of the Procedure

Serum and plasma specimens can be loaded directly onto the Panther system. However, neat urine and whole blood specimens need manual processing before loading. The TMA method amplifies ZIKV RNA using MMLV reverse transcriptase and T7 RNA polymerase.

Warnings and Precautions

Ensure only trained personnel handle the assay. Follow strict procedures for potential spills. Use specified disposable lab ware to prevent contamination.

FAQs

Q: Can negative results from the Aptima Zika Virus assay rule out Zika virus infection?
- A: Negative results should be interpreted in conjunction with clinical observations, patient history, and epidemiological data. They do not definitively exclude Zika virus infection.
Q: What specimens can be directly loaded onto the Panther system for the assay?
- A: Serum and plasma specimens can be loaded directly, while neat urine and whole blood require manual processing before loading onto the system.

Aptima Zika Virus Assay

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General Information

Aptima®

Intended Use
The Aptima® Zika Virus assay is a transcription-mediated amplification test intended for the qualitative detection of RNA from the Zika virus in serum, plasma, processed urine, or processed whole blood K2EDTA (whole blood and urine collected alongside a patient-matched serum or plasma specimen) from individuals meeting CDC Zika virus clinical criteria (e.g., 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 virus transmission at the time of travel, or other epidemiological criteria for which Zika virus testing may be indicated), by laboratories in the United States that are certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. §263a, to perform high complexity tests, or by similarly qualified non-U.S. laboratories.
Specimens are tested using the Panther® System for automated specimen processing, amplification, and detection. Results are for the identification of Zika virus RNA. Zika virus RNA is generally detectable in serum during the acute phase of infection (approximately 14 days following onset of symptoms, if present). Positive results are indicative of current infection. Laboratories are required to report all positive results to the appropriate public health authorities.
Negative results do not preclude Zika virus infection and should not be used as the sole basis for patient management decisions. Negative results must be combined with clinical observations, patient history, and epidemiological information.
The Aptima Zika Virus assay is intended for use by trained clinical laboratory personnel specifically instructed and trained in the techniques of nucleic acid amplification and in vitro diagnostic procedures. This assay is only for use under the Food and Drug Administration’s Emergency Use Authorization.
Summary and Explanation of the Test
Zika virus (ZIKV) is an RNA virus that is a member of the Flaviviridae family and the genus Flavivirus.1 It is transmitted to humans by mosquitoes belonging to the Aedes genus.2 ZIKV was first identified in an infected rhesus macaque in 1947 in the Zika Forest of Uganda, followed by the first reported human cases in Uganda and the United Republic of Tanzania in 1952.3 Since then, sporadic outbreaks of ZIKV have been documented in many areas of Africa and Southeast Asia. The first occurrence of a ZIKV outbreak outside of Asia or Africa occurred in 2007, when a large outbreak occurred on the Pacific island of Yap, in the Federated States of Micronesia.4
In 2013 and 2014, a major outbreak of ZIKV disease, associated with clinical complications, was reported in French Polynesia.5 In May 2015, the first locally acquired cases of ZIKV infection in the Americas were confirmed in Brazil.6,7 As of early 2016, ZIKV had spread to other countries in South America, Central America, Mexico, and the Caribbean, including the U.S. territories of Puerto Rico and the Virgin Islands.7 ZIKV is typically associated with human disease ranging from subclinical infections to mild flu- like illnesses, but ZIKV infection has also been associated with serious and sometimes fatal cases of Guillain-Barré syndrome.8 The virus has also been linked with microcephaly and other birth defects in infants born to infected mothers.9 Although the primary route of infection appears to be through the bite of a mosquito, sexual transmission,10 and possible transfusion- transmission11 of ZIKV have also been reported.

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Aptima®

General Information

Principles of the Procedure
The Aptima Zika Virus assay targets two highly conserved regions in the NS2 and NS4/NS5 regions for increased tolerance to potential mutations. The assay involves three main steps, which take place in a single tube, on the automated Panther system: sample preparation, ZIKV RNA target amplification by Transcription-Mediated Amplification (TMA),12 and detection of the amplification products (amplicon) by Hybridization Protection Assay (HPA).13 The assay incorporates an internal control (IC) to monitor nucleic acid capture, amplification, and detection, as well as operator or instrument error.
Serum and plasma specimens are loaded directly onto the Panther system. However, neat urine and whole blood specimens must be manually processed prior to loading onto the Panther system.
On the Panther system, RNA is isolated from specimens via target capture. The specimen is treated with a detergent to solubilize the viral envelope, denature proteins, and release viral genomic RNA. Oligonucleotides (“capture oligonucleotides”) homologous to highly conserved regions of ZIKV are hybridized to the ZIKV RNA target, if present, in the test specimen. The hybridized target is then captured onto magnetic microparticles that are separated from the specimen in a magnetic field. Wash steps are utilized to remove extraneous components from the reaction tube. Magnetic separation and wash steps are performed with a target capture system.
Target amplification occurs via TMA, which is a transcription-based nucleic acid amplification method that utilizes two enzymes, MMLV reverse transcriptase and T7 RNA polymerase. The reverse transcriptase is used to generate a DNA copy (containing a promoter sequence for T7 RNA polymerase) of the target RNA sequence. The T7 RNA polymerase produces multiple copies of RNA amplicon from the DNA copy template. The Aptima Zika Virus assay utilizes the TMA method to amplify regions of ZIKV RNA.
Detection is achieved by HPA using single-stranded nucleic acid probes with chemiluminescent labels that are complementary to the amplicon. The labeled nucleic acid probes hybridize specifically to the amplicon. The Selection Reagent differentiates between hybridized and unhybridized probes by inactivating the label on unhybridized probes. During the detection step, the chemiluminescent signal produced by the hybridized probe is measured in a luminometer and is reported as Relative Light Units (RLU).
Internal Control is added to each test specimen and assay calibrator via the working Target Capture Reagent. The Internal Control in the Aptima Zika Virus assay controls for specimen processing, amplification, and detection steps. Internal Control signal is discriminated from the ZIKV signal by the differential kinetics of light emission from probes with different labels.13 Internal Control-specific amplicon is detected using a probe with rapid emission of light (flasher signal). Amplicon specific to ZIKV is detected using probes with relatively slower kinetics of light emission (glower signal). The Dual Kinetic Assay (DKA) is a method used to differentiate between the signals from flasher and glower labels.14
The Aptima Zika Virus assay calibrators are used to determine the assay cutoff and assess assay run validity in each run. See Quality Control for details.

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General Information

Aptima®

Warnings and Precautions
A. For in vitro diagnostic use. For use under an Emergency Use Authorization (EUA) only.
B. To reduce the risk of invalid results, carefully read the entire package insert and the Panther/ Panther Fusion Operator’s Manual prior to performing this assay.

Laboratory Related
C. Only personnel adequately trained in the use of the Aptima Zika Virus assay and in handling potentially infectious materials should perform this procedure. If a spill occurs, immediately disinfect following appropriate site procedures.
D. Use only supplied or specified disposable laboratory ware.
E. Use routine laboratory precautions. Do not pipet by mouth. Do not eat, drink, or smoke in designated work areas. Wear disposable, powderless gloves, protective eye wear, and laboratory coats when handling specimens and kit reagents. Wash hands thoroughly after handling specimens and kit reagents.
F. Work surfaces, pipettes, and other equipment must be regularly decontaminated with 2.5% to 3.5% (0.35 M to 0.5 M) sodium hypochlorite solution.
G. Dispose of all materials that have come in contact with specimens and reagents according to local, state, and federal regulations.15,16,17,18 Thoroughly clean and disinfect all work surfaces.
H. Enzyme Reagent contains sodium azide as a preservative. Do not use metal tubing for reagent transfer. If solutions containing sodium azide compounds are disposed of in a plumbing system, they should be diluted and flushed with generous amounts of running water. These precautions are recommended to avoid accumulation of deposits in metal piping in which explosive conditions could develop.

Specimen Related
I. Specimens may be infectious. Use Universal Precautions15,16,17 when performing this assay. Proper handling and disposal methods should be established according to local regulations.18 Only personnel adequately trained in the use of the Aptima Zika Virus assay and trained in handling infectious materials should perform this procedure.
J. Specimen collection, transport, storage and processing procedures outlined in this package insert are required for the optimal performance of this test. Improper collection, transport, or storage of specimens may lead to incorrect results.
K. Maintain proper storage conditions during specimen shipping to ensure the integrity of the specimen. Specimen stability under shipping conditions other than those recommended has not been evaluated.
L. Avoid cross-contamination during the specimen handling steps. Be especially careful to avoid contamination by the spread of aerosols when loosening or uncapping specimens. Specimens can contain extremely high levels of organisms. Ensure that specimen containers do not contact one another, and discard used materials without passing over open containers. Change gloves if they come in contact with specimen.

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General Information

Assay Related
M. Do not use the reagent kit or calibrators after the expiration date.
N. Do not interchange, mix, or combine assay reagents from kits with different master lot numbers. Assay fluids can be from different lot numbers.
O. Avoid microbial and nuclease contamination of reagents.
P. Cap and store all assay reagents at specified temperatures. The performance of the assay may be affected by use of improperly stored assay reagents. See Reagent Storage and Handling Requirements and Panther System Test Procedure for more information.
Q. Do not combine any assay reagents or fluids without specific instruction. Do not top off reagents or fluids. The Panther system verifies reagent levels.
R. Some reagents of this kit are labeled with risk and safety symbols. Note: For information on any hazard and precautionary statements that may be associated with reagents refer to the Safety Data Sheet Library at www.hologicsds.com. For more information on the symbols, refer to the symbol legend on www.hologic.com/package-inserts
US Hazard Information
Selection Reagent
Boric Acid 1 – 5% WARNING Causes skin irritation Causes serious eye irritation Wash face, hands and any exposed skin thoroughly after handling Wear protective gloves/protective clothing/eye protection/face protection

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Reagent Storage and Handling Requirements
A. The following table shows the storage conditions and stability for reagents and calibrators.

Reagent
Amplification Reagent Enzyme Reagent Probe Reagent Internal Control Target Capture Reagent (TCR) Working Target Capture Reagent (wTCR) Selection Reagent NCAL (Negative Calibrator)

Unopened Storage
35°C to 15°C 35°C to 15°C 35°C to 15°C
35°C to 15°C
2°C to 8°C n/a
15°C to 30°C 35°C to 15°C

PCAL (Positive Calibrator) Aptima Blood Processing Medium (BPM)

35°C to 15°C 15°C to 30°C

Open Kit (Thawed)a

Storage

Stability

2°C to 8°C

30 daysb

2°C to 8°C

30 daysb

2°C to 8°C

30 daysb

15°C to 30°C

8 hours prior to combining with TCR

n/a

2°C to 8°C

30 daysb

15°C to 30°C n/a n/a

30 daysb Single use vial Use within 8 hours Single use vial Use within 8 hours

15°C to 30°C

30 days

a Open kit storage and stability conditions are based on similar validated assays. b When reagents are removed from the Panther system, they should be immediately returned to their appropriate storage temperatures.

B. Discard any unused, previously prepared reagents and working target capture reagent after 30 days.

C. After opening a bottle of Aptima Blood Processing Medium, it is stable for 30 days.

D. Reagents stored onboard the Panther system have 120 hours (cumulative) of onboard stability. The Panther system logs each time the reagents are loaded.

E. If a precipitate forms in the Target Capture Reagent (TCR) during storage, see instructions under Preparation of a New Kit. DO NOT VORTEX. DO NOT FREEZE TCR.

F. Do not refreeze Internal Control, Amplification, Enzyme, and Probe Reagents after the initial thaw.

G. Calibrators are single use vials and must be discarded after use.

H. If precipitate forms in the Selection Reagent, Probe Reagent, Negative Calibrator, or Positive Calibrator, see instructions under Panther System Test Procedure.

I. Changes in the physical appearance of the reagent supplied may indicate instability or deterioration of these materials. If changes in the physical appearance of the reagents are observed (e.g., obvious changes in reagent color or cloudiness are indicative of microbial contamination), they should not be used.

J. After thawing the calibrators, the solution must be clear, i.e., not cloudy or have precipitates.

K. The probe reagent is photosensitive. Protect reagent from light during storage and preparation for use.

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General Information

Specimen Collection and Storage

The Aptima Zika Virus assay can be used with serum, plasma, processed whole blood, and processed urine specimens.
A processed urine specimen is neat urine added to urine transport media in an Aptima Urine Specimen Transport Tube.
A processed whole blood specimen is whole blood added to Aptima Blood Processing Medium in an Aptima transport tube.
Note: A urine specimen or whole blood specimen should not be the sole specimen tested from a patient. If a urine specimen or whole blood specimen from a patient is tested, it should be collected alongside a serum or plasma sample from the patient.
Note: Handle all specimens as if they contain potentially infectious agents. Use Universal Precautions.
Note: Take care to avoid cross-contamination during sample handling steps. For example, discard used material without passing over open tubes. False-positive results may occur if crosscontamination of specimens is not adequately controlled during specimen handling and processing. Note: The minimum volume of serum or plasma for primary collection tubes is 1200 L and for specimen aliquot tubes (SATs) the minimum volume is 700 L to obtain the 500 L reaction volume. The minimum volume of processed urine or processed whole blood specimens is 1200 L to obtain the 500 L reaction volume. A. Instructions for Collection
Human serum, plasma, whole blood (K2EDTA), and urine specimens may be used with the Aptima Zika Virus assay on the Panther system.
Refer to the appropriate specimen collection kit package insert for collection instructions.
1. Plasma and Serum Specimens
Whole blood specimens collected in the following glass or plastic tubes may be used according to manufacturer’s instruction:
· Tubes containing ethylenediaminetetraacetic acid (EDTA) or acid citrate dextrose adenine (ACD-A) anticoagulants or sodium citrate (NAC)
· Plasma preparation tubes (PPTs)
· Serum tubes
· Serum separator tubes (SSTs)
For serum, allow the clot to form before further processing.
2. Whole Blood Specimens
Whole blood specimens must be collected in tubes containing K2EDTA.
3. Urine Specimens
Urine specimens must be collected according to manufacturer’s instructions.

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B. Specimen Transport and Storage before Testing
1. Plasma and Serum Specimens
For plasma and serum specimens, whole blood specimens must be centrifuged within 72 hours of collection. Plasma and serum may be stored for a total of 13 days from the time of collection to the time of testing with the following conditions:
· Whole blood can be stored for 72 hours at temperatures up to 25°C, and up to 24 hours during the 72 hours at temperatures up to 30°C. Do not freeze whole blood prior to centrifugation to obtain plasma and/or serum samples.
· After whole blood specimens are centrifuged within 72 hours of collection, plasma and serum specimens should be stored at 2°C to 8°C for up to 10 days unless frozen.
a. If longer storage is needed, freeze plasma and serum separated from the cells and store at 20°C or 70°C.
b. No adverse effect on assay performance was observed when plasma and serum specimens were subjected to three freeze-thaw cycles.
c. Ensure that plasma and serum specimens have sufficient sample volume above the gel separator or red cell interface.
d. Specimens with visible precipitates or fibrinous material should be clarified by centrifugation for 10 minutes at 1000 to 3000g prior to testing.
2. Whole Blood Specimens a. Whole blood may be stored for a total of 14 days from the time of collection to the time of testing with the following conditions: Whole blood can be stored for up to 72 hours at temperatures up to 25°C and up to 24 hours during the 72 hours at temperatures up to 30°C. It can then be stored at 2°C to 8°C for up to 11 days unless frozen.
b. If longer storage is needed, freeze whole blood at 20°C or 70°C.
c. Whole blood should not be frozen or thawed more than once.
d. Whole blood must be processed by adding 1 mL of whole blood to 3 mL of Aptima Blood Processing Medium in an Aptima transport tube. See Panther System Test Procedure, step E.2, for more information.
e. Processed whole blood must be tested within 4 hours of preparation.
3. Urine Specimens a. Urine may be stored at 2°C to 30°C and must be transferred to an Aptima Urine Specimen Transport Tube, which contains urine transport media, and thoroughly mixed within 72 hours. See the appropriate collection kit package insert.
b. Store the mixed, processed urine specimen at 2°C to 30°C and test within 30 days of collection. If longer storage is needed, freeze the processed urine specimen at 20°C or 70°C.
c. No adverse effect on assay performance was observed when processed urine was subjected to three freeze-thaw cycles.
d. Ensure that specimens have sufficient sample volume.
e. Specimens with visible precipitates or fibrinous material should be clarified by centrifugation for 10 minutes at 1000 to 3000g prior to testing.

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C. Specimen Storage after Testing
1. Specimens that have been assayed must be stored upright in a rack. 2. The specimen tubes should be covered with a new, clean plastic film or foil barrier. 3. If assayed samples need to be frozen or shipped, place new caps on the specimen
tubes. If specimens need to be shipped for testing at another facility, recommended temperatures must be maintained. Prior to uncapping previously tested and recapped samples, specimen tubes must be centrifuged briefly (5 minutes at 500g) to bring all of the liquid down to the bottom of the tube. Avoid splashing and cross-contamination.
Note: Specimens must be shipped in accordance with applicable national, international, and regional transportation regulations.

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Panther System
Reagents for the Aptima Zika Virus assay are listed below for the Panther system. Reagent identification symbols are also listed next to the reagent name.

Reagents and Materials Provided

Aptima Zika Virus calibrator kits must be purchased separately. See individual catalog kit number below.
Aptima Zika Virus Assay Kit, 1000 tests (4 x 250 tests) Cat. No. PRD-04037-D (3 assay boxes)

Aptima Zika Virus Assay Box (store at 35°C to 15°C upon receipt)

Symbol Component

Quantity

Amplification Reagent

Non-infectious nucleic acids in buffered solution.

4 x 26 mL

Enzyme Reagent

Reverse transcriptase and RNA polymerase in HEPES buffered

solution.

4 x 13.4 mL

Probe Reagent

Chemiluminescent probes in succinate buffered solution.

4 x 34.7 mL

Internal Control Reagent

4 x 2.8 mL

A HEPES buffered solution containing detergent and an RNA transcript.

Master Lot Barcode Sheet

1 sheet

Aptima Zika Virus Assay Box (store at 15°C to 30°C upon receipt)

Symbol Component

Selection Reagent

600 mM borate buffered solution containing surfactant.

Quantity 4 x 91 mL

Aptima Zika Virus Assay Box (store at 2°C to 8°C upon receipt)

Symbol Component

TCR

Target Capture Reagent A buffered salt solution containing solid phase, non- infectious nucleic acids.

Quantity 4 x 161 mL

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Panther® System

Materials Required but Available Separately
Note: Materials available from Hologic have catalog numbers listed, unless otherwise specified.

Material

Cat. No.

Panther System, Diagnostic

303095

Panther Fusion Module

PRD-04173

Panther System Continuous Fluid and Waste (Panther Plus)

PRD-06067

Aptima Assay Fluids Kit (also known as Universal Fluids Kit)
contains Aptima Wash Solution, Aptima Buffer for Deactivation Fluid, and Aptima Oil Reagent
Aptima Auto Detect Kit

303014 (1000 tests) 303013 (1000 tests)

Multi-tube units (MTUs)

104772-02

Panther Waste Bag Kit

902731

Panther Waste Bin Cover

504405

Or, Panther System Run Kit
contains MTUs, waste bags, waste bin covers, auto detects, and assay fluids

303096 (5000 tests)

Tips, 1000 µL filtered, conductive, liquid sensing, and disposable

901121 (10612513 Tecan)

903031 (10612513 Tecan)

Not all products are available in all regions. Contact your representative for region- MME-04134 (30180117 Tecan)

specific information

MME-04128

Aptima Zika Virus Calibrator Kit
Negative calibrator, buffered solution containing detergent, 15 x 2.2 mL
Positive calibrator, RNA transcript in buffered solution containing detergent, 15 x 2.2 mL

PRD-04039-D

Bleach, 5% to 8.25% (0.7 M to 1.16 M) sodium hypochlorite solution

—

Disposable, powderless gloves

—

Replacement non-penetrable caps

103036A

Reagent replacement caps for 250-test bottles
Amplification and Probe reagents Enzyme reagent TCR and Selection reagents

CL0041 (100 caps) 501616 (100 caps)
CL0040 (100 caps)

Plastic-backed laboratory bench covers

—

Lint-free wipes

—

Pipettor

—

Tips

—

Primary blood collection tubes of the following dimensions may be used:

—

13 mm x 100 mm 13 mm x 75 mm

16 mm x 100 mm

Centrifuge

—

Vortex mixer

—

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Optional Materials
Material Aptima Specimen Aliquot Tubes (SAT) (100 pack) Transport Tube Cap (100 pack)
cap for SAT and Aptima transport tubes
Aptima Transport Tubes
for processed whole blood specimens
Aptima Urine Specimen Collection Kit Or Aptima Urine Specimen Transport Tubes Aptima Blood Processing Medium
Tris buffered solution containing detergent, 2 x 1.6 L
Transfer pipets Cotton-tipped swabs Tube rocker SB100 Reagent Equilibration System (SB100-RES) Water Bath

Aptima®
Cat. No. 503762 504415
101738A
301040 105575
PRD-04744-D
— — — — —

Panther System Test Procedure
Note: See the Panther/Panther Fusion Operator’s Manual for additional procedural information.
A. Work Area Preparation
1. Clean work surfaces where reagents will be prepared. Wipe down work surfaces with 2.5% to 3.5% (0.35 M to 0.5 M) sodium hypochlorite solution. Allow the sodium hypochlorite solution to contact surfaces for at least 1 minute and then follow with a deionized (DI) water rinse. Do not allow the sodium hypochlorite solution to dry. Cover the bench surface with clean, plastic-backed absorbent laboratory bench covers.
2. Clean a separate work surface where samples will be prepared. Use the procedure described above (step A.1).
3. Clean any pipettors. Use the cleaning procedure described above (step A.1).
B. Preparation of a New Kit
Warning: Avoid creating excessive foam in reagents. Foam compromises the level-sensing by the Panther system.
Note: Probe Reagent is photosensitive. Protect it from light during storage and during reagent handling.
Note: Amplification, Enzyme, and Probe Reagents may be thawed up to 24 hours at 2°C to 8°C prior to reagent preparation.
Note: Internal Control may be thawed up to 24 hours at 2°C to 8°C or up to 8 hours at room temperature (15°C to 30°C) prior to wTCR preparation.

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Target Capture Reagent (TCR), Amplification, Enzyme, and Probe Reagents Preparation
1. Remove a new set of reagents from storage. Check the lot numbers on the reagent bottles to make sure that they match the lot numbers on the Master Lot Barcode Sheet.
2. Allow reagents to reach room temperature (15°C to 30°C) using one of three options described below:

SB100-RES Preparation (Option 1)
1. Immediately upon removing from storage (2°C to 8°C), invert TCR bottle vigorously to mix gel into solution (at least 10 inversions and until gel is no longer present on the bottom). DO NOT VORTEX.
2. Prepare the TCR, Amplification, Enzyme, and Probe reagents using the SB100-RES. 3. Upon unload of reagents, record the Thaw Date for the Amplification, Enzyme, and Probe
Reagents in the space provided on the label.

Water Bath Preparation (Option 2)
Warning: Temperature of the water bath should not exceed 30°C.
Note: Refer to room temperature (15°C to 30°C) preparation instructions to prepare TCR. Do not use a water bath to prepare TCR.
1. Upon removing from storage (35°C to 15°C or 2°C to 8°C), place Amplification, Enzyme, and Probe reagents upright in a dedicated room temperature water bath (15°C to 30°C). At least every 10 minutes, gently invert the reagents to mix thoroughly and visually examine to ensure dissolution of precipitates. Continue to gently invert and visually examine until no precipitates are present.
2. Ensure that precipitates are dissolved. Do not use a reagent if gelling, precipitation, or cloudiness is present.
3. Record the Thaw Date for the Amplification, Enzyme, and Probe reagents in the space provided on the label.

Room Temperature Preparation (Option 3) Note: Probe Reagent from 35°C to 15°C storage, may take up to 4 hours to completely thaw at room temperature (15°C to 30°C) with gentle inversion at least every 10 minutes.
1. To prepare TCR, perform the following: a. Immediately upon removing from storage (2°C to 8°C), invert TCR bottle vigorously to mix gel into solution (at least 10 inversions and until gel is no longer present on the bottom). DO NOT VORTEX.
b. Allow the TCR bottle to remain at room temperature (15°C to 30°C) for at least 45 minutes. At least every 10 minutes, gently invert the TCR bottle (at least 10 inversions) to mix thoroughly and visually examine to ensure no gel is present.
c. Ensure gel is dissolved and the magnetic particles are suspended before use.
Note: If gel is present and persists, do not use. Replace TCR bottle into storage (2°C to ?8 ° C ) for subsequent use. Remove a new TCR bottle from storage (2°C to 8°C) and repeat steps 1.a to 1.c.
2. To prepare Amplification, Enzyme, and Probe reagents, perform the following: a. Upon removing from storage (35°C to 15°C or 2°C to 8°C), place reagents upright at room temperature (15°C to 30°C). At least every 10 minutes, gently invert the

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reagents to mix thoroughly and visually examine to ensure dissolution of precipitates. Continue to thaw until no precipitates are present.
3. Ensure that precipitates are dissolved. Do not use a reagent if gelling, precipitation, or cloudiness is present.
4. Record the Thaw Date for the Amplification, Enzyme, and Probe reagents in the space provided on the label.
Internal Control and Working Target Capture Reagent (wTCR) Preparation
Note: Do not use a SB100-RES to prepare Internal Control.
1. To prepare Internal Control, perform the following: a. Remove one tube of Internal Control from storage (35°C to 15°C or 2°C to 8°C).
b. Upon removing from storage (35°C to 15°C or 2°C to 8°C), allow Internal Control to remain at room temperature (15°C to 30°C) for at least 30 minutes.
Option: Internal Control tube may be placed in a room temperature (15°C to 30°C) water bath.
c. At least every 10 minutes, gently invert the Internal Control tube to mix thoroughly and visually examine for presence of gel. Ensure gel is dissolved prior to use.
Option: Internal Control tube may be placed on a tube rocker to mix thoroughly during room temperature preparation.
Note: If gelling occurs, gel must be dissolved prior to use and within the 8 hour thaw period at room temperature (15°C to 30°C). If gel persists, do not use. Discard the tube, obtain a new tube of Internal Control, and repeat steps 1.a to 1.c.
2. To prepare wTCR, perform the following: a. Once the TCR is ready for use, pour the entire contents of the Internal Control tube into the TCR bottle. Cap the TCR bottle and gently invert to mix thoroughly.
b. In the space indicated on the TCR bottle, record the date Internal Control was added, the wTCR expiration date (the date Internal Control was added plus 30 days), the Internal Control lot number (IC LOT), and the operator’s initials.
c. Retain the Internal Control tube as it is required to scan the barcode label into the Panther system.
Selection Reagent Preparation
Note: Do not use if precipitate or cloudiness is present.
1. To prepare Selection Reagent, perform the following: a. Remove a bottle of Selection Reagent from room temperature (15°C to 30°C) storage. Check the lot number on the reagent bottle to make sure it matches the lot number on the Master Lot Barcode Sheet.
b. Gently invert the bottle to mix thoroughly and visually examine to ensure no precipitate or cloudiness is present.
c. Record the date that it was first opened (Open Date) on the space provided on the label.
Note: Selection Reagent Recovery: If Selection Reagent has been inadvertently stored at 2? ° C to 8°C or the temperature of the laboratory falls below 15°C, precipitate may form. If precipitate forms in the Selection Reagent during storage, heat at 60°C ± 1°C for no more

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than 45 minutes and gently mix the bottle frequently (every 5 to 10 minutes). Once all precipitate has gone back into solution, place the bottle in a room temperature (15°C to 30°C) water bath and allow the bottle to equilibrate for at least 1 hour.
C. Calibrator Preparation Note: Avoid creating excessive foam when inverting calibrators. Foam compromises the level-sensing by the Panther system. Note: Do not use SB100-RES to thaw calibrators. 1. Upon removing calibrators from storage (35°C to 15°C), allow calibrators to remain at room temperature (15°C to 30°C) for at least 30 minutes. Option: Calibrators may be placed in a room temperature (15°C to 30°C) water bath to thaw.
2. At least every 10 minutes, gently invert each tube to mix thoroughly. Ensure tube contents are fully thawed prior to use.
Option: Calibrators may be placed on a tube rocker to mix thoroughly during room temperature preparation.
3. If gelling is observed, gently invert the tube until gel is no longer present. Note: If gelling occurs, gel must be dissolved prior to use and within the 8 hour thaw period at room temperature (15°C to 30°C). If gel persists, do not use. Discard the tube(s), obtain new tube(s) of calibrators, and repeat steps C.1 to C.3. 4. When the tube contents have fully thawed, dry the outside of each tube with a clean, dry
disposable wipe. 5. To prevent contamination, do not open the calibrator tubes at this time.
D. Reagent Preparation for Previously Prepared Reagents
wTCR, Amplification, Enzyme, and Probe Reagents Preparation 1. Remove wTCR and previously prepared reagents from storage. 2. Allow reagents to reach room temperature (15°C to 30°C) using one of three options
described below:
SB100-RES Preparation (Option 1) 1. Immediately upon removing from storage (2°C to 8°C), invert TCR bottle vigorously to
mix gel into solution (at least 10 inversions and until gel is no longer present on the bottom). DO NOT VORTEX. 2. Prepare the wTCR, Amplification, Enzyme, and Probe reagents using the SB100-RES.
Water Bath Preparation (Option 2) Warning: Temperature of the water bath should not exceed 30°C. Note: Refer to room temperature (15°C to 30°C) preparation instructions to prepare wTCR. Do not use a water bath to prepare wTCR. 1. Upon removing from storage (2°C to 8°C), place Amplification, Enzyme, and Probe
reagents upright in a dedicated room temperature water bath (15°C to 30°C). At least every 10 minutes, gently invert the reagents to mix thoroughly and visually examine to ensure dissolution of precipitates. Continue to thaw until no precipitates are present.

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2. Ensure that precipitates are dissolved. Do not use a reagent if gelling, precipitation, or cloudiness is present.
Room Temperature Preparation (Option 3) 1. To prepare wTCR, perform the following:
a. Immediately upon removing from storage (2°C to 8°C), invert wTCR bottle vigorously to mix gel into solution (at least 10 inversions and until gel is no longer present on the bottom). DO NOT VORTEX.
b. Allow the wTCR bottle to remain at room temperature (15°C to 30°C) for at least 45 minutes. At least every 10 minutes, gently invert the wTCR bottle (at least 10 inversions) to mix thoroughly and visually examine to ensure no gel is present.
c. Ensure gel is dissolved and the magnetic particles are suspended before use.
Note: If gel is present and persists, do not use. Replace wTCR bottle and matching reagents into storage (2°C to 8°C) for subsequent use.
2. To prepare Amplification, Enzyme, and Probe reagents, perform the following: a. Upon removing from storage (2°C to 8°C), prepare reagents upright at room temperature (15°C to 30°C). At least every 10 minutes, gently invert the reagents to mix thoroughly and visually examine to ensure dissolution of precipitate. Continue to thaw until no precipitates are present.
3. Ensure that precipitates are dissolved. Do not use a reagent if gelling, precipitation, or cloudiness is present.
Selection Reagent Preparation
Note: Do not use if precipitate or cloudiness is present.
1. Remove matching bottle of Selection Reagent from room temperature (15°C to 30°C) storage.
2. Gently invert the bottle to mix thoroughly and visually examine to ensure no precipitate or cloudiness is present.
Note: Selection Reagent Recovery: If Selection Reagent has been inadvertently stored at 2? ° C to 8°C or the temperature of the laboratory falls below 15°C, precipitate may form. If precipitate forms in the Selection Reagent during storage, heat at 60°C ± 1°C for no more than 45 minutes and gently mix the bottle frequently (every 5 to 10 minutes). Once all precipitate has gone back into solution, place the bottle in a room temperature (15°C to 30°C) water bath and allow the bottle to equilibrate for at least 1 hour.
E. Sample Handling
1. Preparation of Serum, Plasma, and Urine Specimens and Calibrators a. Allow the specimens and calibrators to reach 15°C to 30°C prior to processing. Gently invert sample tubes at least 3 times or mix gently on a rocker until the sample is homogeneous.
b. Ensure each specimen tube contains enough volume for each sample type and tube type.
c. Mix fresh or thawed specimens thoroughly.

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d. Prepare samples for loading into a Sample Rack. Do not remove caps. Review each sample for bubbles, fibrinous material, and precipitates. If necessary, prepare as follows:
i. For serum or plasma samples, centrifuge the samples at 1000 to 3000g for 10 minutes.
ii. For whole blood samples, see step E.2.
iii. For processed urine samples, centrifuge if there are bubbles in the tube or liquid in the cap. Bubbles in the tube compromise the level-sensing by the Panther system. Centrifugation times and speeds for pulling down all liquid and precipitates must be validated by the user. If precipitate does not go back into solution, visually ensure that the precipitate does not prevent delivery of the specimen.
2. Whole Blood Specimen Preparation a. Prior to testing whole blood specimens, the specimens must be processed manually as follows:
i. Allow whole blood specimens to reach 15°C to 30°C which takes approximately 30 minutes for refrigerated samples and 1 hour for frozen samples. Do not use water baths or other incubators.
ii. Gently invert whole blood tubes at least 3 times or mix gently on a rocker until blood is homogeneous.
iii. Pipette 3.0 mL of BPM into Aptima transport tubes.
iv. Into the Aptima transport tube, pipette 1.0 mL of whole blood avoiding any clots. Dispense the specimen just below the surface of the BPM and pipette up and down 2 or 3 times to mix.
v. Cap the Aptima transport tube and invert gently at least 10 times or mix gently on a rocker until homogeneous. Avoid creating excessive foam. This is now referred to as a “processed whole blood specimen.” Do not centrifuge processed whole blood specimens.
See System Preparation, step F.2 below, for information about loading the rack and removing the caps.
F. System Preparation
1. Set up the system according to the instructions in the Panther/Panther Fusion Operator’s Manual and Procedural Notes. Make sure that the appropriately sized reagent racks and TCR adapters are used.
2. Load samples into the Sample Rack. Perform the following steps for each sample tube (specimen, and, when necessary, calibrator): a. Loosen one sample tube cap, but do not remove it yet.
Note: Be especially careful to avoid contamination by the spread of aerosols. Gently loosen caps on samples.
b. Load the sample tube into the Sample Rack.
c. Repeat steps 2.a and 2.b for each remaining sample.
d. After the samples have been loaded into the Sample Rack, remove and discard each sample tube cap in one Sample Rack. To avoid contamination, do not pass a cap

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over any other Sample Racks or sample tubes. Pierceable caps from the Aptima Urine Specimen Transport Tube must also be removed and discarded.
e. If necessary, use a new, disposable transfer pipet to remove any bubbles or foam.
f. When the last cap has been removed, load the Sample Rack into a Sample Bay.
Note: If running other assays and sample types at the same time, secure the Sample Retainer prior to loading the Sample Rack into a Sample Bay.
g. Repeat steps 2.a to 2.f for the next Sample Rack.

Procedural Notes
A. Calibrators
1. The calibrator tubes can be loaded in any position in the Sample Rack and in any Sample Bay Lane on the Panther system. Specimen pipetting will begin when one of the following two conditions has been met: a. The calibrators are currently being processed by the system.
b. Valid results for the calibrator are registered on the system.
2. Once the calibrator tubes have been pipetted and are processing for the Aptima Zika Virus assay reagent kit, specimens can be tested with the associated kit for up to 24 hours unless: a. The calibrator results are invalid.
b. The associated assay reagent kit is removed from the system.
c. The associated assay reagent kit has exceeded stability limits.
3. Each calibrator tube can be used once. Attempts to use the tube more than once can lead to processing errors.
B. Glove Powder
As in any reagent system, excess powder on some gloves may cause contamination of opened tubes. Powderless gloves are recommended.

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Quality Control

Acceptance Criteria for the Aptima Zika Virus Assay
A. Run validity
A run (also identified as a worklist) is valid if the minimum number of calibrators meet their acceptance criteria and are valid (see Acceptance Criteria for Calibration and Calculation of Cutoff).
1. In an Aptima Zika Virus assay run, at least four of the six calibrator replicates must be valid. At least two of the three Negative Calibrator replicates and two of the three Positive Calibrator replicates must be valid.
2. Calibrator acceptance criteria are automatically verified by the Panther System software. If less than the minimum number of calibrator replicates is valid, the Panther System software will automatically invalidate the run.
3. In a valid run, cutoff values will be automatically calculated for Internal Control (flasher) and analyte (glower).
4. If a run is invalid, sample results are reported as Invalid and all specimens must be retested.
B. Sample validity
1. In a valid run, a sample result is valid if the IC signal is equal to or above the IC cutoff, with the following exceptions: a. Specimens with an analyte signal (glower signal) greater than the analyte cutoff are not invalidated even if the Internal Control (IC) signal is below the cutoff.
b. Specimens with an IC signal above 750,000 RLU are invalidated by the software and their reactive status cannot be assessed. The software also automatically invalidates Positive Calibrators with an IC signal above 750,000 RLU.
2. A sample may also be invalidated due to instrument and results processing errors. Refer to the Panther/Panther Fusion Operator’s Manual for details.
3. All individual specimen results that are Invalid in a valid run must be retested.
Acceptance Criteria for Calibration and Calculation of Cutoff
A. Negative Calibrator Acceptance Criteria
The Negative Calibrator (NC) is run in triplicate in the Aptima Zika Virus assay. Each individual Negative Calibrator replicate must have an Internal Control (IC) value greater than or equal to 50,000 RLU and less than or equal to 500,000 RLU. Each individual Negative Calibrator replicate must also have an analyte value less than or equal to 40,000 RLU and greater than or equal to 0 RLU. If one of the Negative Calibrator replicate values is invalid due to an IC value or an analyte value outside of these limits, the Negative Calibrator mean (NCx) will be recalculated based upon the two acceptable values. The run is invalid and must be repeated if two or more of the three Negative Calibrator replicate values have IC values or analyte values that are outside of these limits.

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Determination of the mean of the Negative Calibrator values (NCx) for Internal Control [NCx (Internal Control)]

Example:
Negative Calibrator 1 2 3
Total Internal Control RLU

Internal Control Relative Light Units

235,000

200,000

210,000

645,000

NCx (Internal Control)

Total Internal Control RLU 3

= 215,000

Determination of the mean of the Negative Calibrator values (NCx) for Analyte [NCx (Analyte)]

Example:
Negative Calibrator 1 2 3
Total Analyte RLU

Analyte Relative Light Units

14,000

16,000

15,000

45,000

NCx (Analyte)

Total Analyte RLU 3

= 15,000

B. Positive Calibrator Acceptance Criteria

The Positive Calibrator is run in triplicate in the Aptima Zika Virus assay. Individual Positive Calibrator (PC) analyte values must be less than or equal to 4,000,000 RLU and greater than or equal to 400,000 RLU. IC values may not exceed 750,000 RLU. If one of the Positive Calibrator replicate values is outside these limits, the Positive Calibrator mean (PCx) will be recalculated based upon the two acceptable Positive Calibrator replicate values. The run is invalid and must be repeated if two or more of the three Positive Calibrator analyte values are outside of these limits.

Determination of the mean of the Positive Calibrator (PCx) values for Analyte [PCx (Analyte)]

Example:

Positive Calibrator 1 2 3
Total Analyte RLU

Analyte Relative Light Units

1,250,000

1,500,000

1,150,000

3,900,000

PCx (Analyte)

Total Analyte RLU 3

= 1,300,000

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Quality Control

C. Calculation of the Internal Control Cutoff Value Internal Control Cutoff Value = 0.5 X [NCx (Internal Control)] Using values given in the Negative Calibrator example above: Internal Control Cutoff Value = 0.5 X (215,000) Internal Control Cutoff Value = 107,500 RLU

D. Calculation of the Zika Virus Analyte Cutoff Value Analyte Cutoff Value = NCx (Analyte) + [0.03 X PCx (Analyte)] Using values given in the Negative Calibrator and Positive Calibrator examples above: Analyte Cutoff Value = 15,000 + (0.03 X 1,300,000) Analyte Cutoff Value = 54,000 RLU

E. Summary of Acceptance Criteria for the Aptima Zika Virus Assay

Acceptance Criteria Negative Calibrator
Analyte Internal Control

0 and 40,000 RLU 50,000 and 500,000 RLU

Positive Calibrator Analyte
Internal Control

400,000 and 4,000,000 RLU 750,000 RLU

F. Summary of Cutoff Calculations for the Aptima Zika Virus Assay

Analyte Cutoff = NC Analyte Mean RLU + [0.03 X (PC Analyte Mean RLU)]

Internal Control Cutoff =

0.5 X (Negative Calibrator IC Mean RLU)

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Interpretation of Results

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Interpretation of Results

All calculations described above are performed by the Panther System software. Two cutoffs are determined for each assay: one for the Analyte Signal (glower signal) termed the Analyte Cutoff and one for the Internal Control Signal (flasher signal) termed the Internal Control Cutoff. The calculation of these cutoffs is shown above. For each sample, an Analyte Signal RLU value and Internal Control Signal RLU value are determined. Analyte Signal RLU divided by the Analyte Cutoff is abbreviated as the Analyte Signal/Cutoff (S/CO) on the report.

A specimen is Negative if the Analyte Signal is less than the Analyte Cutoff (i.e., Analyte S/CO < 1.00) and the Internal Control (IC) Signal is greater than or equal to the Internal Control Cutoff (IC Cutoff) and less than or equal to 750,000 RLU. A specimen is Positive if the Analyte Signal is greater than or equal to the Analyte Cutoff (i.e., Analyte S/CO 1.00) and the IC Signal is less than or equal to 750,000 RLU. The results will be designated by the software. A specimen is invalid if the Analyte Signal is less than the Analyte Cutoff (i.e., Analyte S/CO < 1.00) and the Internal Control Signal is less than the Internal Control Cutoff. Any specimen with Internal Control values greater than 750,000 RLU is considered invalid.

Summary of Specimen Interpretation

Specimen Interpretation

Criteria

Negative

Analyte S/CO < 1.00 and
IC IC Cutoff and IC 750,000 RLU

Positive

Analyte S/CO 1.00 and IC 750,000 RLU*

Invalid

IC > 750,000 RLU or Analyte S/CO < 1.00 and IC < Cutoff

*For specimens with IC signal greater than 750,000 RLU, the specimen will be invalidated by the software.

A. Any specimen with an interpretation of Invalid in the Aptima Zika Virus assay must be retested in singlet.

B. Specimens with a valid Internal Control value and with an Analyte S/CO less than 1.00 in the Aptima Zika Virus assay are considered Negative for ZIKV RNA.

C. Specimens with an Analyte S/CO greater than or equal to 1.00 with IC Signal less than or equal to 750,000 RLU are considered Positive for ZIKV RNA.

D. A patient-matched serum specimen is currently required for serological follow up testing of negative NAT (nucleic acid testing) results per the CDC testing algorithm (found at http:// www.cdc.gov/zika/index.html).

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Limitations

Limitations
A. Use of this assay is limited to personnel who have been trained in the procedure. Failure to follow the instructions given in this package insert may result in erroneous results.
B. Reliable results are dependent on adequate specimen collection, transport, storage, and processing.
C. Laboratories are required to report all positive results to the appropriate public health authorities.
D. A patient-matched serum specimen is currently required for serological follow up testing of negative NAT results per the CDC testing algorithm (found at http:// www.cdc.gov/zika/index.html).
E. The effect of long term storage of specimens on the performance of the Aptima Zika Virus assay has not been fully evaluated.
F. Though rare, mutations within the highly conserved regions of the viral genome covered by the primers and/or probes in the Aptima Zika Virus assay may result in the failure to detect the virus.
G. This assay has been developed for use with the Panther system only.
H. Performance of this assay with processed whole blood specimens is limited for use on Panther system software version 5.3 only. Performance of the assay with processed whole blood specimens on later Panther system software versions has not been evaluated. Plasma, serum, and urine specimens can be processed with all currently available Panther system software versions.
I. Cross-contamination of samples can cause false positive results.
J. Assays must be performed, and results interpreted, according to the procedures provided.
K. Deviations from these procedures, adverse shipping and/or storage conditions, or use of outdated reagents may produce unreliable results.
L. Failure to achieve expected results is an indication of an invalid run. Possible sources of error include test kit deterioration, operator error, faulty performance of equipment, specimen deterioration, or contamination of reagents.
M. This assay has been tested using only the specimen types indicated. Performance with other specimen types has not been evaluated.
N. Results from the Aptima Zika Virus assay should be interpreted in conjunction with other clinical data available to the clinician.
O. A negative result does not preclude a possible infection because results are dependent on adequate specimen collection. Test results may be affected by improper specimen collection, technical error, specimen mix-up, or target levels below the assay limit of detection.
P. The Aptima Zika Virus assay provides qualitative results. Therefore, a correlation cannot be drawn between the magnitude of a positive assay signal and the number of organisms in a specimen.
Q. Customers must independently validate an LIS transfer process.

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Conditions for Authorization For The Laboratory

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Conditions for Authorization For The Laboratory

The Aptima Zika Virus assay Letter of Authorization, along with the authorized Fact Sheet for Healthcare Providers, the authorized Fact Sheet for Patients and authorized labeling are available on the FDA website: https://www.fda.gov/MedicalDevices/Safety/EmergencySituations/ucm161496.htm
Use of the Aptima Zika Virus assay must follow the procedures outlined in these manufacturer’s Instructions for Use and the conditions of authorization outlined in the Letter of Authorization. Deviations from the procedures outlined are not permitted under the Emergency Use Authorization. To assist clinical laboratories running the Aptima Zika Virus assay, the relevant Conditions of Authorization are listed verbatim below.
· Authorized laboratories will include with reports of the results of the Aptima Zika Virus assay the authorized Fact Sheet for Health Care Providers, the authorized Fact Sheet for Pregnant Women1, and the authorized Fact Sheet for Patients. Under exigent circumstances, other appropriate methods for disseminating these Fact Sheets may be used, which may include mass media.
· Authorized laboratories will perform the Aptima Zika Virus assay on the Panther System or other authorized instruments.
· Authorized laboratories will perform the Aptima Zika Virus assay using the Aptima Auto Detect Reagents kit and Aptima Assay Fluids kit or other authorized ancillary reagents.
· Authorized laboratories will perform the Aptima Zika Virus assay on serum, plasma, or other authorized specimen types.2
· Authorized laboratories will have a process in place for reporting test results to health care providers and relevant public health authorities, as appropriate.3
· Authorized laboratories will collect information on the performance of the test and report to Hologic, Inc., any suspected occurrence of false positive or false negative results of which they become aware.
· All laboratory personnel using the test should be appropriately trained in nucleic acid amplification techniques and use appropriate laboratory and personal protective equipment when handling this kit.
· Hologic, Inc., its authorized distributor(s), and authorized laboratories will ensure that any records associated with this EUA are maintained until notified by FDA. Such records will be made available to FDA for inspection upon request.

1 Please note, subsequent to the original Letter of Authorization the Pregnant Women and Patient Fact Sheets were combined into one Patient Fact Sheet as of September 2, 2016.
2 Please note, as of March 8, 2018 the Aptima Zika Virus assay is authorized for use with processed whole blood.
3 For questions related to reporting Zika test results to relevant public health authorities, it is recommended that Hologic, Inc. and authorized laboratories consult with the applicable country, state or territory health department(s) and/or CDC. According to CDC, Zika is a nationally notifiable condition. http://www.cdc.gov/zika/.

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Performance

Limit of Detection (LoD) for Plasma Specimens

The Limit of Detection (LoD) is defined as the concentration of ZIKV RNA that is detected at 95% or greater probability according to CLSI EP17-A2.19 The LoD was determined by testing a ZIKV positive plasma specimen serially diluted in defibrinated, delipidated human plasma. The positive plasma specimen was collected from a blood donor during the 2015 Zika outbreak in Brazil. The sample was quantified using a validated real-time RT-PCR assay. The LoD of the Aptima Zika Virus assay was also evaluated by testing an in vitro synthesized transcript (corresponding to the appropriate sequence from GenBank accession number AY632535 for Zika isolate MR766). The transcript was serially diluted in buffer. Three Panther instruments were used to test 72 replicates of each target level, except for the panel member with 90 copies/mL, which was tested in 20 replicates. The results are summarized in Table 1 and Table 2.
Table 1: Detection of ZIKV Positive Brazilian Plasma 2015 with the Aptima Zika Virus Assay on the Panther System

Virus

Tested #Positive

copies/mL

0.1

0.3

100

Positivity Lower 95%CI
0 0 12 28 76 95 95 84

Upper 95%CI
5 7 30 50 92 100 100 100

%P = percentage positive, CI = Confidence Interval.

Table 2: Detection of Transcripts with the Aptima Zika Virus Assay on the Panther System

Transcript copies/mL
0 0.3 1 3 10 30 90

Tested #Positive

Positivity

%P Lower 95%CI Upper 95%CI

100

%P = percentage positive, CI = Confidence Interval.

The 95% detection probabilities were determined using Probit analysis. The limit of detection for the ZIKV positive Brazilian donor plasma 2015 in the Aptima Zika Virus assay was determined to be 5.9 copies/mL at 95% detection probability. The limit of detection for the in vitro transcript in

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the Aptima Zika Virus assay was determined to be 13.4 copies/mL at 95% detection probability (Table 3).

Table 3: Probit Analysis Detection

Analytes

95% Detection Probability in copies/mL (95% Fiducial Limits)

Zika Virus (Brazilian plasma 2015)

5.9 (4.38.9)

Transcript (based on Zika isolate MR766)

13.4 (9.920.3)

Inclusivity — in silico

Primer and probe conservation with all publicly available Zika virus strains encompassing intended target regions was assessed by direct comparison to multiple sequence alignments. Distance matrices generated for each oligonucleotide against each isolate indicate that none are likely to be missed as each has at least one set of capture oligonucleotide, forward primer, probe, and reverse primer combination with 100% sequence identity. Based on this analysis, false negative results are not likely to occur with the oligonucleotides included in this system. Inclusivity data analysis is provided in Table 4.

Table 4: Inclusivity Data Analysis

Country Yeara Strain/ Isolate

Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Cambodia Canada Central African Republic Central African Republic Central African Republic Central Africa n Republic China China China

2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2016 2016 2015 2015 2010 2013
1976
1968
N/A
N/A
2016 2016 2016

Bahia01 Bahia07 Bahia09 BeH815744 BeH818995 BeH819015 BeH819966 BeH823339 BeH828305 Brazil-ZKV2015 Natal RGN PE243 Rio-S1 Rio-U1 SSABR1 ZikaSPH2015 FSS13025 From Vero E6 cells
ARB13565
ArB1362
ARB15076
ARB7701
GD01 GDZ16001 GZ01

Oligonucleotide Sequence % Identity

Capture Oligonucleotides

Forward Primers

Probes

TCO_1 TCO_2 TCO_3

100 100 100

100 100

100 100 100

FP_1 95 95 95 95 95 95 95 95 95 95 95 95 95 95 95 95 95 95

FP_2 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

FP_3 91 91 91 91 91 91 91 91 91 91 91 91 91 91 91 91 91 91

FP_4 96 96 96 96 96 96 96 96 96 96 96 96 96 96 96 96 96 96

P_1 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 95 100

P_2 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 95

100 100 95

100 95 96 91

100 100

Reverse Primers
RP_1 RP_2 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100
96 100

100 96

100 100 95 96 91

100 100 100 100

100 100 100 100 95 96 91

100 100 100 100

100 100 95
100 100 100 100 100 100 100 100 100

100 95 96 91
95 100 91 96 95 100 91 96 95 100 91 96

100 100
100 100 100 100 100 100

96 100
100 100 100 100 100 100

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Performance

Table 4: Inclusivity Data Analysis (continued)

Country
China China China China China China China China China China Colombia Colombia Colombia French Polynesia Guatemala Guatemala Haiti Honduras Italy
Italy
Italy
Malaysia
Martinique
Mexico Mexico Mexico Micronesia Nigeria
Nigeria
Panama Panama Panama Panama Philippines Puerto Rico Puerto Rico Senegal Senegal Senegal Senegal Senegal Senegal Senegal Senegal Senegal

Yeara Strain/ Isolate
2016 GZ02/2016 2016 SZ01/2016 2016 SZ02/2016 2016 SZ-WIV01 2016 VE_Ganxian 2016 Z16006 2016 Z16019 2016 Zhejiang04 2016 ZJ03 2016 ZKC2/2016 2015 C1/C2 2015 FLR 2016 UF-1/2016

Oligonucleotide Sequence % Identity

Capture Oligonucleotides

Forward Primers

Probes

TCO_1 TCO_2 TCO_3 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

FP_1 95 95 95 95 95 95 95 95 95 95 95 95 95

FP_2 100 100 100 100 100 100 100 100 100 100 100 100 100

FP_3 91 91 91 91 91 91 91 91 91 91 91 91 91

FP_4 96 96 96 96 96 96 96 96 96 96 96 96 96

P_1 100 100 100 100 100 100 100 100 100 100 100 100 100

P_2 100 100 100 100 95 100 100 100 100 100 100 100 100

2013 H/PF/2013

100 100 100 95 100 91 96

100 100

2015 8375

100 100 100

2015 103344

100 100 100

2014 Haiti/1225/2014

100 100 100

2016 103451

100 100 100

2016 Brazil/2016/INMI1

100 100 100

2016 Dominican Republic/ 100 2016/PD1

100

2016

Dominican Republic/ 2016/PD2

100

1966 P6-740

100 100 100

2015

MRS_OPY_Martinique _PaRi_2015

100

2016 MEX/InDRE/Lm/2016 100 100 100

2016 MEX/InDRE/Sm/2016 100 100 100

2015 MEX_I_7

100 100 100

2007 ZIKV 2007 EC

100 100 100

1968 IbH_30656

100 100 95

1968

IbH-30656_SM21V1V3

100

2016 BEI-259634_V4

100 100 100

2015 CDC-259249_V1-V3 100 100 100

2015 CDC-259359_V1-V3 100 100 100

2015 CDC-259364_V1-V2 100 100 100

2012 CPC-0740

100 100 100

2015 PRVABC59

100 100 100

2015 V3/V2

100 100 100

1984 41525-DAK

100 100 100

1984 41662-DAK

100 100 100

1984 41671-DAK

100 100 100

1984 A1C1/V2

100 100 100

1984 ArD_41519

100 100 100

2000 ArD142623

100 96

2001 ArD157995

100 100 100

2001 ArD158084 1968 ArD7117

100 100 100 100 100 100

95 100 91 96 95 100 91 96 95 100 91 96 95 100 91 96 95 100 91 96
95 100 91 96
95 100 91 96
95 100 91 96
95 100 91 96
95 100 91 96 95 100 91 96 95 100 91 96 95 100 91 96 100 95 100 96
100 95 100 96
95 100 91 96 95 100 91 96 95 100 91 96 95 100 91 96 100 95 91 96 95 100 91 96 95 100 91 96 100 95 100 96 100 95 100 96 100 95 100 96 100 95 100 96 100 95 100 96 100 95 100 96 100 95 100 96 100 95 100 96 100 95 100 96

95 100 95 100 100 100 95 100 100 100
100 100
100 100
100 100
100 100
100 100 100 100 95 100 100 100 100 95
100 95
100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

Reverse Primers
RP_1 RP_2 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100
96 100
100 100 100 100 100 100 100 100 100 100
98 100
100 100
96 100
100 100
100 100 100 100 100 100 100 100 96 100
96 100
100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

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Table 4: Inclusivity Data Analysis (continued)

Country Yeara Strain/ Isolate

Suriname Suriname Thailand Uganda USA USA —

2015 2016 2014 1947 2016 2016 —

Z1106033 ZIKVNL00013 SV0127-14 MR 766 FB-GWUH-2016 Haiti/1/2016 ArD158095

Oligonucleotide Sequence % Identity

Capture Oligonucleotides

Forward Primers

Probes

TCO_1 TCO_2 TCO_3 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

FP_1 FP_2 FP_3 FP_4 95 100 91 96 95 100 91 96 95 100 91 96 100 95 100 96 95 100 91 96 95 100 91 96 100 95 100 96

P_1 100 100 100 100 95 100 100

P_2 100 100 100 100 100 100 100

Reverse
Primers
RP_1 RP_2 100 100 100 100 100 100 100 100 100 100 100 100 100 100

a Year collected.

Repeatability for Plasma Specimens
To assess the repeatability of the Aptima Zika Virus assay, a panel that was made by spiking virus stock into negative plasma (18 copies/mL, 30 copies/mL, 60 copies/mL, and 600 copies/ mL) and transcripts in buffer solution (6000 copies/mL) was tested by two operators using three Panther systems over 2 days. The repeatability panel was tested with 108 replicates for each panel member of 18 copies/mL and 30 copies/mL, and 54 replicates for each panel member of 60 copies/mL, 600 copies/mL, and 6000 copies/mL, for a total of 378 replicates for all five panel members.

Repeatability analyses included evaluation of percent agreement of the observed result to the expected result and mean signal to cutoff (S/CO) ratios for panel members. The results were analyzed to assess total variance as well as variance within each run and between days, operators, and instruments. The standard deviation (SD) and percent coefficient of variation (%CV) of the S/CO ratios are shown in Table 5. The mean analyte S/CO ratios were analyzed for all panel members. The percent agreement between the assay results and the true status of each panel member was calculated using the analyte S/CO for all panel members.

The overall percent agreement of test results was 100% for all panel members. There was no correlation of positive rate to the variance factors tested in this study. With regard to signal variability, intra-run was the largest contributor to total variance (as measured by SD values) in the Aptima Zika Virus assay.
Table 5: Repeatability of the Aptima Zika Virus Assay

Panel

Analyte Inter-Day N #P %A Mean

InterOperator

S/CO SD %CV SD %CV

18 c/mL 108 108 100% 33.12 0.15 0% 0.15 0%

30 c/mL 108 108 100% 33.24 0.24 1% 0.16 0%

60 c/mL 54 54 100% 33.20 0.24 1% 0.19 1%

600 c/mL 54 54 100% 32.96 0.24 1% 0.21 1%

InterInstrument SD %CV
0.25 1% 0.18 1% 0.25 1% 0.27 1%

Intra-Run

Total

SD %CV
1.29 4% 1.32 4% 1.20 4% 1.32 4%

SD %CV
1.33 4% 1.36 4% 1.26 4% 1.38 4%

6000 c/mL 54 54 100% 32.97 0.33 1% 0.21 1%

0.23 1%

1.27 4% 1.35 4%

N= Number of panel members combined for this analysis, P = Number of Positives, A = Agreement, S/CO = Signal to Cutoff Ratio, SD = Standard Deviation, CV = coefficient of variance, c/mL = copies per mL.

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Performance

Cross-Reactivity with Other Blood-Borne Pathogens for Plasma Specimens

Cross-reactivity of the Aptima Zika Virus assay was evaluated by testing clinical specimens from 10 patients with each of the following viral infections: Dengue virus, Hepatitis A virus (HAV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Human Immunodeficiency virus 1 and 2 (HIV-1/ 2), Parvovirus B19, and West Nile virus (WNV). Specimens from 10 individuals that had received HBV vaccine were also tested. The specimens were obtained from a commercial source and characterized by vendors using validated methods. In addition, pooled negative plasma spiked with Hepatitis E virus (HEV) at 1 x 105 copies/mL and pooled negative plasma spiked with Chikungunya virus at 1 x 105 U/mL were evaluated. Each sample described above was split into two aliquots. One aliquot was used for the cross-reactivity evaluation. The other aliquot was spiked with ZIKV positive plasma and used as contrived specimens in the Clinical Evaluation. For cross-reactivity, aliquots from donor samples with naturally occurring infections or who had received the HBV vaccine were tested once. The samples spiked with HEV and Chikungunya were tested in replicates of 10.

Aptima Zika Virus assay results were negative for all samples. No cross- reactivity was observed in the specimens from subjects infected with other blood-borne pathogens or specimens from individuals that had received HBV vaccines or in specimens spiked with virus.

Table 6: Aptima Zika Virus Assay Results Summary of Cross-Reactivity with Other Blood-borne Pathogens

Pathogen

IC S/CO

N #P %P

Mean

Analyte S/CO

Mean

HCV

1.91

0.07

0.00

N/A

WNV

1.95

0.05

0.00

N/A

HAV

1.95

0.04

0.01

0.02

316%

HIV 1-2

1.87

0.09

0.00

0.01

N/A

Dengue

1.91

0.05

0.04

0.08

219%

Parvo B19

1.93

0.04

0.00

N/A

HBV

1.92

0.05

0.01

0.03

316%

HBV Vaccinated 10

1.88

0.06

0.00

N/A

Chikungunya

1.89

0.05

0.01

0.02

316%

HEV

1.93

0.03

0.00

0.01

N/A

Cross-Reactivity and Interference with Other Microorganisms for Plasma Specimens
Negative plasma was used to prepare specimens spiked to 1 x 106 colony forming units (CFU/ mL) or inclusion forming unit per mL (IFU/mL) with each of the following microorganisms: Staphylococcus epidermidis, Staphylococcus aureus, Corynebacterium diphtheriae, Propionibacterium acnes, Candida albicans, Neisseria gonorrhoeae, or Chlamydia trachomatis. Negative urine was used to prepare specimens spiked to 1 x 106 CFU/mL of Escherichia coli or Staphylococcus aureus. Cross-reactivity was tested using ZIKV unspiked specimens, and all results were negative. The potential for microbial interference was tested using an aliquot of each specimen, spiked with ZIKV at 18 copies/mL, and all results were positive. No crossreactivity or interference was observed in the specimens containing bacteria or fungi.

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Performance

Aptima®

Cross-Reactivity Analysis in silico
The sequences of the primers, probes, and capture oligonucleotides in the Aptima Zika Virus assay were subjected to a BLAST (Basic Local Alignment Search Tool) analysis against the species shown in Table 7. The Aptima Zika Virus assay does not appear to be capable of significantly cross-reacting with the examined subject sequences when evaluated by in-silico Blast (blastn, max target seq=10,000, word size=7, e-val threshold=1,000, default penalties) and proximity analysis (correct orientation and common hit proximity of all oligonucleotides within 300bp). Hits that are 45% identical to the query oligonucleotide and within 300bp of one another on the same subject sequence are considered potentially problematic. None of the subjects included in the specific cross-reactivity datasets (751,867 entries and 486,723 entries) or in the GenBank non-redundant viral, plant, bacterial, invertebrate, environmental, other vertebrate or bacteriophage divisions were found to meet all these criteria to a complete set of oligonucleotides to produce false positive results.

The in silico analysis included genomic RNA/DNA sequences of the viruses and organisms listed in Table 7.

Table 7: List of Organisms for Cross-Reactivity Analysis

Flaviviruses
Dengue virus 1, 2, 3 and 4 Hepatitis C virus
Japanese encephalitis virus Spondweni virus
St. Louis encephalitis virus West Nile virus Yellow fever — — — — — — —
—
— — — — — — — — —

Adenovirus

Other Organisms
Acinetobacter lwoffii

Barmah Forest virus

Actinomyces israelii

Borrelia burgdorferi

Alcaligenes faecalis

Chikungunya virus

Atopobium vaginae

Cytomegalovirus

Bacteroides fragilis

Eastern Equine Encephalitis virus

Bifidobacterium adolescentis

Enterovirus

Campylobacter jejuni

Epstein Barr virus

Candida albicans

Group A Streptococcus

Chlamydia trachomatis

Hepatitis B virus

Clostridium difficile

Human Immunodeficiency virus

Corynebacterium genitalium

Leptospirosis

Cryptococcus neoformans

Mayaro virus

Enterobacter cloacae

Measles virus

Enterococcus faecalis

O’nyong-nyong virus (Sindbis virus and Una virus)

Escherichia coli

Parvovirus (B19)

Finegoldia magnus

Plasmodium falciparum

Fusobacterium nucleatum

Plasmodium sp. (Plasmodium vivax)

Gardnerella vaginalis

Rickettsia sp.

Haemophilus ducreyi

Ross River virus

Klebsiella pneumoniae

Rubella virus

Lactobacillus acidophilus

Schistosoma sp.

Lactobacillus crispatus

Trypanosoma cruzi

Leptotrichia buccalis

Varicella Zoster virus

Listeria monocytogenes

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Performance

Table 7: List of Organisms for Cross-Reactivity Analysis (continued)

Flaviviruses
—

Other Organisms

Western Equine Encephalitis virus

Mobiluncus curtisii

—

Herpes simplex virus type 1

Mycoplasma hominis

—

Herpes simplex virus type 2

Mycoplasma pneumoniae

—

Human papillomavirus type 16

Neisseria gonorrhoeae

—

Trichomonas vaginalis

Prevotella bivia

—

Streptococcus agalactiae

Propionibacterium acnes

—

Streptococcus pyogenes

Proteus vulgaris

—

Ureaplasma parvum

Pseudomonas aeruginosa

—

Ureaplasma urealyticum

Staphylococcus aureus

—

Staphylococcus epidermidis

Interference for Plasma Specimens

The potential for interference from endogenous substances was evaluated by testing patient plasma samples with the characteristics shown in Table 8. Ten plasma specimens were included for each characteristic. Each specimen was split into two aliquots. One aliquot was spiked with ZIKV positive plasma to a concentration of 18 copies/mL. The spiked and unspiked aliquots were tested with the Aptima Zika Virus assay. All unspiked samples were negative. One spiked aliquot was negative on initial testing. The negative result was determined to be due to a spiking error. A fresh aliquot of the sample was spiked and retested. The result was positive upon retesting.

Table 8: Aptima Zika Virus Assay Results Summary of Interference from Endogenous Substances

Zika Virus Specimen

IC S/CO N #P %P
Mean SD CV

Analyte S/CO Mean SD CV

Icteric

10 0 0% 1.88 0.03 2%

0.00

N/A

Lipemic

10 0 0% 1.84 0.05 3%

0.00

N/A

Hemolyzed

10 0 0% 1.86 0.05 3%

0.00

N/A

Unspiked Antinuclear antibody 10

0% 1.92 0.06 3%

Multiple Myeloma 10 0 0% 1.87 0.05 3%

0.00

N/A

0.00

N/A

Systemic Lupus Erythematosus

10 0 0% 1.89 0.07 4%

0.00

N/A

Rheumatoid Factor 10 0 0% 1.93 0.04 2%

0.00

N/A

Icteric

10 10 100% 2.25 0.21 9%

31.69 0.69

Lipemic

10 10 100% 2.19 0.26 12%

31.96 1.58

Hemolyzed

10 10 100% 2.12 0.25 12%

31.51 0.49

Spiked (18 Antinuclear Antibody 10 10 100% 2.09 0.42 20%

31.84 5.18

16%

copies/mL)

Multiple Myeloma

10 10 100% 2.14 0.23 11%

31.73 1.28

Systemic Lupus Erythematosus

10 9a 90% 2.35b 0.39b 17%

32.92b 0.69b

Rheumatoid Factor 10 10 100% 2.13 0.34 16%

32.86 0.66

N = number of specimens, #P = number of positives, %P = percentage of positives, IC = Internal Control, S/CO = Signal to Cutoff ratio, SD = Standard Deviation, CV = Coefficient of Variation, N/A = not available. a A fresh aliquot of the sample was spiked and retested. The result on retesting was positive b Calculated using positive results.

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Performance

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Matrix Equivalency for Serum and Plasma Specimens
To compare the performance of serum and plasma specimens in the Aptima Zika Virus assay, blood from 10 normal donors was collected using the following anticoagulants and tube types: 1) dipotassium ethylenediaminetetraacetic acid (K2 EDTA), 2) tripotassium ethylenediaminetetraacetic acid (K3 EDTA), 3) Acid Citrate Dextrose Adenine (ACD-A), 4) Sodium Citrate (NAC), 5) Plasma Preparation Tubes (PPT), 6) Serum Separation Tube (SST), and 7) Serum Tube (Serum). For each of the 10 donors, blood was collected using each of the 7 tube types. Each donor sample was split into two aliquots. One aliquot was spiked with ZIKV positive plasma at 18 copies/mL. Both the spiked and unspiked aliquots were tested with the Aptima Zika Virus assay.

For the unspiked aliquots, all 70 samples were negative in the Aptima Zika Virus assay. The mean IC S/CO ratios ranged from 1.83 to 1.90 with %CVs ranging from 2% to 3% for each tube type (Table 9). For the spiked aliquots, all 70 samples were positive in the Aptima Zika Virus assay. The mean analyte S/CO ratio for each of the 7 tube types ranged from 31.90 to 34.20 with %CVs ranging from 3% to 4% (Table 10).

Table 9: Aptima Zika Virus Assay Results for Unspiked Plasma and Serum Samples Collected in Various Tube Types

Collection Tube

IC S/CO

P %P

Mean

Analyte S/CO

Mean SD

K2EDTA

1.89

0.06

0.00

0.01

N/A

K3EDTA

1.87

0.04

0.00

N/A

ACD-A

1.87

0.04

0.00

N/A

PPT

1.83

0.04

0.00

N/A

NAC

1.84

0.06

0.00

N/A

Serum

1.85

0.06

0.00

N/A

SST

1.90

0.05

0.00

N/A

Table 10: Aptima Zika Virus Assay Results for Spiked Plasma and Serum Samples Collected in Various Tube Types

Collection Tube

IC S/CO

P %P

Mean

Analyte S/CO

Mean SD

K2EDTA

100% 2.05

0.45

22%

32.77

1.23

K3EDTA

100% 1.99

0.39

20%

32.63

0.82

ACD-A

100% 1.88

0.44

23%

32.02

1.32

PPT

100% 1.92

0.25

13%

32.32

1.24

NAC

100% 1.91

0.50

26%

31.90

1.31

Serum

100% 1.78

0.31

18%

34.20

1.34

SST

100% 1.77

0.51

29%

32.52

1.32

N = number of specimens, #P = number of positives, %P = percentage of positives, IC = Internal Control, S/CO = Signal to Cutoff ratio, SD = Standard Deviation, CV = Coefficient of Variation.

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Performance

Clinical Evaluation for Plasma Specimens

Twenty six (26) plasma specimens were obtained from three commercial resources. The 26 specimens were determined by the vendors to be positive for ZIKV based on the results of the CDC TrioPlex Assay (two vendors) or a validated real-time RT-PCR test. The specimens were re-tested using a different validated real-time RT-PCR test and 24 of 26 specimens were confirmed positive. The two specimens that were negative on re-testing are considered negative for the reference result in the analyses below. The Aptima Zika Virus assay was positive for all 26 clinical specimens. Table 11 shows the results for the 24 reference positive specimens.

Table 11: Aptima Zika Virus Assay Results of 24 ZIKV Positive Clinical Specimens

Specimen ID Country of Origin

08847156 08847163 08847229 08847260 08847264 08847284 08847325 08847716 1043-TDS-0112 1043-TDS-0114 1043-TDS-0115 1043-TDS-0119 1043-TDS-0122 1043-TDS-0129 1043-TDS-0130 1043-TDS-0131 1043-TDS-0134 1043-TDS-0135 1043-TDS-0137 1043-TDS-0141 1043-TDS-0143 1043-TDS-0144 1043023924 8798593

Colombia Colombia Colombia Colombia Colombia Colombia Colombia Colombia Dominican Republic Dominican Republic Dominican Republic Dominican Republic Dominican Republic Dominican Republic Dominican Republic Dominican Republic Dominican Republic Dominican Republic Dominican Republic Dominican Republic Dominican Republic Dominican Republic Colombia Colombia

Reference Ct/Cp
34.14 34.90 31.43 32.75 36.32 33.14 36.22 31.76 31.80 35.20 24.74 30.69 35.05 37.24 34.23 29.66 37.30 34.07 29.54 30.71 28.73 34.19 34.69 22.75

Aptima Result
Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive

Aptima S/CO
30.5 31.3 31.3 32.5 32.8 32.5 31.2 29.8 30.9 31.8 32.3 32.1 30.6 31.8 33.4 30.3 31.0 32.1 31.7 32.0 29.6 29.8 30.3 31.7

A total of 90 contrived specimens were prepared by spiking ZIKV positive plasma into individual plasma specimens to a concentration of 18 copies/mL. The 90 specimens include 10 individual plasma specimens from patients who are positive for Parvovirus B19, Dengue, HAV, HBV, HCV, HIV, or WNV; 10 plasma specimens from an HBV vaccinated donor; and 10 plasma specimens from normal donors.

A total of 72 individual plasma samples were used as ZIKV negative specimens. Seventy (70) specimens include 10 individual plasma specimens each that are antinuclear antibody positive, hemolyzed (elevated hemoglobin), Icteric (elevated bilirubin), lipemic (elevated lipid), multiple myeloma, rheumatoid arthritis, or systemic lupus erythematosus. Two specimens positive by initial reference testing but negative on re-testing are also included. These two specimens were

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positive by the Aptima Zika Virus assay. The clinical evaluation results are summarized in Table 12.

Table 12: Clinical Evaluation Results for the Aptima Zika Virus Assay

Specimen Category

Aptima Zika Virus Assay Number Tested ZIKV Positive ZIKV Negative

Natural Zika Positive Specimens

24 / 24

0 / 24

Contrived Zika Positive Clinical Specimens (3 x LoD)

90a

90 / 90

0 / 90

Expected Zika Negative Clinical Specimens Positive Percent Agreement
Negative Percent Agreement

72b

2 / 72

100% (114 / 114)

95% CI: 96.7% to 100%

97.2% (70 / 72)b

95% CI: 90.4 to 99.2%

70 / 72

CI = Confidence Interval. a Includes the Zika spiked aliquots from the 80 plasma specimens evaluated in the Cross-Reactivity study and the Zika spiked aliquots from 10 plasma specimens evaluated in the Matrix Equivalency study. b Includes two patient samples that were positive on initial reference testing and negative on re-testing by an alternate PCR method and was considered a false positive.

Additional Specificity Testing for Plasma and Serum Specimens

The specificity of the Aptima Zika Virus assay on the Panther instrument platform was further evaluated by testing 775 plasma specimens and 240 serum specimens from normal blood donors. All results were negative. The specificity was 100% (1015/1015) for the Aptima Zika Virus assay with a lower 95% confidence interval of 99.6%. There were no invalid results out of 1015 samples tested with the Aptima Zika Virus assay (Table 13).

Table 13: Plasma and Serum Specificity Specimen Type

Plasma

Serum

Total

Number Valid

775

240

1015

Number Negative

775

240

1015

Specificity

100.0

95% CI, Lower Limit

99.5

98.4

99.6

95% CI, Upper Limit

100.0

CI = Confidence Interval.

Invalid Rate for Serum and Plasma Specimens
The invalid rate due to assay chemistry errors for all analytical and clinical specimen testing was 0.09% (3/3375). There were 12 invalid reactions due to hardware or sample issues: 1 CLT (sample clot), 1 RDFS (sample dispense error), 2 VVFS (volume verification failure), 3 QNS (sample quantity not sufficient), and 5 PTF (pipettor arm is unable to pick a tip due to tip loading error by operator). The total invalid rate was 0.44% (15/3375). All invalid replicates were retested with valid results.

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Performance

Clinical Evaluation for Processed Whole Blood Specimens
The clinical evaluation for whole blood for the Aptima Zika Virus assay on the Panther system consisted of evaluating 25 natural Zika positive samples, 25 contrived Zika positive samples, and 50 negative samples.

From 25 individual donors, 25 index plasma samples as well as matched plasma and whole blood samples from a subsequent time point were received from a vendor. These samples were obtained from Puerto Rico and Florida from ZIKV positive donors. At the time of collection, the index plasma specimen from each study donor was determined to be positive for ZIKV RNA using IND assays. For the 25 index plasma positive samples, repeat testing was performed with the primary screening IND assays and an additional validated assay. For the matched plasma and whole blood samples at follow up time points, testing was initially done using an IND assay and a validated assay. For Aptima Zika Virus assay testing, whole blood samples were lysed in order to make processed whole blood. The 25 processed whole blood specimens were tested in one replicate. The plasma specimens were tested neat.

A patient infected status (PIS) was used as the comparator for evaluating the whole blood performance. The PIS was determined based on the combined index plasma testing results of the original IND result and the Aptima Zika Virus assay duplicate testing, where 2/3 is positive and 1/3 is considered indeterminate.

The Aptima Zika Virus assay results are provided in Table 14. Out of 25 index plasma samples tested, 22 were positives. For the 3 negatives seen in the index plasma specimens, the comparator results at time of collection also were negative for repeat testing results. Indeterminate PIS specimens, ARBO8135 and ARBO2165, were excluded from the performance evaluation, giving a total of 23 clinical positives based on the PIS. At the follow up time point, there were 10 Aptima Zika positive plasma results and 24 positive whole blood results. For the 1 out of 25 whole blood sample that tested negative, the matched plasma follow up time point was also negative.
Table 14: Aptima Zika Virus Assay Results of 25 ZIKV Positive Clinical Processed Whole Blood Specimens

Specimen ID ARBO5707

S/CO 31.73

Index Plasma

Result POSITIVE

Patient Infected Status
Positive

Follow up Time Point Plasma

S/CO 0

Result Negative

Follow up Time Point Whole Blood

S/CO

Result

10.50

POSITIVE

ARBO1184

34.00

POSITIVE

Positive

Negative

31.79

POSITIVE

ARBO1501

32.92

POSITIVE

Positive

Negative

21.43

POSITIVE

ARBO8852

18.63

POSITIVE

Positive

Negative

31.89

POSITIVE

ARBO5970

32.35

POSITIVE

Positive

POSITIVE

33.34

POSITIVE

ARBO0560

16.87

POSITIVE

Positive

POSITIVE

31.26

POSITIVE

ARBO3777

17.57

POSITIVE

Positive

Negative

30.98

POSITIVE

ARBO8135a

0.00

Negative

Indeterminate

Negative

30.80

POSITIVE

ARBO7608

32.57

POSITIVE

Positive

POSITIVE

33.27

POSITIVE

ARBO6411 ARBO1358 ARBO2952 ARBO3249 ARBO1389 ARBO3371

32.74 30.61 32.19 32.15 32.26 17.13

POSITIVE POSITIVE POSITIVE POSITIVE POSITIVE POSITIVE

Positive Positive Positive Positive Positive Positive

POSITIVE

Negative

POSITIVE

Negative

20.89 30.75 30.64 30.55 31.42 31.18

POSITIVE POSITIVE POSITIVE POSITIVE POSITIVE POSITIVE

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Table 14: Aptima Zika Virus Assay Results of 25 ZIKV Positive Clinical Processed Whole Blood Specimens

Specimen ID ARBO5191

S/CO 32.62

Index Plasma

Result POSITIVE

Patient Infected Status
Positive

Follow up Time Point Plasma

S/CO 0

Result Negative

Follow up Time Point Whole Blood

S/CO

Result

0.00

Negative

ARBO9066b ARBO8167 ARBO1505 ARBO8314 ARBO6837 ARBO3264 ARBO5760 ARBO8597

0.00 31.41 30.77 31.81 31.91 32.73 15.95 28.31

Negative POSITIVE POSITIVE POSITIVE POSITIVE POSITIVE POSITIVE POSITIVE

Positive Positive Positive Positive Positive Positive Positive Positive

POSITIVE

Negative

POSITIVE

Negative

POSITIVE

32.00 30.87 19.68 31.17 32.93 30.44 30.41 22.62

POSITIVE POSITIVE POSITIVE POSITIVE POSITIVE POSITIVE POSITIVE POSITIVE

ARBO2165c

0.00

Negative

Indeterminate

Negative

30.30

POSITIVE

a Index plasma negative in 2/2 original repeat testing and equivocal in BSRI test. b Index plasma negative in original 1/2 repeat testing and negative in BSRI test. c Index plasma negative in original 2/2 repeat testing and negative in BSRI test.

A total of 25 contrived specimens were prepared by spiking the FDA ZIKV reference S2 positive into individual whole blood specimens. Ten samples were spiked at 10X LOD (1670 RNA NAAT units/mL) and 15 samples were spiked at 2X LOD (334 RNA NAAT units/mL). A total of 50 individual whole blood samples were used as ZIKV negative specimens. The clinical evaluation results based on the PIS are summarized in Table 15.

Table 15: Clinical Evaluation Results for the Aptima Zika Virus Assay: Processed Whole Blood Specimens

Specimen Category

Aptima Zika Virus Assay Number Tested ZIKV Positive ZIKV Negative

Natural Zika Positive Specimens

23a

22/23

1b/23

Contrived Zika Positive Clinical Specimens Expected Zika Negative Clinical Specimens Positive Percent Agreement
Negative Percent Agreement

24/25

0/50

95.3% (46/48)

95% CI: 86.0% to 98.9%

100% (50/50)

95% CI: 92.9% to 100%

1/25c 50/50

a Of the 25 individuals whose index plasma specimen initially tested positive by an IND assay, 2 individuals patient infected status testing resulted in an indeterminate PIS and the samples were excluded from the performance analysis. b The paired plasma result was negative by the Aptima Zika Virus assay; therefore, the negative whole blood result may not be a false negative result. c Negative contrived sample tested positive upon repeat.

Invalid Rate for Processed Whole Blood Specimens
The invalid rate due to assay chemistry errors for all analytical and clinical specimen testing described in this section for processed whole blood specimen was 0.92% (6/655). Upon retesting all invalid results repeated as valid results. There were no invalid reactions due to hardware or sample issues. The total invalid rate was 0.92% (6/655).

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Performance

Performance Evaluation for Urine Specimens
The Aptima Zika Virus assay performance was evaluated using processed urine specimens. For processing, urine was mixed with Aptima Urine Transport Medium at the ratio of 1:1 (2 mL of urine was added into Aptima Urine Specimen Transport Tube, which contained 2 mL of urine transport media).

Limit of Detection (LoD) for Urine Specimens

The Limit of Detection (LoD) is defined as the concentration of ZIKV RNA that is detected at 95% or greater probability according to CLSI EP17-A2.19 The LoD was determined by testing a ZIKV positive plasma specimen serially diluted in pooled negative urine. The highest volume of the positive plasma spiked into urine was 5.5%. The positive plasma specimen was collected from a blood donor during the 2015 Zika outbreak in Brazil. The sample was quantified using a validated real-time RT-PCR assay. The urine sensitivity panel members were prepared by spiking ZIKV positive plasma specimen into the urine at the stated concentration. The spiked panel members were processed by mixing with UTM at a ratio of 1:1 prior to testing. Three Panther instruments were used to test 30 replicates of each target level. The results are summarized in Table 16.

Table 16: Detection of Zika Virus in Processed Urine Specimen with the Aptima Zika Virus Assay on the Panther System

Virus copies/mL

Tested

Positive

Positivity Lower 95%CI Upper 95%CI

0.3

100

%P = percentage positive, CI = Confidence Interval.

The 95% detection probability was determined using Probit analysis. The limit of detection for the Zika virus in processed urine was determined to be 8.5 copies/mL at 95% detection probability (Table 17).

Table 17: Probit Analysis Detection Urine Specimen

95% Detection Probability in copies/mL (95% Fiducial Limits)

Processed Urine

8.5 (6.015.3)

Clinical Evaluation for Urine Specimens
10 paired specimens were obtained (plasma/serum/urine matched specimens collected from 10 symptomatic patients) from a commercial resource. The 10 symptomatic patients were determined by the vendor to be positive for ZIKV based on the results of the serum specimens tested with a validated real-time RT-PCR test. The urine specimens were processed prior to testing. The ten processed urine specimens were tested along with the plasma and serum

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Aptima®

samples from each of 10 patients using the Aptima Zika Virus assay. All specimens were positive upon initial testing. Table 18 shows the results for the 10 matched specimens.

Table 18: Aptima Zika Virus Assay Results of 10 Matched ZIKV Positive Clinical Specimens

Specimen ID

Country of Reference Cp

Plasma

Origin

(Serum) Result S/CO

1043-TDS-0159 Dominican Republic

36.31

Positive

33.1

1043-TDS-0163 Dominican Republic

32.54

Positive

33.4

1043-TDS-0165 Dominican Republic

40.38

Positive

32.8

1043-TDS-0173 Dominican Republic

33.15

Positive

32.6

1043-TDS-0206 Dominican Republic

36.62

Positive

31.6

1043-TDS-0221 Dominican Republic

38.11

Positive

17.8

1043-TDS-0223 Dominican Republic

32.50

Positive

34.0

1043-TDS-0224 Dominican Republic

31.81

Positive

33.8

1043-TDS-0230 Dominican Republic

30.51

Positive

33.8

1043-TDS-0231 Dominican Republic

35.63

Positive

31.6

Ser um Result S/CO

Positive

31.7

Positive

33.4

Positive

32.6

Positive

32.8

Positive

30.8

Positive

33.5

Positive

33.4

Positive

31.6

Positive

33.7

Positive

33.8

Process ed Urine Result S/CO

Positive

32.9

Positive

17.0

Positive

33.7

Positive

34.1

Positive

32.4

Positive

32.8

Positive

31.9

Positive

33.6

Positive

34.7

Positive

34.4

A total of 99 contrived urine specimens were prepared by spiking ZIKV positive plasma into individual urine specimens: 33 specimens were spiked at 20 copies/mL, 33 specimens were spiked at 36 copies/mL, and 33 specimens were spiked at 100 copies/mL. Each spiked urine specimen was processed prior to testing with the Aptima Zika Virus assay. All contrived processed urine specimens tested positive.

A total of 123 individual urine specimens were used as ZIKV RNA negative specimens. 87 urine specimens were collected from a normal population. 36 individual female urine specimens were collected from a patient population (7 patients with breast cancer, 6 patients with chronic kidney disease, 6 patients with systemic lupus erythematosus, 4 patients with pneumonia, 8 patients with diabetes, and 5 patients with urinary tract infection). Each urine specimen was processed prior to testing with the Aptima Zika Virus assay. All specimens tested negative. The clinical evaluation results are summarized in Table 19.

Table 19: Clinical Evaluation Results for Processed Urine Specimens

Specimen Category

Aptima Zika Virus Assay Number Tested ZIKV Positive ZIKV Negative

Natural Zika Positive Specimens Contrived Zika Positive Clinical Specimens Expected Zika Negative Clinical Specimens Positive Percent Agreement
Negative Percent Agreement CI = Confidence Interval

10/10

0/10

99/99

0/99

123

0/123

123/123

100% (109/109)

95% CI: 96.6% to 100%

100% (123/123)

95% CI: 97.0% to 100%

Invalid Rate for Urine Specimens
The invalid rate due to assay chemistry errors for all analytical and clinical specimen testing described in this section for urine specimen was 0% (0/482). There were no invalid reactions due to hardware or sample issues. The total invalid rate was 0% (0/482).

Aptima Zika Virus Assay

AW-15406 Rev. 005

Aptima®

Performance

Comparator Assay Information

The Aptima Zika Virus assay performance with processed whole blood (PWB) specimens was evaluated in comparison to two qualitative RT-PCR tests approved by FDA for the detection of Zika Virus (ZIKV) RNA in plasma specimens (cobas Zika and Procleix Zika Virus Assay approved by CBER) and one RT-PCR lab developed test for the detection of Zika Virus RNA in plasma specimens (Vitalant Research Institute, formerly known as Blood Systems Research Institute).
The Aptima Zika Virus assay performance with plasma specimens was evaluated in comparison to two qualitative RT-PCR tests for use with plasma specimens. The first test was authorized by FDA for use by clinical laboratories for the qualitative detection of RNA from Zika Virus (claimed LoD 10,000 RNA NAAT Detectable Units/mL (FDA Reference Material Testing S1) and 5,000 RNA NAAT Detectable Units/mL (FDA Reference Material Testing S2)). The second test was a RT-PCR lab developed test for the detection of Zika Virus RNA in plasma specimens (Vitalant Research Institute, formerly known as Blood Systems Research Institute). One additional qualitative RT-PCR test authorized by FDA for the detection of Zika Virus RNA in serum was also used. The claimed LoD for the comparator assay with serum specimens was 3,300 RNA NAAT Detectable Units/mL (FDA Reference Material Testing S1), and 1,670 RNA NAAT Detectable Units/mL (FDA Reference Material Testing S2).
The Aptima Zika Virus assay performance with processed urine specimens was evaluated in comparison to a qualitative RT-PCR test that was authorized by FDA for use by clinical laboratories for the qualitative detection of RNA from Zika Virus. The claimed limit of detection for the comparator assay with plasma specimens is 10,000 RNA NAAT Detectable Units/mL (FDA Reference Material Testing S1) and 5000 RNA NAAT Detectable Units/mL (FDA Reference Material Testing S2). Units chose for LoD claims were selected by the comparator assay developers. Not all comparator assays were compared to FDA reference panel S1 and S2.

Analytical Sensitivity using FDA Reference Material
The evaluation of analytical sensitivity was performed using reference material (S1 and S2) and standard protocol provided by the FDA. The study included a range finding study and a confirmatory study for LoD. The results are summarized in the Table 20.

Table 20: Summary of LoD Confirmation Result using the FDA Reference Materials

Reference Materials Provided by FDA
S1 (1×106 RNA NAAT detectable units/mL)
S2 (5×106 RNA NAAT detectable units/mL)

Specimen Type
Plasma Processed Urine Processed Whole Blood
Plasma Processed Urine Processed Whole Blood

Confirmed LoD per FDA Protocol (RNA NAAT Detectable Units/mL)
100 300 1000
150
150
167

Aptima Zika Virus Assay

AW-15406 Rev. 005

Bibliography

Aptima®

Bibliography

1. International Committee on Taxonomy of Viruses. http://www.ictvonline.org/
2. Musso D, Gubler DJ. 2016. Zika virus. Clin Microbiol Rev. Jul;29(3):487-524.
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5. Cao-Lormeau VM, Roche C, Teissier A, Robin E, Berry AL, Mallet HP, Sall AA, Musso D. 2014. Zika virus, French Polynesia, South Pacific, 2013. Emerg Infect Dis 20:10851086.
6. Campos GS, Bandeira AC, Sardi SI. 2015. Zika virus outbreak, Bahia, Brazil. Emerg Infect Dis 21:18851886. doi: 10.3201/ eid2110.150847.
7. Zanluca C, Melo VC, Mosimann AL, Santos GI, Santos CN, Luz K. 2015. First report of autochthonous transmission of Zika virus in Brazil. Mem Inst Oswaldo Cruz 110:569-572.
8. Cao-Lormeau, V.-M. et al. GuillainBarré syndrome outbreak associated with Zika virus infection in French Polynesia: a casecontrol study. Lancet https://doi.org/10.1016/S0140-6736(16)00562-6.
9. Rasmussen SA, Jamieson DJ, Honein MA, Petersen LR. 2016. Zika virus and birth defects — reviewing the evidence for causality. N Engl J Med. 2016 May 19;374(20):1981-1987. doi: 10.1056/NEJMsr1604338. PubMed PMID: 27074377.
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11. U.S. Centers for Disease Control and Prevention. http://www.cdc.gov/zika/transmission/blood-transfusion.html
12. Kacian DL, Fultz T J. 1995. Nucleic acid sequence amplification methods. U.S. Patent 5,399,491.
13. Arnold LJ, Hammond PW, Wiese WA, Nelson NC. 1989. Assay formats involving acridinium-ester-labeled DNA probes. Clin Chem 35:1588-1594.
14. Nelson NC, Cheikh A, Matsuda E, Becker M. 1996. Simultaneous detection of multiple nucleic acid targets in a homogeneous format. Biochem. 35:8429-8438.
15. Clinical and Laboratory Standards Institute. 2005. Collection, Transport, Preparation, and Storage of Specimens for Molecular Methods; Approved Guideline. CLSI Document MM13-A. Wayne, PA.
16. 29 CFR Part 1910.1030. Occupational Exposure to Bloodborne Pathogens; current version.
17. Centers for Disease Control and Prevention/National Institutes of Health. Biosafety in Microbiological and Biomedical Laboratories (BMBL); current version.
18. Clinical and Laboratory Standards Institute. 2002. Clinical Laboratory Waste Management. CLSI Document GP5-A2. Villanova, PA.
19. Clinical and Laboratory Standards Institute (CLSI). 2012. Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline–2nd ed. CLSI Document EP17-A2. Clinical and Laboratory Standards Institute, Wayne, PA.

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AW-15406 Rev. 005 2023-05

Aptima Zika Virus Assay

AW-15406 Rev. 005

References

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HOLOGIC Zika Virus Assay

Product Information

Product Usage Instructions

FAQs

General Information

Specimen Collection and Storage

Reagents and Materials Provided

Quality Control

Performance

Tested #Positive

Tested #Positive

Interference for Plasma Specimens

P %P

P %P

Limit of Detection (LoD) for Urine Specimens

Tested

Positive

Comparator Assay Information

Bibliography

References

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