The Journal of Molecular Diagnostics, Vol. 21, No.

6, November 2019

jmd.amjpathol.org

A Zika Reference Panel for Molecular-Based

Diagnostic Devices as a US Food and Drug
Administration Response Tool to a Public Health Emergency
Mayra García,* Rafaelle Fares-Gusmao,y Kim Sapsford,* Caren Chancey,y Andriyan Grinev,y Stephen Lovell,* Uwe Scherf,* and
Maria Riosy

From the Office of in Vitro Diagnostics and Radiological Devices,* Center for Devices and Radiological Health, and the Office of Blood Research and
Review,y Center for Biologics Evaluation and Research, US Food and Drug Administration, Silver Spring, Maryland

Accepted for publication

June 13, 2019.
Address correspondence to
Mayra García, Ph.D., M.B.A.,
Food and Drug Administration,
10903 New Hampshire Ave,
Bldg 66, Room 3206, Silver
Spring, MD 20993. E-mail:
mayra.garcia@fda.hhs.gov.
In 2015, Zika virus (ZIKV) appeared as an emerging pathogen, generating a global and urgent need for
accurate diagnostic devices. During this public health crisis, several nucleic acid testing (NAT)ebased
Zika assays were submitted to the US Food and Drug Administration (FDA) for Emergency Use Autho-
rization. The FDA’s Center for Devices and Radiological Health, in collaboration with the FDA’s Center for
Biologics Evaluation and Research, responded to this Zika emergency by developing and producing a
reference panel (RP) for Zika RNA (Zika FDA-RP) suitable for performance assessment of ZIKV NAT-based
in vitro diagnostic devices. Reference panels are a fundamental tool for performance assessment of
molecular tests. The panel is composed of five vials: two different heat-inactivated ZIKV strains
(PRVABC59 and FSS13025) in concentrated stocks and three blinded concentrations prepared from
those strains. The Zika FDA-RP was shared with developers who had devices in the final stages of
validation. In vitro diagnostic developers tested the Zika FDA-RP using the FDA-provided protocol.
Depending on sample type, 85% (12/14) of the NAT assays had analytical sensitivities between 500 and
5000 RNA NAT-detectable units/mL (NDUs/mL). One device showed better performance (100 NDUs/mL),
and another one showed lower performance (10,000 to 30,000 NDUs/mL). Vials of the Zika FDA-RP are
available on request to developers who have interacted with the FDA through the review process.
(J Mol Diagn 2019, 21: 1025e1033; https://doi.org/10.1016/j.jmoldx.2019.06.004)

On February 26, 2016, the Secretary of Health and Human
Services declared that circumstances existed justifying the
authorization of the emergency use of in vitro diagnostics
(IVDs) for detection of Zika virus (ZIKV) and/or diagnosis
of ZIKV infection.
ZIKV is an arbovirus member of the Flaviviridae family,
transmitted to individuals primarily through the bite of an
infected Aedes mosquito. In 2015, ZIKV first appeared
outside of Africa and Asia when it was isolated in Brazil,1,2
causing an outbreak that likely originated from an infected
traveler from French Polynesia. From there, the virus spread
through South, Central, and North America, reaching the
Caribbean in the beginning of 2016.3,4 Although it
often causes only arthralgia, myalgia, headache, conjuncti-
vitis, mild rashes, and fever,5 or no symptoms at all,
severe neurologic manifestations, including Guillain-Barré
syndrome and congenital microcephaly, have been associ-
ated with ZIKV infection.6,7 Early and correct diagnosis of
ZIKV infection in pregnant women is critically important to
identify babies with a potential risk of microcephaly and
other brain anomalies, which is complicated by the fact that
80% of ZIKV infections are asymptomatic. Problems from
microcephaly can range from mild to severe, are
often lifelong, and, in some cases, can be life threatening
Supported by US Food and Drug Administration (FDA) Medical
Countermeasure Initiative grant OCET 2016-331 (M.R.). This project was
supported in part by an appointment to the Research Participation Program
at the Office of Blood Research and Review/Center for Biologics Evalua-
tion and Research, FDA, administered by the Oak Ridge Institute for Sci-
ence and Education through an interagency agreement between the US
Department of Energy and the FDA.
Disclosures: None declared.

Published by Elsevier Inc. on behalf of the American Society for Investigative Pathology and the Association for Molecular Pathology.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0).
https://doi.org/10.1016/j.jmoldx.2019.06.004
García et al

(https://www.cdc.gov/pregnancy/zika/testing-follow-up/infa characteristics are summarized on the FDA website in

nts-children.html, last accessed May 13, 2019).
Current diagnostic technologies for ZIKV include virus
isolation, genome detection, antigen detection, and
serology.8 Viral RNA detection by nucleic acid testing
(NAT) is the most sensitive and specific method suitable
for early disease stage diagnosis. ZIKV RNA is typically
detectable in serum during the acute phase of infection
(generally up to 7 days after symptom onset), although it
has been detected in serum up to 13 days after symptom
onset in nonpregnant patients and up to 62 days after
symptom onset in pregnant patients. In addition, ZIKV
RNA has been detected up to 53 days after the last
known possible exposure in an asymptomatic pregnant
patient.7,9
To date, the US Food and Drug Administration (FDA)
has issued Emergency Use Authorizations (EUAs) to 15
NAT-based assays for ZIKV, with 14 currently available
on the market. The authorized tests offer unique charac-
teristics with respect to sample throughput, testing envi-
ronment, claimed sample types, and/or performance that
are taken into account when considering whether to issue
an EUA for an assay. The performance evaluation and key
the Zika Virus Emergency Use Authorization section
(https://www.fda.gov/MedicalDevices/Safety/EmergencySitu
ations/ucm161496.htm, last accessed May 13, 2019). These
authorized NAT-based ZIKV tests used a range of ZIKV
material sources when evaluating the analytical limit of
detection (LoD). In addition, the clinical validation that
was performed for currently authorized NAT-based ZIKV
tests differed with respect to the comparator assay used,
the number of samples, and the population of patients
tested (ie, endemic and nonendemic area samples in
different numbers). All these factors impact the final
performance and make comparison based on the analytical
sensitivity and clinical data between the assays chal-
lenging. Therefore, it is critical to have well-characterized
reference reagents to evaluate performance of all assays
before Emergency Use Authorization, and to compare the
performance of different molecular tests under the same
conditions. The FDA, through the collaborative work
between the FDA/Center for Devices and Radiological
Health (CDRH) and the FDA/Center for Biologics Eval-
uation and Research (CBER), responded to the need to
directly compare the sensitivity of different diagnostic

Source Dilution by
Diluted 1:10 by FDA S1
S1: FSS13025 Stock recipient
in BaseMatrix 1 x 106 NDUs/mL
10-1 S1.1
1 x 105
NDUs/mL

10-7 S1.7
1 x 10-1
NDUs/mL
Diluted 1:10 by FDA in S2
S2: PRVABC59 Stock
BaseMatrix 5 x 106 NDUs/mL
10-1 S2.1
5 x 105
NDUs/mL

10-7 S2.7
5 x 10-1
NDUs/mL

}
S3, S3 10-1
Dilution of S1 and S2
S4 Blinded
S4, S1 and S2 prepared by FDA titer 10-1
in BaseMatrix
S5: S5 10-2

Figure 1 Diagram of S1 through S5 and their original dilutions and/or concentrations as well as the required further dilutions to be performed by the
receiving laboratories. FDA, US Food and Drug Administration; NDUs, nucleic acid testingedetectable units.

1026 jmd.amjpathol.org - The Journal of Molecular Diagnostics

 Panel for ZIKV RNA Diagnostic Devices

A LoD determination LoD confirmation

Dilution Hit rate Dilution Hit rate
10-1 3/3
10-2 3/3 3 x 10-3 20/20
10-3 3/3 1 -3
10 19/20
10-4
2/3 33 x 10-3
00.33 16/20 Figure 2 A: Limit of detection (LoD) deter-
mination: The lowest concentration for which all
10-5 1/3 three tested replicates generated positive results
10-6 0/3 was selected as the estimated LoD for confirma-
10-7 0/3 tion. LoD confirmation: The concentration of each
virus at which repeated testing of this sample
B Standard curve based on S1-S2 range finding would give a positive result approximately 95%
(19/20) of the time and a negative result 5% of
42.0 the time. B: A curve was prepared, plotting the
combined average S1 and S2 CT values obtained
from testing versus the target concentrations in
39.0
nucleic acid testingedetectable units (NDUs)/mL.
Average CT

For each CT value obtained for a blinded material,

36.0 Blinded S3, S4, or S5 C an estimated value of NDUs/mL was obtained by
T comparing the CT values of the unknown with
those in the curve.
33.0

Blinded S3, S4, or S5 concentration

30.0
2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
Log10 NDUs/mL

assays by developing and producing the reference panel
for Zika RNA (Zika FDA-RP). The Zika FDA-RP is
composed of standardized material, suitable for the
determination of analytical sensitivity and for evaluation
of a blinded panel, both required for performance
assessment of ZIKV-NAT IVDs as part of the EUA con-
ditions for authorization.
The ZIKV strains used in this panel have been cultivated,
heat inactivated, and genetically characterized by the FDA/
CBER.

Table 1 Authorized Assays and Applicants

Medical product Date of EUA issuance
CDC Trioplex Real-Time RT-PCR Assay (Trioplex rRT-PCR; CDC, Atlanta, GA) March 17, 2016 (initial issuance)
Zika Virus RNA Qualitative Real-Time RT-PCR (Quest Diagnostics April 28, 2016 (initial issuance)
Infectious Disease, Inc., Walnut Creek, CA)
RealStar Zika Virus RT-PCR Kit U.S. (altona Diagnostics GmbH, Hamburg, Germany) May 13, 2016 (initial issuance)
Aptima Zika Virus assay (Hologic, Inc., San Diego, CA) June 17, 2016 (initial issuance)
Zika Virus Real-time RT-PCR Test (Viracor Eurofins, Lee’s Summit, MO) July 19, 2016 (initial issuance)
VERSANT Zika RNA 1.0 Assay (kPCR) Kit (Siemens Healthcare Diagnostics Inc., Tarrytown, NY) July 29, 2016 (initial issuance)
xMAP MultiFLEX Zika RNA Assay (Luminex Corp., Austin, TX)* August 4, 2016 (initial issuance)
Sentosa SA ZIKV RT-PCR Test (Vela Diagnostics USA, Inc., Fairfield, NJ) September 23, 2016y
Zika Virus Detection by RT-PCR Test (ARUP Laboratories, Salt Lake City, UT) September 28, 2016y
Abbott RealTime ZIKA (Abbott Molecular Inc., Des Plaines, IL) November 21, 2016 (initial issuance)
Zika ELITe MGB Kit U.S. (ELITechGroup Inc. Molecular Diagnostics, Bothell, WA) December 9, 2016y
Gene-RADAR Zika Virus Test (Nanobiosym Diagnostics, Inc., Cambridge, MA) March 20, 2017y
TaqPath Zika Virus Kit (Thermo Fisher Scientific, Pleasanton, CA) August 2, 2017y
CII-ArboViroPlex rRT-PCR Assay (Columbia University, New York, NY) August 11, 2017y
*This EUA was withdrawn in June 2019 after this manuscript was accepted. Therefore, the company data was kept in the analysis. Luminex decided to
discontinue manufacture of the product.
y
No amendments were submitted to update the application.
EUA, Emergency Use Authorization.

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García et al

Table 2 Sensitivity Mean Estimates of the Emergency Use Authorized Assays Using the Zika FDA-RP
FDA-RP results by decreasing sensitivity

Titer, S1 S2
NDUs/ Serum/ Processed Serum/ Processed
mL plasma Urine urine WB plasma Urine urine WB
100 Hologic, Inc.
150 Hologic, Inc. Hologic, Inc.
167 Hologic, Inc.
300 Abbott Molecular Hologic, Inc.
Inc.
500 Abbott Molecular Abbott
Inc. Molecular Inc.
Columbia University Columbia
Thermo Fisher University
Scientific Thermo Fisher
Viracor Eurofins Scientific
Quest Diagnostics Viracor Eurofins
Infectious
Disease, Inc.
1000 Abbott CDC Abbott
Molecular Inc. Columbia University Molecular
Columbia Viracor Eurofins Inc.
University Quest Diagnostics Hologic, Inc.
Quest Infectious
Diagnostics Disease, Inc.
Infectious
Disease, Inc.
Siemens
Healthcare
Diagnostics
Inc.
Viracor Eurofins
1500 Quest Abbott
Diagnostics Molecular
Infectious Inc.
Disease, Inc.
1581 altona
Diagnostics
GmbH
(automated)
1650 ARUP Laboratories
1670 CDC CDC
3000 ARUP Siemens Healthcare
Laboratories Diagnostics Inc.
Luminex Corp.
3160 altona altona Diagnostics
Diagnostics GmbH
GmbH
3300 CDC ARUP Laboratories
ELITechGroup Luminex Corp.
Inc. Molecular Thermo Fisher
Diagnostics Scientific
Nanobiosym
Diagnostics,
Inc.
Thermo Fisher
Scientific
(table continues)

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 Panel for ZIKV RNA Diagnostic Devices

Table 2 (continued )
FDA-RP results by decreasing sensitivity

Titer, S1 S2
NDUs/ Serum/ Processed Serum/ Processed
mL plasma Urine urine WB plasma Urine urine WB
4500 ARUP
Laboratories
5000 altona Diagnostics altona
GmbH Diagnostics
Luminex Corp. GmbH
Nanobiosym (manual)
Diagnostics, Inc. Luminex Corp.
Siemens Healthcare Siemens
Diagnostics Inc. Healthcare
Diagnostics
Inc.
Vela Diagnostics
USA, Inc.
5560 ELITechGroup Inc.
Molecular
Diagnostics
10,000 Vela Diagnostics
USA, Inc.
15,000 Vela Diagnostics
USA, Inc.
30,000 Vela Diagnostics
USA, Inc.
FDA, US Food and Drug Administration; NDUs, nucleic acid testingedetectable units; RP, reference panel; WB, whole blood; Zika FDA-RP, RP for Zika RNA.

Materials and Methods Heat-Inactivated and NR-50722 CBER/FDA PRVABC59 Zika

Production of Reference Reagents
ZIKV strains PRVABC59 [Puerto Rico-2015, GenBank
(https://www.ncbi.nlm.nih.gov/genbank) accession number
KU501215, kindly provided by the CDC] and FSS13025
(Cambodia-2010, GenBank accession number JN860885,
kindly provided by the University of Texas Medical Branch)
were cultivated at multiplicity of infection 0.01 in African
green monkey kidney cells (VeroeWorld Health
Organization seed) using serum-free medium at 37 C and 5%
CO2. Supernatants from each isolate were harvested on day 4,
clarified by centrifugation at 750  g for 5 minutes, and sub-
mitted to heat inactivation at 60 C for 60 minutes. The complete
abolition of infectivity was confirmed by independent titrations
in three sequential Vero cell passages, with no cytopathic effect
observed in any heat-inactivated or negative control cultures,
whereas non-inactivated stocks showed cytopathic effect within
4 days of culture. All the work was performed in a BSL-2
laboratory at FDA/CBER after biosafety practices. FDA/
CBER produced a lyophilized material prepared from the virus
stocks, the FDA ZIKV RNA reference reagents, for use in the
development and validation of ZIKV-NAT assays for blood
screening.10 Lyophilized aliquots of the strains (NR-50723
CBER/FDA FSS13025 Zika Virus RNA Reference Reagent,
Virus RNA Reference Reagent, Heat-Inactivated) have been
deposited and can be found at BEI Resources (Manassas, VA).
The FDA/CDRH has used a variation of these reference re-
agents formulated from dilutions of the culture media virus
stocks in defibrinated human plasma, the Zika FDA-RP, to
evaluate the efficacy of new diagnostic assays before their EUA.
The Zika FDA-RP includes five members (S1 to S5), as
described below, and can be requested from the FDA/CDRH by
qualified IVD developers.
Each culture medium virus stock (10 mL) was diluted 1:10
(Figure 1) up to a concentration of 1  106 NAT-detectable
units (NDUs)/mL for FSS13025 (designated as S1) and
5  106 NDUs/mL for PRVABC59 (designated as S2) in
dialyzed, defibrinated human plasma (BaseMatrix; SeraCare
Life Sciences, Gaithersburg, MD). The diluted stock was
dispensed as 1.1-mL aliquots into 2-mL plastic cryovials and
stored at 80 C. S1 and S2 were further diluted in Base-
Matrix to produce S3, S4, and S5 (Figure 1). S3, S4, and S5
were dispensed as 1.5-mL aliquots into 2-mL plastic cryo-
vials. The concentrations of S1 and S2 were known by the
receiving laboratories. However, the concentrations of ZIKV
RNAs in dilutions S3, S4, and S5 were not provided to ap-
plicants for blinded testing of assays requesting EUA. The
titers of the vials were confirmed by real-time PCR11 by FDA
scientists after an overnight storage at FDA/CBER.

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García et al

Table 3 Detection of Blinded Samples by the Emergency Use Authorized Assays

Applicant S3 S4 S5 Matrix
CDC High High Low Serum
High High Low Urine
Low* Low* Low* WB
Quest Diagnostics Infectious Disease, Inc. High High Low Serum
High High Low Urine
altona Diagnostics GmbH, using Qiagen (Hilden, Germany) High Medium Low Serum
QIAamp Viral RNA Mini Kit (manual) High High Low Urine
altona Diagnostics GmbH, using Roche (Basel, Switzerland) High Medium Low Serum
Magna Pure 96 Instrument/DNA and Viral NA Small High Medium Low Urine
Volume Kit (automatic)
Hologic, Inc. High High High Plasma
High High Medium Processed urine
High High Medium Processed WB
Viracor Eurofins High High Medium Plasma
High High Medium Urine
Siemens Healthcare Diagnostics Inc. High Medium Low Serum
High High Low Urine
Luminex Corp. High Medium Low Serum
High High Low Urine
Vela Diagnostics USA, Inc. High Low Low Serum
High Medium Low Urine
ARUP Laboratories High High Low Plasma
High High Low Urine
Abbott Molecular Inc. High High Low Serum
High High Medium Urine
High High Low WB
ELITechGroup Inc. Molecular Diagnostics High High Low Plasma
Nanobiosym Diagnostics, Inc. High Medium Low Serum
Thermo Fisher Scientific High Medium Low Serum
High Medium Low Urine
Columbia University High High Low Serum
High High Low Urine
Detection rate: low, 0% to <40%; medium, 40% to <80%; and high, 80% to 100%.
*The device did not perform with inactivated virus in WB.
WB, whole blood.

A single NDU is the minimum level of target that will
result in a positive PCR result. In this work, analysis and
reporting of results of qualitative testing were performed on
the basis of the assumptions that a single NDU will result in
a positive test and the overall number of NDUs follows a
Poisson distribution.12 Therefore, it was assumed that a
single NDU will be sufficient to provide a positive test
result. These estimated NDU/mL values are not necessarily
equivalent to a genuine viral copy number per mL and, thus,
it is not appropriate to replace the words viral copy number
with NAT-detectable units.13,14
Study Design
The samples were tested according to the protocol provided
to the applicants by the FDA. Briefly, all applicants were
required to demonstrate that the unspiked matrices specified
for use with their assays generated negative results for all
their virus-specific primers and probes. To this end, each
unspiked clinical matrix of choice (ie, serum, plasma, whole
blood, or urine, according to the manufacturer’s instructions)
was subjected to at least 20 independent RNA extractions
using the method designated by the applicants for use
with their respective assays. Each replicate of extracted RNA
was tested once for ZIKV detection by the assay under
evaluation.
After the testing matrix was demonstrated to be negative
for the virus, the reference panel was used for spiking. The
panel is composed of five vials: heat-inactivated ZIKV
FSS13025 strain in a concentrated stock (S1), heat-
inactivated ZIKV PRVABC59 strain in a concentrated
stock (S2), and three blinded concentrations prepared from
those two strains (S3, S4, and S5).
To prepare the spiked samples for testing, 1 mL of the
provided panel vial S1 was spiked into 9 mL of the testing
matrix (a final volume of 10 mL) and vortex mixed to ho-
mogenize. This dilution was repeated with panel vial S2,
generating two 1:10 dilutions designated S1.1 and S2.1

1030 jmd.amjpathol.org - The Journal of Molecular Diagnostics


(Figure 1). Using this method, a first set of 10-fold dilution
series was prepared in the testing matrix for a total of seven
dilutions for each of S1 and S2, including S1.1 and S2.1
prepared in step 1. The target concentrations for these
dilution series ranged from 105 down to 101 NDUs/mL.
A range finding study was conducted using the dilution
series prepared from the panel samples S1 and S2 to
determine an LoD. The viral RNA from each diluted
sample was extracted three times, and each extraction was
tested once using the assay method under evaluation. The
lowest concentration for which all three tested S1 or S2
replicates generated positive results was selected as the
estimated LoD for confirmation of the concentration of
each virus (Figure 2A).
For the sensitivity confirmation study, a second set of
dilution series for each sample S1 and S2 was then prepared,
composed of the estimated LoD identified in the previous
step plus one threefold dilution above and below the
estimated LoD. Seventeen additional replicates of each
estimated LoD dilution were tested from extraction to
amplification/detection and added to the results from the
range finding study, for a total of 20 replicates. For each of
the two dilutions bracketing the targeted level for confir-
mation, 20 replicates were tested from extraction to ampli-
fication/detection. Each individually extracted nucleic acid
sample was tested once by the assay under study. If needed
to reach a sensitivity confirmation, threefold dilutions were
continued further down until at least one of the 20 samples
generated a negative result in the abbreviated dilution
window (Figure 2A).
For an independent validation of the range finding
study, 1:10 dilutions of the provided materials S3 and S4
and a 1:100 dilution of the provided material S5 were also
prepared (Figure 1). The concentrations of these diluted
materials were not provided to the developers to use the
panel as a tool for evaluation of proficiency and blinded
validation of performance. Each diluted material was
extracted 10 times, according to the manufacturer’s in-
structions, and tested by the assay under evaluation. For
each CT value obtained for a blinded material (10 values in
total), an estimated value of NDUs/mL was obtained by
comparing the CT values of the material with those in the
curve prepared by plotting the combined S1 and S2
CT values obtained from testing versus the target con-
centrations in NDUs/mL in the range finding study
(Figure 2B).
Results
A list of the authorized assays and their applicants is
shown in Table 1. Although 15 assays were authorized,
one product was later withdrawn by the applicant and is
not included in the list or the analysis. All 14 NAT-based
ZIKV tests have been authorized for use with serum, 9
with plasma, 12 with urine, and 3 with whole blood. For
the purposes of the Zika FDA-RP testing, EUA holders
The Journal of Molecular Diagnostics - jmd.amjpathol.org
Panel for ZIKV RNA Diagnostic Devices
that claimed both serum and plasma could select one of
those matrices for testing, whereas evaluation in urine and/
or whole blood had to be performed with all claimed
matrices separately. A total of 4 devices tested the Zika
FDA-RP panel with plasma, 10 with serum, 3 with whole
blood, and 12 with urine. One device was unable to obtain
results when using whole blood in combination with the
inactivated virus used in the Zika FDA-RP; however, it
was authorized on the basis of the LoD studies with live
virus in whole blood, which provided satisfactory
performance.
Results from qualitative analysis for each assay were
analyzed by Probit regression using JMP version 11 (SAS
Institute Inc., Cary, NC) or 95% positivity, resulting in
means of 100 (one test) to 30,000 (one test) NDUs/mL, with
the remaining 12 currently authorized assays showing
analytical sensitivities between 500 and 5000 NDUs/mL for
PRVABC59 and FSS13025 (Table 2).
Blinded samples (S3, S4, S5) were tested as a validation
of the measured LoD and proficiency of handling the panels
(Table 3). Detection rates were calculated for each device
and its claimed sample types, and category rates of low (0%
to <40%), medium (40% to <80%), and high (80% to
100%) were assigned.
Two scores for detection of these samples were also
calculated: the percentage of devices that detected each
unknown and the average of the percentage of total positive
results detected for each unknown.
S3 and S4 were detected by all devices regardless of the
matrix that was evaluated, although the number of detect-
able replicates ranged from 8/10 to 10/10 for S3 and from
2/10 to 10/10 for S4. S5 was detectable at a frequency
1/10 for 100.0% of devices that claimed plasma, only
30.0% of those that claimed serum, 75.0% of those that
claimed urine, and 50.0% of the two that successfully
evaluated whole blood.
The average percentage of total positive results equaled
the following:
PN h x i
iZ1 10  100
i
% ð1Þ
N
where x is the number of positive results scored in the 10
replicates tested per device, and N is the number of
different devices tested with that matrix. For S3, the per-
centage of replicates detected is an average of 96.0% in
serum and 100.0% in plasma, whole blood, and urine. For
S4, the average percentage of replicates detected is 69.5%
in serum, 95.0% in plasma, 100.0% in whole blood, and
92.6% in urine. For S5, an average of only 4.0% of rep-
licates is detected in serum, 45.0% in plasma, 20.0% in
whole blood, and 20.9% in urine (Figure 3). For altona
Diagnostics GmbH, only the manual version of the assay
was included in the calculations to avoid counting the
same device twice.
1031
García et al
Figure 3 Average percentage of replicates detected (frequency) for the
blinded panel members by matrix type. Blinded panel members (S3, S4, and
S5) are evaluated in 10 replicates each. Average percentage of time that
each unknown is detected is calculated as,
PN h x i developers who have interacted with the FDA through the
iZ1 10  100 i
% Results reported by each company using blinded S3, S4,
N
where x is the number of positive results scored in the 10 replicates for
each device, and N is the number of devices tested with that matrix. Bars
represent error, calculated as SD/ON.
The usefulness of the Zika FDA-RP was demonstrated by
the fact that two additional NAT assays submitted to FDA
for review could not establish an assay performance within
the same ranges of NAT-based ZIKV tests that had received
EUA. Neither company has obtained EUA for their
NAT-based ZIKV assay to date.
Discussion
According to the CDC, vector-borne diseases are a large and
growing public health problem in the United States. During
2004 to 2016, a total of 642,602 cases were reported in the
United States. Transmission in Puerto Rico, the US Virgin
Islands, and American Samoa accounted for most reports of
dengue, chikungunya, and Zika diseases; West Nile virus
emerged in the United States in 1999 and has been endemic
since 2002.15 Beside mosquito bites, additional transmission
routes have been documented for arboviruses, such as intra-
uterine, perinatal, sexual, and blood transfusion. Vector con-
trol programs, such as those suggested by the World Health
Organization (http://www.who.int/en/news-room/fact-sheets/
detail/vector-borne-diseases, last accessed May 13, 2019),
and accurate IVD devices are important tools to respond to
this challenge.
Molecular diagnostic techniques usually yield rapid re-
sults and have high specificity. However, available assays
have a wide range of performances, depending on sample
throughput, testing environment, claimed sample types, in-
struments, extraction methods, and the comparator of
choice. These caveats highlight the need to confirm the
1032
performance of different ZIKV RNA detecting devices with
a common, independent, and well-characterized reference
material. Use of the same reference material across manu-
facturers allows a direct comparison of analytical sensitivity
performance. In collaboration with FDA/CBER, FDA/
CDRH has produced an RP for ZIKV device evaluations.
The importance of such panels became evident during the
Ebola public health emergency that took place in 2014,
where no comparison was possible between assays. With
this new strategy and the requirement of evaluation of the
Zika FDA-RP as a condition of the EUA, the FDA has been
able to provide a comparative analysis of the performance of
alternative devices, which may be useful to health care
providers and laboratories implementing these tests. Even
when no formal bars have been set for the acceptability of
the assays, the performance with the reference panel guides
developers regarding the suitability of their devices for
EUA. The Zika FDA-RP has been shared on request to
review process.
and S5 provide an indication of their proficiency in deter-
mining LoD as well as sample integrity and handling. Thus, a
company that has an assay with excellent sensitivity would
have no problem detecting S3 through S5. However, a com-
pany with a somewhat less sensitive assay might not detect S5
but would have no problem with S4 or S3. The overall results
for S3 through S5 are presented in Figure 3 and Table 3.
Further details (ie, the concentration of blinded panel mem-
bers) cannot be provided as these preparations are still being
used in regulatory decisions based on blind testing. The
authorized assays have shown consistency between reported
LoDs with S1 and S2 and the detectability of S3, S4, and S5.
The EUA authority allows the FDA to facilitate tempo-
rary availability of a device because of the existence of a
public health emergency. EUAs will no longer be available
when the Secretary of Health and Human Services’ decla-
ration terminates, unless the FDA revokes it sooner. It is
important that companies with well-performing diagnostic
devices submit their assays for clearance, a premarket
application that when granted allows permanent marketing
of the device. At this time, there are two NATs approved by
FDA/CBER for blood donor testing (https://www.fda.gov/
NewsEvents/Newsroom/PressAnnouncements/ucm579313.h
tm and https://www.fda.gov/BiologicsBloodVaccines/
BloodBloodProducts/ApprovedProducts/LicensedProducts
BLAs/BloodDonorScreening/InfectiousDisease/ucm61269
6.htm, both last accessed May 13, 2019), but no FDA/
CDRH approved/cleared IVD device is available in the
United States that can detect ZIKV in clinical specimens.
The Zika FDA-RP will also be useful to evaluate
diagnostic devices seeking a de novo/510(k).
In conclusion, we have established a well-characterized
reference panel for ZIKV for use in the evaluation
of analytical performance of IVDs. Vials of the Zika FDA-RP
are available to qualified companies on request.
jmd.amjpathol.org - The Journal of Molecular Diagnostics

Acknowledgments
We thank the CDC and the University of Texas Medical
Branch for providing Zika virus strains KU501215 and
JN860885, respectively.
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