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A Zika Reference Panel for Molecular-Based Diagnostic Devices as a US Food and Drug Administration Response Tool to a Public Health Emergency

2019; Elsevier BV; Volume: 21; Issue: 6 Linguagem: Inglês

10.1016/j.jmoldx.2019.06.004

1943-7811

Mayra García, Rafaelle Fares‐Gusmao, Kim E. Sapsford, Caren Chancey, Andriyan Grinev, Stephen Lovell, Uwe Scherf, María Rios,

COVID-19 epidemiological studies

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)–based Zika assays were submitted to the US Food and Drug Administration (FDA) for Emergency Use Authorization. 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. 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)–based Zika assays were submitted to the US Food and Drug Administration (FDA) for Emergency Use Authorization. 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. 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,1Campos G.S. Bandeira A.C. Sardi S.I. Zika virus outbreak, Bahia, Brazil.Emerg Infect Dis. 2015; 21: 1885-1886Crossref PubMed Scopus (824) Google Scholar, 2Zanluca C. Merlo V.C. Mosimann A.L. Santo G.I. Santos C.N. Luz K. First report of autochthonous transmission of Zika virus in Brazil.Mem Inst Oswaldo Cruz. 2015; 110: 569-572Crossref PubMed Scopus (837) Google Scholar 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 2016.3Faria N.R. Azevedo R.D.S.D.S. Kraemer M.U.G. Souza R. Cunha M.S. Hill S.C. et al.Zika virus in the Americas: early epidemiological and genetic findings.Science. 2016; 352: 345-349Crossref PubMed Scopus (716) Google Scholar, 4World Health Organization Zika virus outbreaks in the Americas.Wkly Epidemiol Rec. 2015; 90: 609-610PubMed Google Scholar Although it often causes only arthralgia, myalgia, headache, conjunctivitis, mild rashes, and fever,5Musso D. Nhan T.-X. Emergence of Zika virus.Clin Microbiol Open Acces. 2015; 04: 222Google Scholar or no symptoms at all, severe neurologic manifestations, including Guillain-Barré syndrome and congenital microcephaly, have been associated with ZIKV infection.6Moore C.A. Staples J.E. Dobyns W.B. Pessoa A. Ventura C.V. Fonseca E.B. Ribeiro E.M. Ventura L.O. Neto N.N. Arena J.F. Rasmussen S.A. Characterizing the pattern of anomalies in congenital Zika syndrome for pediatric clinicians.JAMA Pediatr. 2017; 171: 288-295Crossref PubMed Scopus (578) Google Scholar, 7Driggers R.W. Ho C.Y. Korhonen E.M. Kuivanen S. Jaaskelainen A.J. Smura T. Rosenberg A. Hill D.A. DeBiasi R.L. Vezina G. Timofeev J. Rodriguez F.J. Levanov L. Razak J. Iyengar P. Hennenfent A. Kennedy R. Lanciotti R. du Plessis A. Vapalahti O. Zika virus infection with prolonged maternal viremia and fetal brain abnormalities.N Engl J Med. 2016; 374: 2142-2151Crossref PubMed Scopus (646) Google Scholar 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 (https://www.cdc.gov/pregnancy/zika/testing-follow-up/infants-children.html, last accessed May 13, 2019). Current diagnostic technologies for ZIKV include virus isolation, genome detection, antigen detection, and serology.8Charrel R.N. Leparc-Goffart I. Pas S. de Lamballerie X. Koopmans M. Reusken C. Background review for diagnostic test development for Zika virus infection.Bull World Health Organ. 2016; 94: 574D-584DCrossref PubMed Scopus (95) Google Scholar 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.7Driggers R.W. Ho C.Y. Korhonen E.M. Kuivanen S. Jaaskelainen A.J. Smura T. Rosenberg A. Hill D.A. DeBiasi R.L. Vezina G. Timofeev J. Rodriguez F.J. Levanov L. Razak J. Iyengar P. Hennenfent A. Kennedy R. Lanciotti R. du Plessis A. Vapalahti O. Zika virus infection with prolonged maternal viremia and fetal brain abnormalities.N Engl J Med. 2016; 374: 2142-2151Crossref PubMed Scopus (646) Google Scholar, 9Meaney-Delman D. Meaney-Delman D. Oduyebo T. Polen K.N. White J.L. Bingham A.M. Slavinski S.A. Heberlein-Larson L. St George K. Rakeman J.L. Hills S. Olson C.K. Adamski A. Culver Barlow L. Lee E.H. Likos A.M. Munoz J.L. Petersen E.E. Dufort E.M. Dean A.B. Cortese M.M. Santiago G.A. Bhatnagar J. Powers A.M. Zaki S. Petersen L.R. Jamieson D.J. Honein M.A. US Zika Pregnancy Registry Prolonged Viremia Working GroupProlonged detection of Zika virus RNA in pregnant women.Obstet Gynecol. 2016; 128: 724-730Crossref PubMed Scopus (93) Google Scholar 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 characteristics with respect to sample throughput, testing environment, 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 characteristics are summarized on the FDA website in the Zika Virus Emergency Use Authorization section (https://www.fda.gov/MedicalDevices/Safety/EmergencySituations/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 challenging. 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 Evaluation and Research (CBER), responded to the need to directly compare the sensitivity of different diagnostic 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 conditions for authorization. The ZIKV strains used in this panel have been cultivated, heat inactivated, and genetically characterized by the FDA/CBER. 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 (Vero–World 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 submitted 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.10Fares-Gusmao R. Chancey C. Volkova E. Grinev A. Sippert E. Jiang Z. Rios M. Production and characterization of Zika virus RNA reference reagents as a response to a public health emergency.Transfusion. 2018; 58: 2171-2174Crossref PubMed Scopus (2) Google Scholar Lyophilized aliquots of the strains (NR-50723 CBER/FDA FSS13025 Zika Virus RNA Reference Reagent, Heat-Inactivated and NR-50722 CBER/FDA PRVABC59 Zika 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 reagents 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 BaseMatrix to produce S3, S4, and S5 (Figure 1). S3, S4, and S5 were dispensed as 1.5-mL aliquots into 2-mL plastic cryovials. 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 applicants for blinded testing of assays requesting EUA. The titers of the vials were confirmed by real-time PCR11Lanciotti R.S. Kosoy O.L. Laven J.J. Velez J.O. Lambert A.J. Johnson A.J. Stanfield S.M. Duffy M.R. Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007.Emerg Infect Dis. 2008; 14: 1232-1239Crossref PubMed Scopus (1599) Google Scholar by FDA scientists after an overnight storage at FDA/CBER. 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.12Collet D. Modeling Binary Data. Chapman & Hall, London1991Crossref Google Scholar 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.13Saldanha J. Shead S. Heath A. Drebot M. the West Nile Virus Collaborative Study GroupCollaborative study to evaluate a working reagent for West Nile virus RNA detection by nucleic acid testing.Transfusion. 2004; 44: 97-102PubMed Google Scholar, 14Saldanha J. Lelie N. Heath A. WHO Collaborative Study GroupEstablishment of the first international standard for nucleic acid amplification technology (NAT) assays for HCV RNA.Vox Sang. 1999; 76: 149-158Crossref PubMed Google Scholar 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 homogenize. This dilution was repeated with panel vial S2, generating two 1:10 dilutions designated S1.1 and S2.1 (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 10−1 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 confirmation, 20 replicates were tested from extraction to amplification/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 instructions, 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 concentrations in NDUs/mL in the range finding study (Figure 2B). 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 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.Table 1Authorized Assays and ApplicantsMedical productDate of EUA issuanceCDC 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 Infectious Disease, Inc., Walnut Creek, CA)April 28, 2016 (initial issuance)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)∗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.August 4, 2016 (initial issuance)Sentosa SA ZIKV RT-PCR Test (Vela Diagnostics USA, Inc., Fairfield, NJ)September 23, 2016†No amendments were submitted to update the application.Zika Virus Detection by RT-PCR Test (ARUP Laboratories, Salt Lake City, UT)September 28, 2016†No amendments were submitted to update the application.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, 2016†No amendments were submitted to update the application.Gene-RADAR Zika Virus Test (Nanobiosym Diagnostics, Inc., Cambridge, MA)March 20, 2017†No amendments were submitted to update the application.TaqPath Zika Virus Kit (Thermo Fisher Scientific, Pleasanton, CA)August 2, 2017†No amendments were submitted to update the application.CII-ArboViroPlex rRT-PCR Assay (Columbia University, New York, NY)August 11, 2017†No amendments were submitted to update the application.EUA, Emergency Use Authorization.∗ 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.† No amendments were submitted to update the application. Open table in a new tab EUA, Emergency Use Authorization. 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).Table 2Sensitivity Mean Estimates of the Emergency Use Authorized Assays Using the Zika FDA-RPFDA-RP results by decreasing sensitivityTiter, NDUs/mLS1S2Serum/plasmaUrineProcessed urineWBSerum/plasmaUrineProcessed urineWB100Hologic, Inc.150Hologic, Inc.Hologic, Inc.167Hologic, Inc.300Abbott Molecular Inc.Hologic, Inc.500Abbott Molecular Inc.Columbia UniversityThermo Fisher ScientificViracor EurofinsQuest Diagnostics Infectious Disease, Inc.Abbott Molecular Inc.Columbia UniversityThermo Fisher ScientificViracor Eurofins1000Abbott Molecular Inc.Columbia UniversityQuest Diagnostics Infectious Disease, Inc.Siemens Healthcare Diagnostics Inc.Viracor EurofinsCDCColumbia UniversityViracor EurofinsQuest Diagnostics Infectious Disease, Inc.Abbott Molecular Inc.Hologic, Inc.1500Quest Diagnostics Infectious Disease, Inc.Abbott Molecular Inc.1581altona Diagnostics GmbH (automated)1650ARUP Laboratories1670CDCCDC3000ARUP LaboratoriesLuminex Corp.Siemens Healthcare Diagnostics Inc.3160altona Diagnostics GmbHaltona Diagnostics GmbH3300CDCELITechGroup Inc. Molecular DiagnosticsNanobiosym Diagnostics, Inc.Thermo Fisher ScientificARUP LaboratoriesLuminex Corp.Thermo Fisher Scientific4500ARUP Laboratories5000altona Diagnostics GmbHLuminex Corp.Nanobiosym Diagnostics, Inc.Siemens Healthcare Diagnostics Inc.altona Diagnostics GmbH (manual)Luminex Corp.Siemens Healthcare Diagnostics Inc.Vela Diagnostics USA, Inc.5560ELITechGroup Inc. Molecular Diagnostics10,000Vela Diagnostics USA, Inc.15,000Vela Diagnostics USA, Inc.30,000Vela Diagnostics USA, Inc.FDA, US Food and Drug Administration; NDUs, nucleic acid testing–detectable units; RP, reference panel; WB, whole blood; Zika FDA-RP, RP for Zika RNA. Open table in a new tab FDA, US Food and Drug Administration; NDUs, nucleic acid testing–detectable units; RP, reference panel; WB, whole blood; Zika FDA-RP, RP for Zika RNA. 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.Table 3Detection of Blinded Samples by the Emergency Use Authorized AssaysApplicantS3S4S5MatrixCDCHighHighLowSerumHighHighLowUrineLow∗The device did not perform with inactivated virus in WB.Low∗The device did not perform with inactivated virus in WB.Low∗The device did not perform with inactivated virus in WB.WBQuest Diagnostics Infectious Disease, Inc.HighHighLowSerumHighHighLowUrinealtona Diagnostics GmbH, using Qiagen (Hilden, Germany) QIAamp Viral RNA Mini Kit (manual)HighMediumLowSerumHighHighLowUrinealtona Diagnostics GmbH, using Roche (Basel, Switzerland) Magna Pure 96 Instrument/DNA and Viral NA Small Volume Kit (automatic)HighMediumLowSerumHighMediumLowUrineHologic, Inc.HighHighHighPlasmaHighHighMediumProcessed urineHighHighMediumProcessed WBViracor EurofinsHighHighMediumPlasmaHighHighMediumUrineSiemens Healthcare Diagnostics Inc.HighMediumLowSerumHighHighLowUrineLuminex Corp.HighMediumLowSerumHighHighLowUrineVela Diagnostics USA, Inc.HighLowLowSerumHighMediumLowUrineARUP LaboratoriesHighHighLowPlasmaHighHighLowUrineAbbott Molecular Inc.HighHighLowSerumHighHighMediumUrineHighHighLowWBELITechGroup Inc. Molecular DiagnosticsHighHighLowPlasmaNanobiosym Diagnostics, Inc.HighMediumLowSerumThermo Fisher ScientificHighMediumLowSerumHighMediumLowUrineColumbia UniversityHighHighLowSerumHighHighLowUrineDetection rate: low, 0% to <40%; medium, ≥40% to <80%; and high, ≥80% to 100%.WB, whole blood.∗ The device did not perform with inactivated virus in WB. Open table in a new tab Detection rate: low, 0% to <40%; medium, ≥40% to <80%; and high, ≥80% to 100%. WB, whole blood. 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 detectable 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: ∑i=1N[x10∗100]iN%(1) 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 percentage 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 replicates 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. 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. 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 2002.15Rosenberg R. Lindsey N.P. Fischer M. Gregory C.J. Hinckley A.F. Mead P.S. Paz-Bailey G. Waterman S.H. Drexler N.A. Kersh G.J. Hooks H. Partridge S.K. Visser S.N. Beard C.B. Petersen L.R. Vital signs: trends in reported vectorborne disease cases—United States and territories, 2004–2016.MMWR Morb Mortal Wkly Rep. 2018; 67: 496-501Crossref PubMed Scopus (426) Google Scholar Beside mosquito bites, additional transmission routes have been documented for arboviruses, such as intrauterine, perinatal, sexual, and blood transfusion. Vector control 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 results and have high specificity. However, available assays have a wide range of performances, depending on sample throughput, testing environment, claimed sample types, instruments, extraction methods, and the comparator of choice. These caveats highlight the need to confirm the performance of different ZIKV RNA detecting devices with a common, independent, and well-characterized reference material. Use of the same reference material across manufacturers 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 developers who have interacted with the FDA through the review process. Results reported by each company using blinded S3, S4, and S5 provide an indication of their proficiency in determining 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 company 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 members) 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 temporary 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' declaration 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.htm and https://www.fda.gov/BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/LicensedProductsBLAs/BloodDonorScreening/InfectiousDisease/ucm612696.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. We thank the CDC and the University of Texas Medical Branch for providing Zika virus strains KU501215 and JN860885, respectively.