Rapid Implementation of Severe Acute Respiratory Syndrome Coronavirus 2 Emergency Use Authorization RT-PCR Testing and Experience at an Academic Medical Institution

An epidemic caused by an outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in China December 2019 has since rapidly spread internationally, requiring urgent response from the clinical diagnostics community. We present a detailed overview validation and implementation first laboratory-developed real-time RT-PCR test offered NewYork-Presbyterian Hospital system following Emergency Use Authorization issued US Food Drug Administration. Nasopharyngeal sputum specimens (n = 174) were validated using newly designed dual-target (altona RealStar SARS-CoV-2 Reagent) for detecting upper tract lower specimens. Accuracy testing demonstrated excellent assay agreement between expected observed values comparable diagnostic performance to reference tests. The limit detection was 2.7 23.0 gene copies per reaction nasopharyngeal specimens, respectively. Retrospective analysis 1694 1571 patients revealed increased positivity older males compared with females, increasing rate approximately 20% at start 50% end 3 weeks later. Herein, we demonstrate that accurately sensitively identifies multiple specimen types setting summarize data early New York City. novel is member Betacoronavirus genera subfamily Coronavirinae, which are known cause illness gastroenteritis humans.1Lorusso A. Calistri P. Petrini Savini G. Decaro N. Novel epidemic: veterinary perspective.Vet Ital. 2020; 56: 5-10PubMed Google Scholar,2Wei X. Li Cui J. Evolutionary perspectives on coronaviruses identified pneumonia cases China.Natl Sci Rev. 7: 239-242Crossref PubMed Scopus (25) Scholar disease SARS-CoV-2, detected Wuhan, China, 2019, other countries, including United States.3Chen L. Q. Zheng D. Jiang H. Wei Y. Zou Feng Xiong Sun Wang Zhao Qiao Clinical characteristics pregnant women Covid-19 China.N Engl J Med. 382: e100Crossref (338) Scholar,4Chen Zhou M. Dong Qu Gong F. Han Qiu Liu Xia Yu T. Zhang Epidemiological 99 China: descriptive study.Lancet. 395: 507-513Abstract Full Text PDF (12997) City particular became epicenter pandemic.5Goyal Choi J.J. Pinheiro L.C. Schenck E.J. Chen R. Jabri Satlin M.J. Campion Jr., T.R. Nahid Ringel J.B. Hoffman K.L. Alshak M.N. H.A. Wehmeyer G.T. Rajan Reshetnyak E. Hupert Horn E.M. Martinez F.J. Gulick R.M. Safford M.M. COVID-19 City.N 2372-2374Crossref (1448) Given devastating impact health care need accurate quick diagnosis infection, Administration (FDA) established rapid pathway tests outlined guidance document published February 29, 2020 (https://www.fda.gov/media/135659/download, last accessed October 22, 2020). According this guidance, could be performed Laboratory Improvement Amendments–certified high-complexity laboratories under (EUA), according set recommendations regarding minimum required ensuring analytical validity test. Details had submitted laboratory FDA through EUA application within 15 days initiating testing, after continue provisionally until decision rendered. CDC State Department Health manufactured new kits SARS-CoV-2. However, few able get access these reagents or instruments, also not available our institution. Limited RNA control material presented another significant hurdle process. announcement procure World Reference Center Emerging Viruses Arboviruses (WRCEVA) NIH Biodefense Infections Research Resources Repository. scale demand shortage supplies led high-throughput readily implemented variety laboratories. describe tract, (NP) research use only Reagent Diagnostics GmbH, Hamburg, Germany). detail workflow considerations results (COVID-19) (March 11, 2020, March 31, 2020) (URT) peak number (April 17, May 15, (LRT) WRCEVA obtained University Texas Medical Branch (strain USA_WA1/2020, lot TVP 23156) evaluation (LOD) studies. samples used initial residual NP routine suspected having infections pooled archived frozen (NP sputum) as matrix generating contrived Reactive included four SARS-CoV-2–positive tested Mental Hygiene. presence 21 common viruses BioFire FilmArray Respiratory Pathogen panel (BioFire Diagnostics, LLC, Salt Lake City, UT). Moreover, further evaluate test, total 30 20 patient analyzed parallel Roche cobas 6800 (Roche Indianapolis, IN) Cepheid GeneXpert (Cepheid, Sunnyvale, CA) Hologic Panther Fusion (Hologic, Inc., Marlborough, MA), Additional retrospective consecutive URT 1694) LRT 141) high suspicion who treated campuses April Institutional Review Board Committee Weill Cornell Medicine approved study. All initially processed Biosafety level biosafety measures (CDC, https://www.cdc.gov/coronavirus/2019-ncov/lab/lab-biosafety-guidelines.html, off-board lysis viral inactivation step 200 ?L swab transport media followed automated extraction nucleic acid QIAsymphony DSP Virus/Pathogen Mini Kit coupled SP (Qiagen, Germantown, MD) produce resulting eluate volume 60 ?L. For inactivation, mixed ACL buffer, vortexing mix consisting internal (IC), carrier RNA, proteinase K, AVE ATL buffers, then incubated 68°C minutes. IC consisted proprietary artificial RNA/DNA molecules no homologies any sequences, reagents. sputum, 100 0.3% dithiothreitol solution (1:1 ratio) 37°C minutes reduce viscosity. One-step reverse transcription cDNA targets E (Envelope) S (Spike) genes performed, 10 1.0 GmbH) Rotor-Gene Q Thermocyler (Qiagen) PCR amplification multicolor fluorescent dye–labeled probes identification differentiation B-betacoronavirus, SARS-CoV-2–specific single reaction. Samples both 40 cycles considered positive. sole target classified B-betacoronavirus detected. Failure detect signal indeterminate. specified distinct characteristics, LOD, inclusivity (analytical sensitivity), cross-reactivity specificity), evaluation. LOD studies, six 10-fold serial dilutions (1 × 101 107) three replicates each concentration spiking (6 105 plaque-forming units/?L stock material; 6 107 genomic copies/?L) into eluates pooled-negative specimens.6Mitchell S.L. St. George K. Rhoads D.D. Butler-Wu S.M. Dharmarha V. McNult Miller M.B. Understanding, verifying, implementing emergency authorization molecular RNA.J Clin Microbiol. 58 (e00796-20)Crossref (40) confirmed additional type sample (sputum NP). accuracy 104 positive (54 swabs 70 negative (40 used. Positive either real orthogonal methods generated eluates, described above. Twenty spiked 1× 2× remainder spanning range. defined acceptance criteria 95% 100% all concentrations Inclusivity studies altona GmbH. high-priority pathogens same genetic family FDA. Data analyses, statistics plot generation, R programming language version 3.6.0.7Team CoreR: A Language Environment Statistical Computing. Foundation Computing, Vienna, Austria2019Google determined probit regression model glm function CLSI (Clinical Standards Institute, Annapolis Junction, EP17A2E Guidance Application Quantitative Molecular Measurement Procedures.8Chen Procedures. Function Library, Copenhagen, Denmark2016Google Dilution series material, across range 1 copy 1,000,000 log10), linear five logs (Table Figure 1). Probit applied 0.8, 0.6, 0.5, 0.4, 0.2 reaction, narrowed (Figure 2). similar 80, 60, 50, 40, showed sensitivity rate. 23 respective LODs resulted positive.Table 1Limit Detection Studies Were Performed SPU Specimen Types Three Replicates Each DilutionDilutionGene reactionRun, nDetected, %Mean CtS geneE geneIC detectedNPSPUNPSPUNPSPUNPSPUNPSPUNPSPU1:10106333310010014.613.115.214.530.633.01:102105333310010018.016.518.717.929.030.41:103104333310010020.919.921.621.428.330.81:104103333310010024.623.825.225.329.429.71:10510232632610010027.931.128.431.030.229.81:10610233230100032.3ND32.0ND30.829.71:1071330000NDNDNDND29.829.8An (SPU) estimated respectively.IC, control; ND, detected; NP, media; SPU, sputum. Open table tab 2Limit analysis. dilution (A) (B) determine LOD. Five (A, B, C, D, E) starting 1000 ending 0.1 specimens; (C) (D). Red dashed lines represent copies/mL (x-axis) (y-axis).View Large Image ViewerDownload Hi-res image Download (PPT) IC, silico homology forward primers 563 whole-genome sequences Global Initiative Sharing Influenza (https://www.gisaid.org, National Biotechnology Information 16, 2020. In <80% vast majority different (125 strains) tested. There >80% E-gene primer strain Streptococcus pneumoniae (81.82%); S-gene strains Legionella pneumophila subspecies Pascuellei (80.95%), Pneumocystis jirovecii (85.71%), Pseudomonas aeruginosa (90.48%), Staphylococcus epidermidis (85.71%); S. Candida albicans (80.95%). Cross-reactivity rare concern primer, but never primers, affected, thus rendering impossible. addition, human coronavirus-positive [NL63 2), 229E OC43 4), HKU1 2)] assay. 20) SARS-CoV-2–negative Hygiene Supplemental Table S1). high-positive run successive 10) remained throughout (1:2 1:1024; cycle threshold range, 22 31) Similar most challenging FDA, concordance S2). 2).Table 2Summary TypeSpecimen typeCt, mean (range)Tested (POS/NEG), NClassification %S geneInternal controlNP swab?NP dilutions.29.3 (20.6–37.2)29.2 (20.8–37.7)30.1 (28.6–30.8)30100Sputum?NP dilutions.24.1 (16.2–30.0)24.2 (17.0–29.2)30.2 (29.4–32.3)30100NP swab†NP part validation.26.8 (20.6–37.2)27.4 (20.8–37.7)29.5 (28.8–30.1)4100NP swab‡Authentic assayed going live.24.0 (9.2–39.6)25.9 (10.2–37.7)29.2 (27.9–30.4)20/4096/100Sputum§Sputum authentic diluted NEG (2/20 samples).16.0 (9.5–28.0)17.2 (10.8–28.9)30.2 (29.4–32.3)20/30100Clinical extraction, targeting genes. Mean Ct shown POS samples. (either actual samples) noted, along percentage correctly NEG.NEG, negative; nasopharyngeal; POS, positive.? dilutions.† validation.‡ Authentic live.§ Sputum samples). NEG. NEG, went live 39 (19/20 specimens) (E gene/S gene) 25.9/24.0 17.2/16.0 samples, respectively 3). Of 11 (55%) low >30. discordant indeterminate (pan-Sarbecovirus detected) suggesting load. Orthogonal sent Associated Regional Pathologists, Inc. (ARUP Laboratories), Utah, specificity 100%.Table 3Summary Testing Specimens Different PlatformsSample no.Sample typealtona resultaltona valuesOrthogonal platformOrthogonal resultOrthogonal valuesConcordant (Y/N)1NP swabPOS16.9/18.2cobas 6800POS19.5/19.7Y2NP swabPOS30.4/32.6cobas 6800POS31.8/34.7Y3NP swabPOS26.4/28.5cobas 6800POS29.5/30.8Y4NP swabPOS30.0/32.5cobas 6800POS31.1/32.5Y5NP swabPOS18.6/20.4cobas 6800POS23.6/24.5Y6NP swabPOS23.1/25.0cobas 6800POS26.6/27.4Y7NP swabPOS25.6/26.5cobas 6800POS27.4/28.1Y8NP swabPOS15.6/17.4cobas 6800POS19.4/19.7Y9NP swabPOS23.6/23.6cobas 6800POS25.3/26.2Y10NP swabNEG–/–cobas 6800IND–/33.4N11NP 6800NEG–/–Y12NP 6800NEG–/–Y13NP 6800NEG–/–Y14NP 6800NEG–/–Y15NP 6800NEG–/–Y16NP 6800NEG–/–Y17NP 6800NEG–/–Y18NP 6800NEG–/–Y19NP 6800NEG–/–Y20NP swabPOS30.3/31.2cobas 6800POS31.0/32.1Y21NP swabPOS9/2/10.2CepheidPOS11.7/13.9Y22NP swabPOS15.4/16.3CepheidPOS19.1/21.2Y23NP swabPOS21.8/22.8CepheidPOS24.7/26.8Y24NP swabPOS30.9/31.3CepheidPOS33.4/35.5Y25NP swabPOS–/39.6CepheidPOS33.3/35.8N26NP swabPOS29.4/29.5CepheidPOS33.2/35.3Y28NP swabPOS26.0/26.8CepheidPOS28.6/30.9Y29NP swabPOS32.1/32.3CepheidPOS28.7/31.2Y30NP swabPOS27.0/27.7CepheidPOS34.8/36.3Y27NP swabNEG–/–CepheidNEG–/–Y31NP swabPOS23/23.2NYS-DOHPOSN/A32NP swabPOS20.6/20.8NYS-DOHPOSN/A33NP swabPOS26.3/27.7NYS-DOHPOSN/A34NP swabPOS37.2/37.7NYS-DOHPOSN/A35SputumPOS9.5/10.8HologicPOSN/AY36SputumPOS9.7/11.0HologicPOSN/AY37SputumPOS10.5/11.9HologicPOSN/AY38SputumPOS11.9/13.4HologicPOSN/AY39SputumPOS13.6/14.9HologicPOSN/AY40SputumPOS13.7/15.0HologicPOSN/AY41SputumPOS14.6/15.9HologicPOSN/AY42SputumPOS15.9/17.2HologicPOSN/AY43SputumPOS16.1/17.4HologicPOSN/AY44SputumPOS17.1/18.4HologicPOSN/AY45SputumPOS18.9/20.1HologicPOSN/AY46SputumPOS20.7/21.8HologicPOSN/AY47SputumPOS15.5/16.9HologicPOSN/AY48SputumPOS16.2/17.4HologicPOSN/AY49SputumPOS17.2/18.5HologicPOSN/AY50SputumPOS18.6/19.9HologicPOSN/AY51SputumPOS17.8/19.2HologicPOSN/AY52SputumPOS18.8/20.1HologicPOSN/AY53SputumPOS13.5/15.2HologicPOSN/AY54SputumPOS28.0/28.9HologicPOSN/AYThirty orthogonally (EUA) assays platforms. reported gene/E GmbH); specific/target pan-Sarbecovirus (cobas 6800); gene/N2 (Cepheid). NYS-DOH ARUP (Hologic System). 18 (1:10 1:12,800) have infection leftover Two (numbers 49 50) samples.IND, indeterminate; N, no; N/A, applicable; NYS-DOH, Health; positive; Y, yes. Thirty IND, During pandemic 1354 (41% positive), 32 oropharyngeal (OP) (31% 308 combined + OP (24% positive) patients. significantly gene, OP, NP/OP (Supplemental 5 4, respectively, (range, 11.1 40.7), 22.5 10.3 40.6), 29.6 27.0 38.4), Using value surrogate burden, groups: (Ct < 20; n 222, 34%), medium 30; 335, 52%), > 89, 13.8%).9Craney A.R. Velu Fauntleroy K.A. Callan Robertson La Spina Lei B. Alston Rozman Loda Rennert Cushing Westblade L.F. Comparison two transcription-polymerase chain systems 2.J 58: e00890-20Crossref (42) Over >75% burden. 135 repeated 17 second 13 subsequently virus one OP) other. converted positive, 12 department (ED). On days, 13th inpatient burden 7 note, although symptoms being obstetrics gynecology labor delivery wards universally screened preprocedural measure if personal protective equipment would during interactions workers. This group 102 female 7% Means turnaround times fro