key: cord-0912389-mb784dam authors: Velu, P.; Craney, A.; Ruggiero, P.; Sipley, J.; Cong, L.; Hissong, E.; Loda, M.; Westblade, L. F.; Cushing, M.; Rennert, H. title: Rapid implementation of SARS-CoV-2 emergency use authorization RT-PCR testing and experience at an academic medical institution date: 2020-06-08 journal: nan DOI: 10.1101/2020.06.05.20109637 sha: 8e711780fd1caffd33f78fda3d6e6d48dc1a9a09 doc_id: 912389 cord_uid: mb784dam An epidemic caused by an outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in China in December 2019 has since rapidly spread internationally, requiring urgent response from the clinical diagnostics community. We present a detailed overview of the clinical validation and implementation of the first laboratory-developed real-time reverse-transcription-PCR (rRT-PCR) test offered in the NewYork-Presbyterian Hospital system following the emergency use authority (EUA) guidance issued by the US Food and Drug Administration. Validation was performed on nasopharyngeal and sputum specimens (n=124) using newly designed dual-target rRT-PCR (altona RealStar SARS-CoV-2 Reagent) for detecting of SARS-CoV-2 in upper respiratory and lower respiratory tract specimens, including bronchoalveolar lavage and tracheal aspirates. Accuracy testing demonstrated excellent assay agreement between expected and observed values. The limit of detection (LOD) was 2.7 and 23.0 gene copies/reaction for nasopharyngeal and sputum specimens, respectively. Retrospective analysis of 1,694 tests from 1,571 patients revealed increased positivity in older patients and males compared to females, and an increasing positivity rate from approximately 20% at the start of testing to 50% at the end of testing three weeks later. Our findings demonstrate that the assay accurately and sensitively identifies SARS-CoV-2 in multiple specimen types in the clinical setting and summarizes clinical data from early in the epidemic in New York City. The novel coronavirus SARS CoV-2 is a member of the Betacoronavirus genera in the 49 subfamily Coronavirinae, which is known to cause respiratory illness and gastroenteritis 50 in humans and other mammals [1, 2] . Two other Betacoronavirus that have met with 51 global attention are SARS-CoV (2002) and MERS-CoV (2012) . An outbreak of IC within one reaction, allowing for higher throughput testing compared to the CDC assay. 6 2 inactivated virus or RNA spiked into artificial or real clinical matrix was acceptable, as 141 long as the matrix was from the most difficult specimen type accepted for testing on the 142 clinical assay (in decreasing order of difficulty: sputum, other lower respiratory tract 143 specimens, and NP or oropharyngeal (OP) swabs collected and transported in viral 144 transport media). NP and sputum samples were tested on the altona RealStar® rRT-PCR 145 assay to ensure the absence of SARS-CoV-2 and pooled for use as a matrix for spiking 146 in RNA for LOD studies and accuracy studies. Six ten-fold serial dilutions (1 x 10 1 to 1 x 147 10 7 ) were performed with three replicates at each concentration by spiking WRCEVA 148 RNA reference material (60,000 pfu/L stock WRCEVA RNA reference material; ~6:10 7 149 genomic copies/L) into NP and sputum eluates obtained from pooled-negative patient 150 NP or sputum specimens. The dilutions at the LOD were performed by spiking 1 L of 151 1:10 5 dilution to 60 L eluate, yielding a concentration of 0.01 pfu/uL=10pfu/mL 152 (~10,000copies/mL). Assay performance at the determined LOD was confirmed with at 153 least 20 additional replicates for each type of sample (sputum and NP). For the accuracy studies, a total of 64 positive (34 NP and 30 sputum specimens, 156 respectively) specimens and 60 negative (30 NP swabs and 30 sputum) specimens that 157 tested negative for SARS-CoV-2 were used. Positive specimens were either contrived 158 positive samples generated by spiking WRCEVA RNA reference material into pooled 159 leftover negative NP or sputum specimens or real patient specimens, as described above. 160 Twenty of the contrived clinical specimens were spiked at a concentration of 1x-2x LoD, 161 with the remainder of samples spanning the assay testing range. FDA defines the 162 acceptance criteria for the performance as 95% agreement at 1x-2x LOD, and 100% 163 agreement at all other concentrations and negative specimens [6]. 164 165 Inclusivity and cross-reactivity studies used a combination of in silico and in vitro 166 approaches. As the primer and probe sequences were proprietary to the kit 167 manufacturer, we included the results of their in silico analysis in our EUA application. 168 Additional studies to determine cross-reactivity were performed in our laboratory by 169 testing 10 NP samples that were positive by the RP2 for the four human coronaviruses 170 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2020. Dilution series studies on pooled negative NP specimens spiked with WRCEVA RNA 183 reference material, with three replicates across a viral range of 1 gene copy/reaction to 184 1,000,000 gene copies/reaction (1 to 6 log10), demonstrated an accurate and linear 185 response across five logs of detection for NP and four logs of detection for sputum (Table 186 1, Figure 1 ). Probit analysis was applied to the NP data after an additional five replicates 187 of testing were performed at 0.8, 0.6, 0.5, 0.4, and 0.2 gene copies/reaction, and 188 narrowed the LOD to 2.7 gene copies/reaction at 95% detection rate (Figure 2) The in silico analysis for inclusivity that was performed by the manufacturer of the kit 197 found 100% homology of the E gene and S gene forward and reverse primers and probes 198 with 563 whole-genome sequences of SARS-CoV-2 published in GISAID and NCBI as of 199 3/16/2020 [9] . The in silico analysis for cross-reactivity that was performed by the 200 manufacturer found that the S gene and E gene forward and reverse primers and probes 201 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2020. . https://doi.org/10.1101/2020.06.05.20109637 doi: medRxiv preprint had less than 80% homology with the vast majority of 40 different pathogens (125 strains 202 total) tested (see Supplementary Material). In cases with greater than 80% homology, 203 cross-reactivity was not a concern as only the forward or reverse primer, but never both 204 primers were affected, thus rendering amplification impossible. Additionally, all ten 205 samples that had human coronaviruses NL63, 229E, OC43, or HKU1 detected by RP2 206 panel tested negative for SARS-CoV-2 on the RealStar® rRT-PCR assay. All 30 NP specimens that tested negative on the RP2 panel also tested negative on the 210 SARS-CoV-2 rRT-PCR assay ( Table 2) . Leftover VTM from these negative specimens 211 was pooled to create a sample matrix for the LOD and contrived positive sample studies. Means of turn-around-times from test order to result and time-in-lab to result were 19.8 261 (13.1-26.2) hours and 11.9 (7.0-24.0) hours, respectively. The percentage of tests with 262 detected SARS-CoV-2 increased as the weeks progressed, and settled at approximately 263 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2020. . https://doi.org/10.1101/2020.06.05.20109637 doi: medRxiv preprint 50% from 3/21/2020 to 3/30/2020 (Figure 4a) . Most of the samples were from the ED 264 (n=911), followed by inpatient wards (n=492) and outpatient clinics (n=113) ( Table 4) , 265 and the highest positivity rate was in the ED with 50% of patients with detected SARS- 266 CoV-2 (p=0.0005). There was a significant difference in the age (p=0.0005) and gender (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2020. In the cohort of patients tested over three weeks by our assay, positive results were seen 348 more frequently in older males compared to younger and female patients, which has been 349 supported by several studies [14, 15] . Post-menopausal women have been reported to 350 have a greater risk of hospitalization compared to non-menopausal women attributed to 351 the protective effect of estrogen [16] . In this study, older women were more likely to test 352 positive for SARS-CoV-2 compared to younger female patients. However, the difference 353 in detection rate between older and younger women was diminished after removing 354 obstetrics patients screened universally regardless of symptoms, highlighting the 355 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2020. . https://doi.org/10.1101/2020.06.05.20109637 doi: medRxiv preprint ACKNOWLEDGMENTS 387 We would like to thank all the dedicated medical technologists and health care 388 professionals who performed and assisted in testing at the clinical laboratories of NYPH-389 WCM. We also thank Dr. Scott C. Weaver, World Reference Center for Emerging Viruses (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2020. performed starting at 1,000,000 gene copies/reaction and ending at 1 gene copy/reaction. 463 The apparent LOD was between 1 and 10 gene copies/reaction for NP specimens and 464 between 10 and 100 gene copies/reaction for sputum specimens. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2020 . . https://doi.org/10.1101 0.5, 0.4, and 0.2 gene copies/reaction for NP and 80, 60, 50, 40, and 20 gene 478 copies/reaction for sputum. Probit analysis showed LOD to be (C) 2.7 gene 479 copies/reaction for NP and (D) 23.0 gene copies/reaction for sputum. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2020. . specimen types with three replicates at each dilution. An additional twenty (NP) and 503 twenty-three (SPU) specimens were tested at the estimated LOD of 10 gene 504 copies/reaction and 100 gene copies/reaction, respectively. 505 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. CoV-2 rRT-PCR assay using automated QIAsymphony total nucleic acid (TNA) extraction 508 followed by rRT-PCR targeting the E and S coronavirus genes on an in-house validation 509 panel consisting of patient and contrived samples for NP (124 ) and sputum (62). All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2020. Ct values are shown for positive samples. The number of positive (either contrived 516 through spiking RNA into a negative matrix or actual patient samples) and negative 517 specimens are also noted along with the percent of specimens that were correctly 518 classified as positive or negative. 519 520 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 8, 2020. . https://doi.org/10.1101/2020.06.05.20109637 doi: medRxiv preprint Novel coronavirus (SARS-CoV-2) epidemic: a veterinary 401 perspective Evolutionary perspectives on novel coronaviruses 403 identified in pneumonia cases in China Clinical Characteristics of Pregnant Women with Covid-19 in 405 Epidemiological and clinical characteristics of 99 cases of 2019 407 novel coronavirus pneumonia in Wuhan, China: a descriptive study Clinical Characteristics of Covid-19 in New York City Detection of 2019 novel coronavirus (2019-nCoV) by real-430 time RT-PCR Detection of SARS-CoV-2 in Different Types of Clinical 432 Specimens Characteristics of COVID-19 infection in Beijing Asymptomatic carrier state, acute respiratory disease, and 436 pneumonia due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-437 2): Facts and myths China：Protective Effects of Non-menopause and Female Hormones on 442 SARS-CoV-2 infection Universal Screening for SARS-CoV-2 in Women Admitted for