key: cord-0878960-rstmd0va authors: Matsumura, Yasufumi; Shimizu, Tsunehiro; Noguchi, Taro; Nakano, Satoshi; Yamamoto, Masaki; Nagao, Miki title: Comparison of 12 molecular detection assays for SARS-CoV-2 date: 2020-06-25 journal: bioRxiv DOI: 10.1101/2020.06.24.170332 sha: 10e66b2bf3f9896fd2337db3a68b974aaebc79d1 doc_id: 878960 cord_uid: rstmd0va Molecular testing for SARS-CoV-2 is the mainstay for accurate diagnosis of the infection, but the diagnostic performances of available assays have not been defined. We compared 12 molecular diagnostic assays, including 8 commercial kits using 155 respiratory samples (65 nasopharyngeal swabs, 45 oropharyngeal swabs, and 45 sputum) collected at 2 Japanese hospitals. Sixty-eight samples were positive for more than one assay and one genetic locus and were defined as true positive samples. All the assays showed a specificity of 100% (95% confidence interval, 95.8 to 100). The N2 assay kit of the US Centers for Disease Control and Prevention (CDC) and the N2 assay of the Japanese National Institute of Infectious Disease (NIID) were the most sensitive assays with 100% sensitivity (95% confidence interval, 94.7 to 100), followed by the CDC N1 kit, E assay by Corman, and NIID N2 assay multiplex with internal control reactions. These assays are reliable as first-line molecular assays in laboratories when combined with appropriate internal control reactions. shows the molecular assays evaluated in this study. Real-time RT-PCRs 54 were performed using N1, N2, and RNaseP (RP) internal control assays developed by the (4) (with/without EAV), N and E assays developed by Charité in Germany (1) (Corman) 59 with TaqPath™ 1-Step RT-qPCR Master Mix, CG (Thermo Fisher Scientific). We also 60 tested the LightMix® Modular assays (Roche) for E, RdRP, and N genes multiplexed LoopampEXIA® real-time turbidimeter (Eiken Chemical, Tokyo, Japan). Analytical sensitivity 72 We determined the limit of detection (LOD) of each assay using a minimum of 73 four replicates of two-fold serial dilutions of recombinant Sindbis virus containing a 74 partial SARS-CoV-2 genome (AccuPlex™ SARS-CoV-2 Reference Material Kit, 5,000 75 copies/mL; SeraCare, Milford, MA, USA). We calculated the 95% limit of detection 76 (LOD) using probit analysis. At the time manuscript preparation, no gold standard exists. In this study, to 79 ensure the presence of SARS-CoV-2 RNA and to avoid false-positives, a sample was 80 defined as positive when positive test results were obtained for more than one genetic 81 locus and assay and the others were defined as negative. is available as Dataset S1. All the assays exhibited a specificity of 100%, while sensitivity varied ( Table 2 ). The 100 CDC N1, CDC N2, NIID N2 (with/without EAV), and Corman E assays were the most 101 sensitive assays with ≥95.6% sensitivity. These 5 assays displayed high overall 102 agreement compared with the reference standard (kappa values of ≥0.96) and between 103 any two of them (kappa values of ≥0.95). The CDC N2 and NIID N2 assays exhibited 104 100% sensitivity; thus, their results were equal to the defined reference standard. The 105 sensitivities of the remaining 7 assays (Corman N, Roche E, Roche RdRP, Roche N, 106 Thermo Combo, BGI and LAMP assays; ≤88.2%) were significantly lower than those of 107 the most sensitive assays. The CDC protocol requires both N1 and N2 assays, and a sample will be 109 considered positive if both produced positive results. In this study, one true positive 110 6 nasopharygeal sample was positive only for the N2 assay even after retesting. The 111 sample was considered inconclusive and the performance of the CDC protocol was 112 considered the same as the CDC N1 assay. The NIID protocol includes both NIID N2 and 113 Corman N assays, and a sample will be considered positive if either assay produces a 114 positive result. In this study, 69.1% of samples were positive for both assays, and 30.9% Table 3 shows diagnostic performances for each specimen type. Nasopharyngeal 123 swabs tended to have a higher sensitivity than the other samples. The sensitivity of 124 Corman N assay for sputum samples and that of Roche N assay for oropharyngeal swabs 125 and sputum samples were significantly lower than those for nasopharyngeal swabs. The LODs of the Roche RdRP and N assays were high (>5,000 copies/mL). The current diagnosis of COVID-19 mainly relies on RT-PCR tests (6). We 141 performed manufacturer-independent evaluation of the molecular assays, including 142 commercial kits that utilize otherwise-extracted RNA templates. We found that the 143 specificity was perfect for all the assays and that the CDC N1, CDC N2, NIID N2, and 144 Corman E assays were the most sensitive and highly concordant (7). Genetic variations 145 that may compromise sensitivity of the CDC N1, N2, and Corman E assays have been 146 rarely observed as of week 21 of 2020 (8). False negatives by the other assays occurred 147 among low-copy number samples (presenting high Ct values by the CDC N2 or NIID N2 148 assay; Dataset S1), suggesting a lack of sensitivity of these assays. The Roche assays were based on Corman's assays (1) but had lower sensitivity 150 for their E and N assays. This is likely due to lower Ct cutoffs for the Roche assays, 151 rather than differences in reagents and reaction conditions (Table 1 and Dataset S1). Previous studies reported that N assay was less sensitive than the E and RdRP assays (1) 153 and the RdRP assay was less sensitive than the Roche E assay (3). The low sensitivity of 154 the Roche RdRP and N assays were concordant with their high LODs (Table 4) To avoid false negatives due to technical errors such as extraction problems or 164 8 PCR inhibition, it is recommended to include internal control reactions. The CDC assays 165 were designed to be combined with a separate internal control reaction (Table 1) . 166 Different from multiplex assays that incorporate internal controls such as Roche, Thermo, 167 or BGI kits, this approach needs extra reagents, time, and space in a reaction plate but 168 can be combined with other in-house assays (NIID N2 or Corman E) without any 169 modification. For the multiplex approach, we selected the NIID N2 assay to be Seegene. These reports are in agreement with our findings. The study limitations included a relatively small sample size of each specimen 180 type and lack of clinical information, measurements by multiple investigators, and 181 genomic variation analysis. In conclusion, we validated the NIID N2 assay with EAV control reaction and showed that 4 samples (2 true positive sputum samples, 1 true positive pharyngeal sample, and 1 263 true negative sputum sample) were negative for all genes, and these were considered negative. Detection of 2019 202 novel coronavirus (2019-nCoV) by real-time RT-PCR RT-PCR for Severe Acute Respiratory Syndrome Coronavirus 2 Improved Molecular Diagnosis of COVID-19 by the Novel, Highly Sensitive and 210 Specific COVID-19-RdRp/Hel Real-Time Reverse Transcription-PCR Assay 211 Validated Genetic Diagnostic Methods for Novel Coronavirus 2019-nCoV) Real-Time RT-PCR Diagnostic Panel Interpreting Diagnostic Tests for 220 SARS-CoV-2 The measurement of observer agreement for 222 categorical data 05 in comparison with the sensitivity for nasopharyngeal swabs CDC N1 NIID N2 with EAV 271 CI, confidence interval.