key: cord-348209-rkkhv4mw authors: Noerz, Dominik; Fischer, Nicole; Schultze, Alexander; Kluge, Stefan; Mayer-Runge, Ulrich; Aepfelbacher, Martin; Pfefferle, Susanne; Luetgehetmann, Marc title: Clinical evaluation of a SARS-CoV-2 RT-PCR assay on a fully automated system for rapid on-demand testing in the hospital setting date: 2020-04-11 journal: nan DOI: 10.1101/2020.04.07.20056234 sha: doc_id: 348209 cord_uid: rkkhv4mw The ongoing SARS-CoV-2 pandemic presents a unique challenge for diagnostic laboratories around the world. Automation of workflows in molecular diagnostics is instrumental for coping with the large number of tests ordered by clinicians, as well as providing fast-tracked rapid testing for urgent cases. In this study we evaluated a SARS-CoV-2 LDT for the NeuMoDx 96 system, a fully automated (sample to result) RT-PCR platform offering random-access capabilities and good clinical performance for SARS-CoV-2 testing. Background: The ongoing SARS-CoV-2 pandemic presents a unique challenge for diagnostic 27 laboratories around the world. Automation of workflows in molecular diagnostics are instrumental for 28 coping with the large number of tests ordered by clinicians, as well as providing fast-tracked rapid 29 testing for highly urgent cases. In this study we evaluated a SARS-CoV-2 LDT for the NeuMoDx 96 30 system, a fully automated device performing extraction and real-time PCR. Methods: A publicly 31 available SARS-CoV-2 RT-PCR assay was adapted for the automated system. Analytical performance 32 was evaluated using in-vitro transcribed RNA and clinical performance was compared to the cobas 33 6800-based reference assay within the lab. Results: The NeuMoDx-sarbeco-LDT displayed good 34 analytical performance with an LoD of 95.55 cp/ml and no false positives during evaluation of cross-35 reactivity. A total of 176 patient samples were tested with both the Sarbeco-LDT and the reference 36 assay. Positive and negative agreement were 100% and 99.2% respectively. Invalid-rate was 6.3%. 37 The NeuMoDx-sarbeco-LDT showed analytical and clinical performance comparable to the 38 cobas6800-based reference assay. Due to its random-access workflow concept and rapid time-to-39 result of about 80 minutes, the device is very well suited for providing fast-tracked SARS-CoV-2 40 diagnostics for urgent clinical samples in the hospital setting. 41 2 Introduction: In early January 2020, SARS-CoV-2 was first identified as the likely causative agent of a cluster of cases 43 of viral pneumonia in the city of Wuhan, China (1). The novel virus is situated in the 'sarbecovirus' 44 subgenus along with its genetically distinct relative, the original SARS-coronavirus (2). SARS-CoV-2 saw 45 rapid spread worldwide eventually prompting the WHO to declare a 'global health emergency' by the 46 end of January (3). 47 Outbreak scenarios present a unique challenge for diagnostic laboratories. Particularly in the case of 48 respiratory viruses such as SARS-CoV-2, clinical symptoms can be largely indistinguishable from other 49 common respiratory pathogens such as e.g. Influenza (4) and polymerase chain reaction (PCR) assays 50 are necessary to confirm or rule out the novel virus (5). A variety of suitable assays were made available 51 early on during the outbreak, notably by Corman et al. (6) and the CDC, which were swiftly adopted by 52 many labs in Europe and around the world. However, their overall testing capacity remained limited 53 (7). We and others have previously demonstrated how automation in molecular diagnostics enables 54 easy scaling of testing capacity by substantially cutting back hands-on time for 9) . 55 For the assay presented in this study, we used a fully automated random-access platform for molecular 56 diagnostics, handling everything from extraction, amplification, signal detection to reporting of results 57 (10). For RNA targets, the time-to-result is approximately 80 minutes, given optimal conditions. The 58 availability of an open mode allows for the rapid implementation of lab developed tests (LDT). The aim 59 of this study was to adapt and evaluate a previously published SARS-CoV-2 PCR assay (by Corman et 60 al. (6) In accordance with instructions issued by the manufacturer, a 6x Primer/Probe mix was prepared and 68 5µL of the mix were loaded into the LDT-Strip well by well for each reaction (e.g. 400nM primers, 75 69 nM probe per reaction). For a complete run protocol see the test-summary displayed in table 1. 70 71 72 All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is Step Denature: 6 sec, at 95°C, No Detect Step Anneal: 19 sec, at 60°C, Detect 73 Table 1 : NeuMoDx-Software run-protocol summary displaying settings and PCR protocol. 74 75 Clinical specimens used for this study were oropharyngeal and nasopharyngeal swabs (E-Swab 76 collection kits, Copan, Italy). Prior to analysis, 1ml Roche cobas PCR medium (≤ 40% guanidine 77 hydrochloride in Tris-HCL buffer) was added to the sample in order to inactivate potential pathogens 78 within and facilitate further handling. Samples were then briefly vortexed before being loaded into the 79 instrument. 80 All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is (2, 12). TaqMan based RT-PCR-assays for the novel virus were available online mere days after the 119 initial sequence of SARS-CoV-2 had been published (6). However, while these assays can be 120 implemented relatively swiftly by local diagnostic laboratories, their reliance on manual PCR setups 121 sets narrow limits to overall capacity. A study by Reusken et al. reported Coronavirus by the end of January 2020 in almost all countries of the European union, but with a 123 capacity of 250 tests per week or less for the vast majority of them (7). Similar issues were reported 124 early on in China, where testing could not be performed for all suspected cases due to limitations in 125 capacity (13). 126 In a recent study we demonstrated that a previously published TaqMan based SARS-CoV-2 RT-PCR 127 assay, endorsed by ECDC and WHO, can be adapted to run on an automated batch-based high-128 All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is . https://doi. org/10.1101 org/10. /2020 and signal detection without requiring any human interaction. it provides random-access capabilities 136 and turn-around times of 80 minutes for RNA targets. In this study we have adapted the SARS-CoV-2 137 RT-PCR assay by Corman et al. (6) for use on the NeuMoDx 96 automated system. Analytical and clinical 138 performance was comparable to the cobas6800-based reference assay (11), showing an LoD of 139 approximately 100 copies/ml and positive and negative agreement of 100% and 99.2% respectively. 140 The relatively high inhibition rate of 6.3% suggests that sample preparation procedures can be 141 optimized further. The NeuMoDx-based Sarbeco-LDT represents a valuable complement to routine 142 SARS-CoV-2 testing by offering the ability to run individual high-priority samples at any time and 143 reporting results within two hours if necessary. 144 In this study we have adapted a publicly available SARS-CoV-2 screening assay for use on the open 146 channel of the NeuMoDx 96/288 system (Qiagen). The assay demonstrates comparable analytical and 147 clinical performance to established LDTs currently in use for SARS-CoV-2 diagnostics. Due to its 148 random-access capabilities and short turn-around times (80 minutes), the system is well suited for 149 automating medium-throughput routine SARS-CoV-2 testing, or as an addition to high-throughput 150 systems to allow fast-tracking for highly urgent clinical samples. 151 All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.04.07.20056234 doi: medRxiv preprint the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.04.07.20056234 doi: medRxiv preprint the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10. 1101 /2020 Novel Coronavirus-Infected Pneumonia A Novel Coronavirus from Patients with 158 Pneumonia in China Emergency Committee regarding the outbreak of novel coronavirus (2019-nCoV). World Health 161 Organisation Website (whoint) 2020. 164 5. WHO. Laboratory testing for coronavirus disease 2019 (COVID-19) in suspected human cases. 165 . World Health Organisation Website (whoint) Detection of 2019 novel 167 coronavirus (2019-nCoV) by real-time RT-PCR Laboratory readiness 169 and response for novel coronavirus (2019-nCoV) in expert laboratories in 30 EU/EEA countries Clinical evaluation of 172 multiplex RT-PCR assays for the detection of influenza A/B and respiratory syncytial virus using a high 173 throughput system Ten years of R&D and full automation in molecular 175 diagnosis NeuMoDx Group B Streptococcus Assay on the NeuMoDx 288 Molecular System Evaluation of a quantitative RT-PCR assay for 180 the detection of the emerging coronavirus SARS-CoV-2 using a high throughput system War stories Characteristics of and Important Lessons From the Coronavirus Disease 184 2019 (COVID-19) Outbreak in China: Summary of a Report of 72314 Cases From the Chinese Center for 185 Disease Control and Prevention Generating timely molecular 187 diagnostic test results: workflow comparison of the cobas(R) 6800/8800 to Panther. Expert Rev Mol 188 Diagn