Background: The ongoing SARS-CoV-2 pandemic presents a unique challenge to diagnostic 31 laboratories. There are preliminary studies correlating qRT-PCR results from different materials to 32 clinical outcomes, yet, comparability is limited due to the plethora of different assays used for 33 diagnostics. In this study we evaluate clinical performance and linear range for the SARS-CoV-2 IVD 34 (cobas6800/8800 system, a fully automated sample-to-result platform) in different clinically relevant 35 matrix materials outside official specifications. Methods: Assay performance was assessed in human 36 plasma, BAL/BL and transport medium following chemical inactivation. For analytical evaluation, 37 respective matrix materials were spiked with SARS-CoV-2 RNA in ten-fold dilution series. The efficacy 38 of chemical inactivation by guanidine hydrochloride solution was confirmed in cell culture infectivity 39 experiments. For correlation, a total of 235 predetermined clinical samples including respiratory 40 swabs, plasma and BAL/BL were subjected to the SARS-CoV-2 IVD test and results were compared. 41
The SARS-CoV-2 IVD showed excellent linearity over five to seven log steps depending on 42 matrix material. Chemical inactivation resulted in a reduction in plaque forming units of at least 3.5 log 43 steps, while having no significant impact on assay performance. Inter-run consistency from three 44 different testing sites demonstrated excellent comparability of RT-PCR results (maximum deviation 45 was 1.53 CT). Clinical evaluation for respiratory swabs showed very good agreement with the 46 comparator assay (Positive agreement 95.7%, negative agreement 98.9%). Conclusion: The SARS-CoV-47 2 Introduction: 51 The novel coronavirus SARS-CoV-2 has been subject of extensive study since its emergence in 52 late December 2019 (1, 2) . Diagnostic RT-PCR was quickly determined as the gold standard for 53 detecting the new pathogen in patients, in large parts due to the rapid dissemination of 54 complete virus sequences from the assumed origin of the outbreak in China and consecutive 55 publication of specific PCR assays (3, 4) . Besides merely confirming the diagnosis, there exists 56 evidence for a correlation between viral loads and clinical outcomes for various respiratory 57 viruses, including Influenza and the original SARS-CoV (5, 6). In the case of SARS-CoV-2 there 58 has already been a flurry of publications describing viral load dynamics in different clinical 59 specimens (7, 8) . It can be assumed that this topic will further grow in relevance in the coming 60 months. 61 Reliable quantification of RT-PCR signals is highly dependent on assay specifications and 62 reference materials (9). In the absence of an international standard to serve as reference, 63 quantification of SARS-CoV-2 RNA is ultimately based on a variety of different methods and 64 standards used in the individual labs (8, 10), resulting in inherently poor comparability and 65 reproducibility between testing sites. However, the increasing availability of commercial SARS-66 CoV-2 RT-PCR kits for widely used fully automated systems offers the opportunity to generate 67 highly consistent PCR results which can then be used for quantification. 68 The cobas6800 system is a fully automated sample-to-result high-throughput RT-PCR 69 platform, capable of performing 384 tests in an 8 hour shift and requiring minimal hands-on 70 time (11). Until very recently, laboratory developed tests (LDT) using the open mode (utility 71 channel) had to be employed to use the system for SARS-CoV-2 testing (10). However, Roche 72 has since received "Emergency use authorization" by the FDA for their own SARS-CoV-2 IVD 73 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 May 30, 2020 . . https://doi.org/10.1101 /2020 assay and have made the test available commercially (12). This provides a common ground 74 across different testing sites for quantification of RT-PCR data, especially when taking the 75 remarkable inter-run consistency of the instrument into account (13). 76 The aim of this study was to provide clinical evaluation of the new SARS-CoV-2 IVD assay by 77 Roche and further validate clinical specimen types outside specifications for use in clinical 78 studies. In this context, efficiency and linear range was to be determined in different clinical 79 materials. Lastly, comparability of results was to be evaluated using a multicenter approach 80 for inter-run variability. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 98 In order to determine the virus inactivation capability of the Roche PCR Media Kit (≤ 40% 99 guanidine hydrochloride in Tris-HCl), 100 µl of the SARS-CoV-2 stock solution (with an 100 estimated 3x10 7 PFU/ml) were diluted 1:10 in either UTM or E-swab medium (modified Amies 101 medium in Tris-HCl). 500 µl of each dilution were supplemented 1:1 with Roche PCR-Media, 102 the remaining 500µl were supplemented 1:1 with either UTM or E-swab medium. After 30 min 103 of incubation at room temperature, ten-fold serial dilution series of each mixture were used 104 for plaque assays as described above. 107 The SARS-CoV-2 IVD dual-target test for the cobas6800/8800 system was obtained from Roche 108 and used according to instructions issued by the manufacturer. Target-1 (RdRp gene) and 109 Target-2 (E gene) properties were analysed separately, though the entire assay was deemed 110 positive as long as one Target returned a positive result. Apart from loading the ready-to-use 111 SARS-CoV-2 IVD-test cartridges onto the device, there are no manual steps required in the 112 assay setup. 113 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 May 30, 2020. . https://doi.org/10.1101/2020.05.28.20115469 doi: medRxiv preprint 114 For determining linear range in different matrix materials, an initial 1:20 dilution of cell culture 115 supernatant containing SARS-CoV-2 (isolate HH-1) was prepared using normal human plasma 116 (SARS-CoV-2 RNA negative) as diluent. For BAL/BL, clinical specimens previously tested PCR-117 negative for SARS-CoV-2 were pooled to serve as matrix material. The initial stock was used 118 to generate tenfold dilution series, which were subjected to the SARS-CoV-2 IVD test in 8 119 repeats for each step. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 
Inter-run variability was compared between a total of four different instruments, located at 3 138 different testing sites. Triplicates of a solution containing diluted SARS-CoV-2 infected cell 139 culture supernatant were prepared as described above. Concentrations of SARS-CoV-2 RNA 140 were estimated to be within linear range of the assay. Spiked sample sets were sent to "Labor 141 Limbach", Heidelberg and "Labor Stein", Moenchengladbach for analysis with the SARS-CoV-142 2 IVD test using their own cobas6800 instruments. Ct values were reported and compared. 143 The total number of measurements for each series was 11. (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 May 30, 2020. 169 show excellent correlation with the comparator assay 170 For clinical validation, a set of predetermined samples was subjected to testing with the SARS-171 CoV-2 IVD and results were compared to the comparator assay. The SARS-CoV-2 IVD result 172 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 May 30, 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 May 30, 2020. . https://doi.org/10.1101/2020.05.28.20115469 doi: medRxiv preprint (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 May 30, 2020 . . https://doi.org/10.1101 /2020 performance. The aim of this study was to go beyond specifications and validate the assay on 212 relevant clinical materials such as BAL/BL and blood, as well as evaluating the efficacy and 213 performance impact of chemical inactivation procedures. 214 Safety has been a serious concern when handling clinical samples potentially containing SARS- (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 May 30, 2020. . https://doi.org/10.1101/2020.05.28.20115469 doi: medRxiv preprint assay by the Hong-Kong University (24) seem to stand out from the crowd, featuring both a 235 maximum of analytical sensitivity and wide linear range. However, other factors such as 236 extraction methods, and choice of enzymes and cyclers will render comparability between 237 sites difficult, even when using the same assays. 238 In this study we demonstrate that the SARS-CoV-2 IVD dual-target assay is highly efficient and 239 offers a wide linear range in different types of matrix materials outside official specifications. 240 Based on our data, it seems preferable to use the Target-2 (E gene) CT for quantification 241 purposes as it performed slightly better than Target-1 (RdRp gene) in this trial. Compared to 242 the inhouse assay SARS-CoV-2 UCT, the SARS-CoV-2 IVD assay showed good agreement for a 243 total of 180 respiratory swab samples, which is in line with previously reported data (12). (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 May 30, 2020 . . https://doi.org/10.1101 /2020 
In this study we provide evaluation for different matrix materials and clinical performance of 258 the SARS-CoV-2 IVD for the cobas6800/8800 systems. We observed excellent linearity over 259 five to seven log steps in different clinically relevant materials outside assay specifications, (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 May 30, 2020. to Vero cells grown to fluency in a 24 well plate. 350 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 May 30, 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 May 30, 2020 . . https://doi.org/10.1101 /2020 