key: cord-289367-zna8qkkv
authors: Wirden, Marc; Feghoul, Linda; Bertine, Mélanie; Nere, Marie-Laure; Le Hingrat, Quentin; Abdi, Basma; Boutolleau, David; Ferre, Valentine Marie; Jary, Aude; Delaugerre, Constance; Marcelin, Anne-Genevieve; Descamps, Diane; Legoff, Jérôme; Visseaux, Benoit; Chaix, Marie-Laure
title: Multicenter comparison of the Cobas 6800 system with the RealStar RT-PCR kit for the detection of SARS-CoV-2
date: 2020-06-30
journal: bioRxiv
DOI: 10.1101/2020.06.29.179184
sha: 
doc_id: 289367
cord_uid: zna8qkkv

Background RT-PCR testing is crucial in the diagnostic of SARS-CoV-2 infection. The use of reliable and comparable PCR assays is a cornerstone to allow use of different PCR assays depending on the local equipment. In this work, we provide a comparison of the Cobas® (Roche) and the RealStar® assay (Altona). Methods Assessment of the two assays was performed prospectively in three reference Parisians hospitals, using 170 clinical samples. They were tested with the Cobas® assay, selected to obtain a distribution of cycle threshold (Ct) as large as possible, and tested with the RealStar assay with three largely available extraction platforms: QIAsymphony (Qiagen), MagNAPure (Roche) and NucliSENS-easyMag (BioMérieux). Results Overall, the agreement (positive for at least one gene) was 76%. This rate differed considerably depending on the Cobas Ct values for gene E: below 35 (n = 91), the concordance was 99%. Regarding the positive Ct values, linear regression analysis showed a determination correlation (R2) of 0.88 and the Deming regression line revealed a strong correlation with a slope of 1.023 and an intercept of -3.9. Bland-Altman analysis showed that the mean difference (Cobas® minus RealStar®) was + 3.3 Ct, with a SD of + 2.3 Ct. Conclusions In this comparison, both RealStar® and Cobas® assays provided comparable qualitative results and a high correlation when both tests were positive. Discrepancies exist after 35 Ct and varied depending on the extraction system used for the RealStar® assay, probably due to a low viral load close to the detection limit of both assays.

In this study, we compared two different widely used tests in three major Parisian university 78 hospital laboratories. These are the RealStar® SARS-CoV-2 RT-PCR Kit 1.0 (Altona 79 diagnostics, France) which can be associated to different extraction and amplification devices, 80 and the Cobas® SARS-CoV-2 kit used on the Cobas® 6800 system (Cobas 6800; Roche 81 Diagnostics, Mannheim, Germany). 82

Samples 85

In April 2020, 140 patients were included in this prospective study performed in 3 virological 86 laboratories located in Paris (Saint Louis hospital (n=45), Bichat hospital (n=49) and La Salpêtrière hospital (n=46)). Then, each laboratory selected 45 to 49 samples firstly detected 88 using the Cobas 6800 with a stratification according to the Ct of the E gene Cobas results, 89 allowing to cover the whole linear range of the assays. Thus, three categories were retained: 90

Ct < 25, Ct between 25 and 34 and with a Ct > 35. Rapidly, in the same day or within 48 91 hours, the leftover samples stored at +4°C were tested with the RealStar assay. Thirty 92 nasopharyngeal swab samples collected in 2019 (in the pre-epidemic Covid 19 period) were 93 also tested with both techniques (10 in each laboratory). 94 95

The Cobas ® SARS-CoV-2 test is a single-well dual target assay, which targets the non-97 structural ORF1a/b region specific of SARS-CoV-2 and the structural protein envelope E 98 gene for pan-sarbecovirus detection. The test used RNA internal control for sample extraction 99 and PCR amplification process control. To take into account the available sample volume and 100 the security conditions required for this virus before loading on the Cobas 6800 system, the 101 pre-analytical protocol has been adapted as recommended by the manufacturer as follows: 102 400 μl of each sample were transferred at room temperature into barcoded secondary tubes 103 containing 400 μl of Cobas lysis buffer for the SARS-CoV-2 neutralization. Then, the tube 104 was loaded on the Cobas 6800 where 400 μl from those 800 μl were used for RNA extraction, 105 and eluted in 50 μl of which 27 µl were used for the RT-PCR. The test was performed in 7 positive results either for both ORF1a/b and E genes or for the ORF1a/b gene only. In the 109 case of single E gene positivity, the result should be reported as SARS-CoV-2 presumptive 110 positive and repeated, but were considered as positive for this study. 111

The RealStar® SARS-CoV-2 RT-PCR Kit 1.0 assay targets the E gene specific for 114 sarbecoviruses, and the S gene specific for SARS-CoV-2. It includes a heterologous the Cobas 6800 assay. This is in accordance with the Cobas 6800 insert information reporting 204 a higher sensitivity for the E gene detection than for the ORF1/a, and also a drop in the 205 positivity rate above 35 Ct for the E target. This may explain why the RealStar test yielded 206 many negative results in such cases as both tests probably reached their detection limits. This 207 is a limitation of our study as we did not assessed comparatively the limit of detection of the 208 two methods but the reliability of their Ct values among COBAS 6800 positive samples, 209 excluding those that could be negative with COBAS 6800 and positive with RealStar in this 210 range of low viral loads. Our work highlights the impact of the extraction system on the 211 sensitivity of the RealStar assay. 212

Overall, we demonstrated the good performances and concordance between the two assays, at 214 least for viral loads above the detection limit of both assays. This concordance allows to 215 reliably compare Ct values obtained from both methods. However, the variations observed 216 between the Ct values of the two assays, evaluated here as about 3.5 additional Ct with the 217

Cobas 6800 assay, has to be taken into account for Ct values follow-up done for the most 218 severe patients in case of successive use of the two methods, depending of reagent and 219 analyser availability. 

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Emergency Committe Regarding the outbreak 253 of Novel Coronavirus

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The authors declare no conflict of interest. 235

We acknowledge all the laboratory staff of Saint Louis hospital virology department, Bichat 238 hospital virology department and La Pitié-Salpêtrière hospital virology department. 239