key: cord-1034130-xp3b2uei authors: Martin Ramirez, A.; Zurita Cruz, N. D.; Gutierrez-Cobos, A.; Rodriguez Serrano, D. A.; Gonzalez Alvaro, I.; Roy Vallejo, E.; Gomez de Frutos, S.; Fontan Garcia-Rodrigo, L.; Cardenoso Domingo, L. title: Evaluation of two RT-PCR techniques for SARS-CoV-2 RNA detection in serum for microbiological diagnosis date: 2020-11-16 journal: nan DOI: 10.1101/2020.11.15.20231795 sha: a04a38aa5a5bda5fcec9f4e21a2089d91670db47 doc_id: 1034130 cord_uid: xp3b2uei Presence of SARS-CoV-2 RNA in serum (viraemia) in COVID-19 patients has been related to poor prognosis and death. The aim of this study was to evaluate the ability of two commercial reverse real-time-PCR (rRT-PCR) kits, cobas SARS-CoV-2 (Cobas test) and TaqPath COVID-19 CE-IVD RT-PCR Kit (Taqpath test), to detect viraemia in COVID-19 patients and their implementation as routine diagnosis in microbiology laboratory. This retrospective cohort study was conducted with 203 adult patients admitted to Hospital Universitario de La Princesa, (89 Intensive Care Unit and 114 ward) with at least one serum sample collected in the first 48 hours from admission. A total 265 serum samples were included for study. Evaluation of both rRT-PCR techniques was performed comparing with the gold standard, a Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit; considering at least one target as a positive result. Comparison of Cobas test and Taqpath test with the gold standard method, showed high values of specificity (93.75 and 92.19 respectively) and Positive Predictive Value (92.92 and 99.88 respectively). Nevertheless, sensitivity (53.72 and 73.63 respectively) and Negative Predictive Value (32.53 and 42.99 respectively) were lower; Kappa values were 0.35 for cobas test and 0.56 for Taqpath test. For both techniques, differences of viraemia detection between the ICU and non-ICU patients were significant (p<0.001). Consequently, SARS-CoV-2 viraemia positive results obtained by both rRT-PCR should be considered good tools and may help in handling COVID-19 patients. Moreover, these methods could be easily integrated in the routine laboratory COVID-19 diagnosis and may open new strategies based on an early COVID-19 treatment. serum samples, which were collected during their hospital admission as part of their 96 routine management. A total of 265 serum samples were included for this study. All 97 samples were conserved at -20 ºC until they were tested. 98 Serum samples were tested with two rRT-PCR: cobas® SARS-COV-2 test (cobas® 100 test), a qualitative assay for detection of SARS-CoV-2 RNA; and TaqPath™ COVID-101 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 16, 2020. ; https://doi.org/10.1101 https://doi.org/10. /2020 Results obtained by both techniques were compared with results obtained by Novel 105 Coronavirus ( Test assay of all the three rRT-PCR was carried out with 400 µL of serum, treated 115 previously for virus inactivation with lysis buffer. 116 The assay detects a fragment of the orf-1ab region, specific of SARS-COV-2; and a 118 conserved region of e gene, a structural enveloped gene, for pan-sarbecovirus detection. 119 Test was performed by cobas® 6800 System (Roche Diagnostics, USA); an automatic 120 platform of nucleic acids extraction and RT-PCR amplification and detection. Serum 121 samples were processed according to manufacturer's indications, following the same 122 protocol used for SARS-CoV-2 detection in respiratory samples. Results were analyzed 123 and interpreted automatically by the cobas® 6800/8800 Software version 1.02.12.1002. 124 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 16, 2020. ; https://doi.org/10.1101 https://doi.org/10. /2020 from sample, which was performed by the automatic eMAG® Nucleic Acid Extraction 127 System (Biomerieux, France). Extraction was carried out according to eMAG® 128 manufacturer's directions, obtaining purified RNA in 60 µL of elution buffer, which 129 was used to performed both assays. 130 TaqPath™ test detects three specific SARS-CoV-2 genomic regions: orf-1ab, s, and n 131 genes and was carried out using 5 µL of purified RNA, according to the manufacturer's 132 Gold standard test detects two specific regions of SARS-CoV-2 genome: orf-1ab and n 134 genes. The nucleic acid amplification was performed according to the kit 135 manufacturer's indications using 10 µL of purified RNA. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint or Non-ICU patients, respectively. No significant differences between both groups were 170 found (p=0.14). 171 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 16, 2020. ; https://doi.org/10.1101/2020.11.15.20231795 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 16, 2020. ; https://doi.org/10.1101/2020.11.15.20231795 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 16, 2020. ; https://doi.org/10. 1101 /2020 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 16, 2020. ; https://doi.org/10.1101/2020.11.15.20231795 doi: medRxiv preprint (11/53), were positive by detection of both, n and orf1ab genes. There was agreement in 208 45 (85%) of the false negatives obtained by TaqPath™ test with cobas® test. 209 210 Discussion 211 To our knowledge, this is the first study which assesses the performance and accuracy 212 of these techniques in serum samples for SARS-CoV-2. The goal of this study is to 213 assess two commercial rRT-PCR assays for SARS-CoV-2 RNA detection in serum 214 samples, to provide clinicians a useful tool in the management of the Although these commercial assays have not been validated for their use in serum or 216 plasma samples, the extraction methods performed in this study are commonly carried 217 out in serum or plasma samples for other viruses (10-12). is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 16, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 16, 2020. ; https://doi.org/10.1101 https://doi.org/10. /2020 On the other hand, the negative predictive values obtained with both cobas® test and 258 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 16, 2020. ; https://doi.org/10. 1101 /2020 The present study has some limitations. First, the intrinsic analytical variability of PCR 279 can have influenced in the results obtained. 280 Another limitation is the type of sample used in this study. Although serum samples are 281 accepted in the analysis of viraemias, the common samples used in most microbiology 282 laboratories are plasma samples. 283 In conclusion, this study shows that cobas® SARS-CoV-2 Test and TaqPath™ is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 16, 2020. ; https://doi.org/10. 1101 /2020 Coronavirus disease (COVID-19) -World Health Organization Detectable serum SARS-CoV-2 viral load (RNAaemia) is 308 closely correlated with drastically elevated interleukin 6 (IL-6) level in critically ill 309 COVID-19 patients Relationship Between serum SARS-CoV-2 nucleic 312 acid(RNAemia) and Organ Damage in COVID-19 Patients: A Cohort Study Molecular and serological investigation of 2019-nCoV 328 infected patients: implication of multiple shedding routes Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit Probing) Instructions for use. Version 00 Consistent 349 Detection of 2019 Novel Coronavirus in Saliva SARS-CoV-2 RNA in serum as predictor of severe outcome in 353 Clinical Infectious Diseases ciaa1285 High 356 Frequency of SARS-CoV-2 RNAemia and Association With Severe Disease