Comparison of Two Commercial Platforms and a Laboratory-Developed Test for Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) RNA

Abstract

Mitigation of the ongoing coronavirus disease 2019 (COVID-19) pandemic requires reliable and accessible laboratory diagnostic services. In this study, the performance of one laboratory-developed test (LDT) and two commercial tests, cobas SARS-CoV-2 (Roche) and Amplidiag COVID-19 (Mobidiag), were evaluated for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in respiratory specimens. A total of 183 specimens collected from suspected COVID-19 patients were studied with all three methods to compare their performance. In relation to the reference standard, which was established as the result obtained by two of the three studied methods, the positive percent agreement was highest for the cobas test (100%), followed by the Amplidiag test and the LDT (98.9%). The negative percent agreement was lowest for the cobas test (89.4%), followed by the Amplidiag test (98.8%), and the highest value was obtained for the LDT (100%). The dilution series of positive specimens, however, suggests significantly higher sensitivity for the cobas assay in comparison with the other two assays, and the low negative percent agreement value may be due to the same reason. In general, all tested assays performed adequately. Clinical laboratories need to be prepared for uninterrupted high-throughput testing during the coming months to mitigate the pandemic. To ensure no interruption, it is critical that clinical laboratories maintain several simultaneous platforms in their SARS-CoV-2 nucleic acid testing. Mitigation of the ongoing coronavirus disease 2019 (COVID-19) pandemic requires reliable and accessible laboratory diagnostic services. In this study, the performance of one laboratory-developed test (LDT) and two commercial tests, cobas SARS-CoV-2 (Roche) and Amplidiag COVID-19 (Mobidiag), were evaluated for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in respiratory specimens. A total of 183 specimens collected from suspected COVID-19 patients were studied with all three methods to compare their performance. In relation to the reference standard, which was established as the result obtained by two of the three studied methods, the positive percent agreement was highest for the cobas test (100%), followed by the Amplidiag test and the LDT (98.9%). The negative percent agreement was lowest for the cobas test (89.4%), followed by the Amplidiag test (98.8%), and the highest value was obtained for the LDT (100%). The dilution series of positive specimens, however, suggests significantly higher sensitivity for the cobas assay in comparison with the other two assays, and the low negative percent agreement value may be due to the same reason. In general, all tested assays performed adequately. Clinical laboratories need to be prepared for uninterrupted high-throughput testing during the coming months to mitigate the pandemic. To ensure no interruption, it is critical that clinical laboratories maintain several simultaneous platforms in their SARS-CoV-2 nucleic acid testing. Mitigation of the ongoing coronavirus disease 2019 (COVID-19) pandemic requires reliable and accessible laboratory diagnostic services. The specific diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection relies on molecular methods, especially on reverse transcription PCR, although other technologies, including serologic immunoassays, are emerging.1Cheng M.P. Papenburg J. Desjardins M. Kanjilal S. Quach C. Libman M. Dittrich S. Yansouni C.P. Diagnostic testing for severe acute respiratory syndrome-related coronavirus-2: a narrative review.Ann Intern Med. 2020; 172: 726-734Crossref PubMed Scopus (441) Google Scholar,2Jääskeläinen A.J. Kekäläinen E. Kallio-Kokko H. Mannonen L. Kortela E. Vapalahti O. Kurkela S. Lappalainen M. Evaluation of commercial and automated SARS-CoV-2 IgG and IgA ELISAs using coronavirus disease (COVID-19) patient samples.Euro Surveill. 2020; 25: 2000603Crossref PubMed Scopus (86) Google Scholar The first methods for SARS-CoV-2 detection were laboratory-developed reverse transcription PCR tests (LDTs), and one of the first methods published was described by Corman et al.3Corman V.M. Landt O. Kaiser M. Molenkamp R. Meijer A. Chu D.K. Bleicker T. Brünink S. Schneider J. Schmidt M.L. Mulders D.G. Haagmans B.L. van der Veer B. van den Brink S. Wijsman L. Goderski G. Romette J.-L. Ellis J. Zambon M. Peiris M. Goossens H. Reusken C. Koopmans M.P. Drosten C. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR.Euro Surveill. 2020; 25: 2000045Crossref PubMed Scopus (5087) Google Scholar This method was later endorsed by the World Health Organization and was widely implemented in clinical laboratories. Roche Molecular Systems (Branchburg, NJ) cobas SARS-CoV-2 test was the first commercial test to get emergency use authorization from the Food and Drug Administration on March 12, 2020. Since then (as of June 25, 2020), more than 100 commercial molecular in vitro diagnostic tests have been granted Food and Drug Administration or other national authorities' emergency use authorization and/or CE-mark (Kalorama Information, https://kaloramainformation.com/covid19diagnosticstracker, last accessed June 25, 2020). Both LDTs and commercial tests have been set up under high time pressure. Therefore, it is of great importance to evaluate the tests in the clinical laboratory settings. In this study, the performance of one LDT and two commercial tests, namely, cobas SARS-CoV-2 (Roche) and recently, CE/in vitro diagnostic–marked Amplidiag COVID-19 (Mobidiag, Espoo, Finland), were evaluated for the detection of SARS-CoV-2 RNA in respiratory specimens. Altogether, 237 respiratory tract specimens referred to Helsinki University Hospital Laboratory (HUS Diagnostic Center, HUSLAB), Department of Virology and Immunology, Finland, were included in this study (Figure 1). The study was approved by the local review board (HUS/157/2020-29). Fifty-four specimens collected from patients with respiratory symptoms in 2018 to 2020 were used to verify the analytical specificity of the tests. The specimens from 2020 were negative for SARS-CoV-2 RNA by the LDT. A total of 183 specimens collected from suspected COVID-19 patients in 2020 were studied with all three methods to compare the performance of the tests in detecting SARS-CoV-2 RNA. Although most of the specimens tested were nasopharyngeal swabs (89%), oropharyngeal (9%) and nasal (2%) swabs also were included due to the global shortage of the swab sticks needed for nasopharyngeal sampling. The specimens were collected either in Copan UTM tubes (Copan, Brescia, Italy) or, due to the global shortage of Copan UTM tubes, in tubes containing 0.9% saline. The suitability of the 0.9% saline tubes as an alternative to the viral transport media in SARS-CoV-2 testing has been shown previously.4Rodino K.G. Espy M.J. Buckwalter S.P. Walchak R.C. Germer J.J. Fernholz E. Boerger A. Schuetz A.N. Yao J.D. Binnicker M.J. Evaluation of saline, phosphate-buffered saline, and minimum essential medium as potential alternatives to viral transport media for SARS-CoV-2 testing.J Clin Microbiol. 2020; 58 (e00590-20)Crossref Scopus (80) Google Scholar The specimens (n = 183) used to compare the performance of the three studied RT-PCR methods comprised two sets of specimens collected within two time frames. The first set of specimens (n = 37) was part of the material used for the initial verification of the cobas SARS-CoV-2 and Amplidiag COVID-19 tests. These specimens were collected between March 5 and March 18, 2020, and the SARS-CoV-2–positive specimens (n = 18) represent virus strains from the early epidemic. The second set of specimens (n = 146) were collected between May 4 and May 8, 2020, and the positive specimens (n = 90) thus represent strains from a declining phase of the epidemic in Finland. Specimens were stored at −20°C/−70°C after the initial analysis and were thawed upon analysis. Specimens were not thawed more than twice before RT-PCR. In addition, QCMD 2020 Coronavirus Outbreak Preparedness EQA Pilot Study proficiency samples (Glasgow, Scotland, UK) were used to evaluate the performance of the methods. All specimens were inactivated in a biosafety cabinet in a biosafety level 2 laboratory that has negative pressure. At the beginning of the epidemic, an FFP3 mask, protective glasses, and protective clothing were worn when working in the laboratory. Later in the epidemic, a visor and a surgical mask replaced the FFP3 masks and protective glasses. The possible SARS-CoV-2 in the specimen was inactivated by adding 250 μL of MagNA Pure Lysis/Binding Buffer (Roche Diagnostics, Mannheim, Germany) to 250 μL of patient specimen. The lysates were incubated for a minimum of 10 minutes at room temperature before being processed further. If needed, the specimens were first equilibrated to room temperature, after which the possible SARS-CoV-2 in the specimen was inactivated by adding 350 μL of MagNA Pure Lysis/Binding Buffer (Roche Diagnostics) to 350 μL of patient specimen. The lysates were incubated for a minimum of 10 minutes at room temperature before being processed further. The possible SARS-CoV-2 in the specimen was inactivated either by adding 600 μL of the specimen to 1 mL of eNAT medium (Copan), or 360 μL to 1.2 mL of mNAT Medium (Mobidiag). The eNAT tubes were incubated for a minimum of 30 minutes, and the mNAT-tubes, for a minimum of 5 minutes at room temperature before being processed further. Due to the different protocols, the inactivation step dilutes the specimens unevenly. The dilution factor for the LDT and cobas tests is 1:2, whereas it is 1:2.7 (eNAT tube) or 1:4.3 (mNAT tube) for the Amplidiag test. The real-time LDT SARS-CoV-2 RT-PCR used in this study is a modification of the method published by Corman et al.3Corman V.M. Landt O. Kaiser M. Molenkamp R. Meijer A. Chu D.K. Bleicker T. Brünink S. Schneider J. Schmidt M.L. Mulders D.G. Haagmans B.L. van der Veer B. van den Brink S. Wijsman L. Goderski G. Romette J.-L. Ellis J. Zambon M. Peiris M. Goossens H. Reusken C. Koopmans M.P. Drosten C. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR.Euro Surveill. 2020; 25: 2000045Crossref PubMed Scopus (5087) Google Scholar The test is suitable for the detection of SARS-CoV-2 RNA from sputum; nasopharyngeal/tracheal aspirates; nasal, nasopharyngeal, and oropharyngeal swab specimens; and feces. Initially, all target genes (E, RdRP, and N) were included in the diagnostic assay. In addition, a PCR for the beta-globin gene5Nummi M. Mannonen L. Puolakkainen M. Development of a multiplex real-time PCR assay for detection of Mycoplasma pneumoniae, Chlamydia pneumoniae and mutations associated with macrolide resistance in Mycoplasma pneumoniae from respiratory clinical specimens.Springerplus. 2015; 4: 684Crossref PubMed Scopus (21) Google Scholar was performed to verify successful sampling, extraction, and PCR. A full-length SARS-CoV in vitro transcript was used as a positive control. When the epidemic spread, and there was suddenly a high demand of testing in combination with the global shortage of supplies, the PCR was first of the positive obtained from negative R. A. B. A. B. A. of laboratory for the novel coronavirus SARS-CoV-2 in Surveill. 2020; 25: Scopus Google Scholar also was and the were with the PCR The PCR was a dilution series sensitivity for the PCR PCR, although were for PCR. from specimens a sensitivity for RT-PCR not were from μL of the respiratory specimen using the MagNA Pure (Roche with the MagNA Pure and in μL of the RT-PCR was performed using the Germany) with 600 of the of the reverse and of the V.M. Landt O. Kaiser M. Molenkamp R. Meijer A. Chu D.K. Bleicker T. Brünink S. Schneider J. Schmidt M.L. Mulders D.G. Haagmans B.L. van der Veer B. van den Brink S. Wijsman L. Goderski G. Romette J.-L. Ellis J. Zambon M. Peiris M. Goossens H. Reusken C. Koopmans M.P. Drosten C. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR.Euro Surveill. 2020; 25: 2000045Crossref PubMed Scopus (5087) Google Scholar The RT-PCR was performed on the PCR and negative were included in of the was to PCR with the 1 of for minutes, 1 of for minutes, followed by of for and for The cobas SARS-CoV-2 test (Roche Molecular is a test for automated cobas The test is to SARS-CoV-2 RNA from nasal, nasopharyngeal, and oropharyngeal swab specimens. The test two SARS-CoV-2 which is specific for and a of the which is specific and also SARS-CoV and other to In addition, the test an RNA which is to the specimens before The test also positive and negative which are processed the same as the A total of 600 μL of the specimen was to the cobas The testing was performed to the from the inactivation The Amplidiag COVID-19 test is a test for the detection of SARS-CoV-2 RNA in nasopharyngeal specimens. The test two SARS-CoV-2 and which are specific for In addition, the test the which as a control. The test positive and negative The eNAT or the mNAT tubes were to the Amplidiag which nucleic and the PCR The PCR was then to a real-time which the Amplidiag (Mobidiag). The Amplidiag from the Amplidiag and the result The testing was performed to the from the specimen for nasopharyngeal and the inactivation step with the A of the Amplidiag and Amplidiag COVID-19 was by the during the The not from the first 10 and the obtained are 10 than with the The was to get of the with the target of the the not analysis of the specimens from this were from the same mNAT tubes within 5 of the first The and of the tests were performed all three tests and the time needed for by a The specimen inactivation step was from this analysis. The an specimen was using the test and were not included in the of the are and are by the laboratory. When the epidemic the specimens were collected in Copan UTM tubes later in tubes containing mL of 0.9% saline, of the global shortage in the of the Copan To verify that the used methods were at the same two dilution series were A patient specimen to SARS-CoV-2 RNA collected in a Copan was in negative specimens. A patient specimen to SARS-CoV-2 RNA collected in 0.9% saline was in 0.9% negative specimens. The value for SARS-CoV-2 detection by RT-PCR was for the and the positive specimens. A dilution series of to for specimens Copan UTM or in the specimen were and with all three studied methods. Due to the shortage of the eNAT tubes, a to mNAT tubes was This was evaluated with a of specimens. specimens positive by the cobas test were in by the specimen the mNAT and the eNAT tubes, and in the same PCR The analytical specificity of the tests was studied by patient specimens containing other respiratory Altogether, specimens were not all samples were with all three methods. of the specimens several (n = In specimens, the of respiratory virus nucleic acid was by Diagnostics, and in specimens by the of The specimens were to of viral nucleic value for specimens by the test or a high in the The specimens were collected during the 2018 to 2020 thus virus strains in Finland. The specimens are described in The positive percent agreement and negative percent agreement including were with an last accessed 2020). The agreement of the assays was evaluated by the which was a with an last accessed 2020). were as no agreement agreement to agreement to agreement to agreement to and agreement to 1). All were to the reference standard, which was established as the result obtained by two of the three studied methods. The of the of the obtained by Amplidiag test from the eNAT the mNAT tubes was by was Altogether, 183 specimens were by all three evaluated methods. specimen an result by the cobas SARS-CoV-2 test two specimens an of specimen in the LDT and were and specimens by the Amplidiag COVID-19 test The specimens were from the agreement analysis. In relation to the reference standard, the positive percent agreement was highest for the cobas test (100%), followed by the Amplidiag test and the LDT (98.9%). percent agreement was lowest for the cobas test (89.4%), followed by the Amplidiag test (98.8%), and the specificity value was obtained for the LDT (100%). The agreement as by value was for all studied tests and of the to the was used as a reference value and was as the result obtained by at two of the three studied laboratory-developed negative percent positive percent was used as a reference value and was as the result obtained by at two of the three studied methods. in a laboratory-developed negative percent positive percent The time for the cobas test was 30 minutes for for the Amplidiag 30 minutes for and for the LDT 4 30 minutes for The time was for the cobas test. To get a for the studied tests, the of the tests were by the of the test. The LDT was the most test for the laboratory with a of followed by the cobas test with a of The Amplidiag test was the with a of and SARS-CoV-2 COVID-19 one to laboratory specimens were during the time the is specimens in an in an was with one and one set of one 30 30 30 test and coronavirus disease laboratory-developed severe acute respiratory syndrome coronavirus Due to laboratory specimens were during the time the is specimens in an was with one and one set of test and in a coronavirus disease laboratory-developed severe acute respiratory syndrome coronavirus dilution series of two positive specimens were in 0.9% saline and Copan UTM in a respiratory specimen to verify that specimens collected in Copan and saline at the same level with the evaluated methods. The performance of all three methods of the medium used and the a higher sensitivity for the cobas test the dilution was positive for RT-PCR in the cobas whereas to was the last dilution positive test in the LDT and in the Amplidiag test. The was with In the the last dilution with a positive result with the LDT and the Amplidiag test was for samples in saline and whereas a positive result was obtained for in dilution by the cobas of from of Coronavirus 2 (SARS-CoV-2) in Copan UTM and laboratory-developed test. in a laboratory-developed test. the specimens positive by the cobas were positive with the Amplidiag test of the used for the the specimens positive by the Amplidiag the in and target were and for the the was from all specimens. The in the obtained was for the = = for the and for the = QCMD 2020 Coronavirus Outbreak Preparedness EQA Pilot Study proficiency samples were used to the performance of the evaluated methods. The cobas test and the LDT were in agreement with the QCMD The Amplidiag test to one SARS-CoV-2–positive result with a SARS-CoV-2 of the test containing SARS-CoV-2 RNA The Amplidiag test samples as the EQA samples the target needed for negative with the of QCMD 2020 Coronavirus Outbreak Preparedness EQA Pilot Study with the with the cobas SARS-CoV-2 with the Amplidiag COVID-19 with the transport coronavirus disease laboratory-developed severe acute respiratory syndrome coronavirus in a transport coronavirus disease laboratory-developed severe acute respiratory syndrome coronavirus of the studied SARS-CoV-2 tests positive for the patient specimens containing other respiratory Altogether, there were In one which a positive result with the Amplidiag the positive was obtained for the target with a value of in the by the Amplidiag no specific be of the in the a in the was not one a positive result was obtained with the cobas and the Amplidiag tests, a negative result with the LDT. In positive were obtained with the cobas test and the whereas the Amplidiag test a negative In that the specimen two before it was by the Amplidiag one or when by the cobas or the In specimens, a positive result was obtained by the cobas test. a positive result was obtained for two of specimens when with the Amplidiag COVID-19 In all of the specimens, a positive was obtained for of the cobas the were for of the specimens in the between the and in the between Specimens with the and the of Amplidiag of SARS-CoV-2 test COVID-19 test of the by Amplidiag specimen two when by Amplidiag and one with cobas test and with specimens positive by cobas a from one all a value of the specimens a positive result when specimens were with the Amplidiag of result with result with of the by the Amplidiag Amplidiag the result as negative there is from positive by cobas that 10 be to the value obtained with the of the to compare the to the obtained by the laboratory-developed test. in a that 10 be to the value obtained with the of the to compare the to the obtained by the laboratory-developed test. the specimens with the and the of the Amplidiag COVID-19 specimens a result with 1 or 2 of the or (n = and were from the analysis. A total of positive and negative were obtained by the and positive and negative by the were The specimen that a result by the of the Amplidiag was negative in the with the Amplidiag specimens that were positive with the negative by the there was of the two specimens were positive with the and negative with the of the specimens that a negative result by the a positive result when with the In the comparison of the three methods, two specimens were positive by the cobas assay In general, all tested assays performed adequately. Both the time from the to the result and the time were the for the cobas test. Although the were not the LDT test was the most for the followed by the cobas whereas the Amplidiag test was the The cobas and the Amplidiag assays for an of the to the laboratory the other the LDT and the Amplidiag test for the of the which is for the of the especially when the tests have been set up in a the three RT-PCR methods evaluated in this study, the cobas SARS-CoV-2 test the performance in the detection of SARS-CoV-2 RNA in the clinical respiratory specimens. In the of a standard, a reference value was as a of two methods. The agreement between the different methods tested was the positive percent agreement was highest for the cobas SARS-CoV-2 test (100%), whereas negative percent agreement was the lowest All of the specimens positive with the cobas SARS-CoV-2 test high with one of and were The dilution series for specimens collected in Copan UTM 0.9% saline, however, a significantly higher sensitivity for the cobas assay in comparison with the other two assays, and the low negative percent agreement value may be due to the same reason. also high sensitivity for the cobas SARS-CoV-2 M. M. M. T. T. M. Clinical of the cobas SARS-CoV-2 test and a diagnostic during in the of the COVID-19 Clin Microbiol. 2020; 58 Scopus Google E. A. T. B. A. C. J. of SARS-CoV-2 detection from nasopharyngeal swab samples by the Roche cobas SARS-CoV-2 test and a laboratory-developed real-time RT-PCR 2020; PubMed Scopus Google Scholar The second commercial test the Amplidiag COVID-19 obtained the same positive percent agreement as the negative percent agreement was one specimen that was positive by the Amplidiag COVID-19 this to be a positive by the of the of the positive target This specimen was negative by the Amplidiag which not of negative in this it that is in specificity is in the three specimens positive by the negative specific from the target be All the Amplidiag test to on the same level with the from the high of the Amplidiag test The comparison of the from the eNAT tubes the mNAT tubes suggests that the Amplidiag test is for for the when using the mNAT The in the obtained was for the 2 than for the specific virus This was also in the percent to not when the to the mNAT tubes was of this study is the inactivation which in of the specimen in the Amplidiag test in comparison to the cobas test and the LDT. This may the sensitivity of the Amplidiag test. the is not this in the initial of the specimen in 1 in the PCR The QCMD 2020 Coronavirus Outbreak Preparedness EQA Pilot samples were with all of the evaluated methods. The cobas test and the LDT all samples The Amplidiag test one specimen with a PCR reference value of although it with a PCR reference value of In addition, the Amplidiag test a test result for samples samples the target needed for a negative result with the test. The of in the EQA samples is a in The performance of the test the target virus may be different in the in comparison to the virus nucleic not a result in a PCR, may the and/or the have in the or the LDT specimens more than in the specimens with included not is that RNA mutations at high RNA there is that SARS-CoV-2 also is and mutations may in the target of the molecular tests A suggests that a has in the target of the cobas SARS-CoV-2 M. S. M. R. C. A at of SARS-CoV-2 is associated with of the reverse in a commercial diagnostic Clin Microbiol. 2020; 58 PubMed Scopus Google Scholar In this study, all specimens a positive for target genes by the cobas test. the test a which that the to be in the target of the cobas test not This the importance of a A. M. Mannonen L. Kurkela S. Lappalainen M. M. Puolakkainen M. The of Chlamydia with a in the target detection by the 2 Scopus Google L. R. A. B. of two real-time tests the for viral of PubMed Scopus (21) Google C. A. of a real-time PCR and for the detection of PubMed Scopus Google Scholar are from to and in their COVID-19 which requires a for and reliable SARS-CoV-2 laboratory testing. Clinical laboratories need to be prepared for uninterrupted high-throughput testing during the coming To laboratories need to have testing methods in the of as as for the ongoing global shortage in testing Therefore, it is of critical importance for clinical laboratories to maintain several simultaneous platforms in their SARS-CoV-2 nucleic acid testing and the performance of the assays the laboratory the SARS-CoV-2 at for their for this with

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