key: cord-0855924-a6thk21n
authors: Mannonen, Laura; Kallio-Kokko, Hannimari; Loginov, Raisa; Jääskeläinen, Anu; Jokela, Pia; Antikainen, Jenni; Väre, Paula; Kekäläinen, Eliisa; Kurkela, Satu; Jarva, Hanna; Lappalainen, Maija
title: Comparison of two commercial platforms and a laboratory developed test for detection of SARS-CoV-2 RNA
date: 2021-01-21
journal: J Mol Diagn
DOI: 10.1016/j.jmoldx.2021.01.005
sha: 97d153fbb73e2d7cd97c4324d65095541ee6fdce
doc_id: 855924
cord_uid: a6thk21n

Mitigation of the ongoing 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 SARS-CoV-2 RNA in respiratory specimens. 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 (PPA) was highest for the cobas test (100%), followed by the Amplidiag test and the LDT (98.9%). The negative percent agreement (NPA) 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 NPA 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 in mitigation of the pandemic. To secure that, it is of critical importance for clinical laboratories to maintain several simultaneous platforms in their SARS-CoV-2 nucleic acid testing.

Mitigation of the ongoing COVID-19 pandemic requires reliable and accessible laboratory 44 diagnostic services. The specific diagnosis of SARS-CoV-2 infection relies on molecular 45 methods, especially on RT-PCR, although other technologies including serological 46 immunoassays are emerging 1, 2 . The first methods for SARS-CoV-2 detection were laboratory 47 developed RT-PCR tests (LDTs) and one of the first methods published was described by 48 Corman et al. 3 . This method was later endorsed by WHO and was widely implemented in 49 clinical laboratories. Roche Molecular Systems (Branchburg, NJ, USA) cobas SARS-Cov-2 50 test was the first commercial test to get EUA from FDA on March 12, 2020 . Since then (as of 51 June 25, 2020) more than 100 commercial molecular IVD-tests have been granted the FDA 52 or other national authorities' EUA and/or CE-mark 53 (https://kaloramainformation.com/covid19diagnosticstracker/, last accessed 25.6.2020). Both 54 LDTs and commercial tests have been set up in a high time pressure. Therefore, it is of great 55 importance to evaluate the tests in the clinical laboratory settings. In this study the 56 performance of one LDT and two commercial tests, namely cobas SARS-CoV-2 (Roche) 57 and recently CE/IVD marked Amplidiag COVID-19 (Mobidiag, Espoo, Finland) were 58 evaluated for the detection of SARS-CoV-2 RNA in respiratory specimens. If needed, the specimens were first equilibrated to room temperature, after which the possible 106 SARS-CoV-2 in the specimen was inactivated by adding 350 µl of MagNA Pure 107 Lysis/Binding Buffer (Roche Diagnostics GmbH, Mannheim, Germany) to 350 µl of patient 108 specimen. The lysates were incubated for a minimum of 10 minutes at RT before processed 109 further.

The real-time LDT SARS-CoV-2 RT-PCR used in this study is a modification of the method 125 published by Corman et al. 3 . The test is suitable for the detection of SARS-CoV-2 RNA from 126 sputum, nasopharyngeal/tracheal aspirates, nasal, nasopharyngeal and oropharyngeal swab 127 specimens and feces. Initially, all target genes (E, RdRP and N) were included in the 128 diagnostic assay. In addition, a PCR for the beta-globin gene 5 was performed in order to 129 verify successful sampling, extraction and PCR. A full-length SARS-CoV in vitro transcript 130 was used as a positive control. When the epidemic spread and there was suddenly a high controls/specimens 6 . Later also RdRP was excluded and the diagnostics were continued with sensitivity for the N-gene PCR over RdRP PCR, although earlier ct-values were gained for 136 RdRP PCR. Furthermore, findings from low positive specimens suggested a better sensitivity 137 for N-gene RT-PCR (data not shown). Supplemental Table S1 . The positive percent agreement (PPA) and negative percent agreement (NPA) including two-241 sided 95% confidence intervals (CI) were calculated with an on-line MEDCALC tool 242 (https://www.medcalc.org/calc/diagnostic_test.php, last accessed 20.9.2020). The overall 243 agreement of the assays was evaluated by the kappa value, which was calculated (including 244 two-sided 95% CI) with an on-line QuickCalcs tool shortest for the cobas test. In order to get a cost efficiency ratio for the studied tests, the costs 273 of the tests were divided by the cost of the cheapest test. The LDT was the most cost efficient 274 test for the laboratory with a cost efficiency ratio of 1, followed by the cobas test with a cost 275 efficiency ratio of 1.25. The Amplidiag test was the least cost efficient with a cost efficiency 276 ratio of 1.38 (Table 2) . Two independent dilution series of two positive specimens were constructed to 0.9% saline 282 and copan UTM in a respiratory specimen matrix to verify that specimens collected to Copan 283 and saline perform at the same level with the evaluated methods. The results indicate similar 284 performance of all three methods independent of the collection media used (Table 3 and 285 Figure 2 ). However, the data suggest a higher overall sensitivity for the cobas test as the 286 dilution 1:20,000 was still positive for both RT-PCR targets in the cobas test, while 1:200- None of the studied SARS-CoV-2 tests gave positive results for the patient specimens 318 containing other respiratory viruses (Supplemental Table S1 ). test, but only one or none when analyzed by the cobas or the LTD test, respectively. In nine 332 specimens a positive result was obtained only by the cobas test. However, a positive result 333 was obtained for two of these specimens, when rerun with the new Amplidiag COVID-19 334 software version. In all of the nine specimens a positive signal was obtained for both 335 amplification targets of the cobas test, but the ct-values were high, >30 for 8/9 specimens. (Table 5) . even though specific amplification from the orf1ab target could be seen. All things 384 considered, the Amplidiag test seems to perform on the same level with the LDT, apart from 385 the relatively high failure rate of the Amplidiag test (3.8%).

suggests that the Amplidiag test is for some reason less sensitive (later ct-value) for the 389 sampling control (RNase P gene) when using the mNAT tubes. The difference in the obtained 390 ct-values was greater for the RNase P gene (~2 ct-cycles) than for the specific virus targets 391 (<1 ct-cycle). This was also observed in the rising failure percent (from 3.8 % to 8.4%, data 392 not shown) when the change to the mNAT tubes was implemented. One limitation of this 393 study is the inactivation step, which results in 0.46 × less specimen in the Amplidiag test in It is common that RNA-viruses accumulate mutations at high frequency since 413 RNA-polymerase lacks proofreading activity. Indeed, there is evidence that also SARS-CoV-414 2 is evolving during time and mutations may occur in the target area of the molecular tests 415 used. A previous report suggests that a mutation has already occurred in the target region of 416 the cobas SARS-CoV-2 E-gene test 9 . In this study, all specimens gave a positive signal for 417 both target genes by the cobas test. Fortunately, the test uses a dual target approach, which 418 means that the mutation proposed to be in the target area of the cobas E-gene test does not 419 compromise correct results. This highlights the importance of a dual target approach 10-12 . 

Diagnostic Testing for Severe Acute Respiratory Syndrome-Related 443

Coronavirus-2: A Narrative Review

ELISAs using coronavirus disease (COVID-19) patient samples

Detection of 2019 novel 455 coronavirus (2019-nCoV) by real-time RT-PCR

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Saline, and Minimum Essential Medium as Potential Alternatives to Viral Transport 461

The Finnish New Variant of 493

Chlamydia trachomatis with a Single Nucleotide Polymorphism in the 23S rRNA 494

Target Escapes Detection by the Aptima Combo 2

Comparison of two quantitative real-time CMV-PCR tests 498 calibrated against the 1st WHO international standard for viral load monitoring of 499 renal transplant patients

Comparison of a UL111a real-502 time PCR and pp65 antigenemia for the detection of cytomegalovirus

) pos (33.3/32.77) pos (27.41) pos (27.70) 1:100 pos (30.11/30.90) pos (29.28/29.77) pos