key: cord-0967479-8bcag1zh
authors: Ehret, Robert; Breuer, Stefan; Dhein, Jens; Reinhardt, Birgit; Obermeier, Martin
title: Clinical evaluation of the automated Abbott RealTime SARS-CoV-2, Alinity m SARS-CoV-2, and Alinity m Resp-4-Plex assays
date: 2021-10-22
journal: J Virol Methods
DOI: 10.1016/j.jviromet.2021.114338
sha: 9663098f24e83e619bad83526eecdc1914d32c31
doc_id: 967479
cord_uid: 8bcag1zh

BACKGROUND: Detection of SARS-CoV-2 infections relies on the use of sensitive, accurate and high throughput RT-PCR assays. OBJECTIVES: We assessed the analytical performance of the Abbott RealTime SARS-CoV-2 (RT-SARS), Alinity m SARS-CoV-2 (AlinSARS) assays and compared the clinical performance of the RT-SARS, AlinSARS, and Alinity m Resp-4-Plex (Alin4Plex) assays to the Seegene Allplex assay (Allplex) and an inhouse test (Inhouse). RESULTS: We found 100% positive percent agreement (PPA) and 100% negative percent agreement (NPA) comparing RT-SARS and Allplex. RT-SARS, AlinSARS and Inhouse showed 100% NPA and 100% PPA across all assays, except for the RdRp target of Inhouse (PPA = 84%). Similarly, Alin4Plex and Allplex showed high agreement with specimens containing either SARS-CoV-2, influenza A, influenza B, or RSV. Detection rates of 100% for SARS-CoV-2 at 50 copies/mL, high precision, and no cross-reactivity with non-SARS-CoV-2 respiratory pathogens were observed for RT-SARS and AlinSARS. AlinSARS detected SARS-CoV-2 in spiked throat washes and in specimens infected with SARS-CoV-2 Alpha or Beta variants. CONCLUSIONS: The newly developed RT-SARS, AlinSARS, and Alin4Plex assays proved to be useful for detecting SARS-CoV-2 RNA in clinical samples.

Rapid and accurate diagnostic testing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is essential for the management of the COVID-19 pandemic [1] . Reverse transcriptase-polymerase chain reaction (RT-PCR) test methods targeting SARS-CoV-2 RNA are the gold standard for diagnosing suspected cases of COVID-19, patient care, contact tracing and outbreak investigations and are expected to reliably detect also newly emerging variants of concern (VOC) [2, 3] . Testing strategies are increasingly complemented by active surveillance of individuals at high risk of infection, like healthcare workers or teachers and children at school [4, 5] . Recommendations include saliva or throat wash as alternative specimen types besides oro-and nasopharyngeal swabs, especially when testing children [5] . As case definitions for influenza and acute respiratory infections overlap with those of COVID-19, the use of J o u r n a l P r e -p r o o f multiplex RT-PCR assays, e.g. for SARS-CoV-2, influenza, and RSV, can be considered [6] .

Testing of specimens for the presence and discrimination of respiratory viruses relies on the use of accurate molecular diagnostic assays. High throughput molecular diagnostic analyzers, with high level automation returning the RT-PCR test results within 24 hours, support coping with the high testing demand for SARS-CoV-2 [7] . We evaluated the analytical performance of the Abbott RealTime™ SARS-CoV-2 assay for use on the automated m2000™ batch analyzer system and the Abbott Alinity m SARS-CoV-2 assay for use on the Alinity m system, a fully automated continuous and random-access analyzer reporting approximately 1000 results in 24 h. We also compared the clinical performance of the RealTime SARS-CoV-2, the Alinity m SARS-CoV-2, and the Alinity m Resp-4-Plex assays to the Seegene Allplex assay and an inhouse test.

The Abbott RealTime SARS-CoV-2 ("RT-SARS") and the Alinity m SARS-CoV-2 ("AlinSARS") assays (both Abbott Molecular Inc., Des Plaines, IL, USA) are intended for the qualitative detection of SARS-CoV-2 in nasopharyngeal and oropharyngeal swabs from patients suspected of COVID-19 infection [8, 9] . The Alinity m Resp-4-Plex assay ("Alin4Plex"; Abbott Molecular Inc., Des Plaines, IL,

CoV-2 from nasopharyngeal swabs [10] . All three assays detect highly conserved and SARS-CoV-2-specific target regions in the RdRp and N genes of the SARS-CoV-2 genome. Additionally, Alin4Plex targets the Matrix genes of RSV and FluA and the Nonstructural 1 gene of FluB.

The comparator method Allplex™ SARS-CoV-2 ("Allplex", Seegene Inc., Seoul, Korea) targets the E gene for the detection of the sarbecovirus group and additionally two SARS-CoV-2-specific sequences in the RdRp and N genes [11] . Extraction and mastermix preparation was performed on the Seegene NIMBUS automated liquid handling workstation. Abbott and Seegene assays were performed according to the manufacturers´ instructions. Furthermore, the TIB MolBiol LightMix® SarbecoV Egene plus EAV control assay [12] (TIB Molbiol Syntheselabor GmbH, Berlin, Germany) was extended to an inhouse multiplex assay by adding primers and probe targeting the RdRp gene according to Corman et al [13] ("Inhouse"). It was run on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories GmbH, Feldkirchen, Germany)

and was used as a second molecular comparator test. All samples positive for either one or two targets of Inhouse were retested with Allplex for confirmation.

Practice and conducted in adherence with the Declaration of Helsinki. Only residual samples from routine SARS-CoV-2 testing were used.

To assess the detection rates of RT-SARS, an RT-SARS dilution panel with target concentrations of 1000, 500, 250, 100, 50, 10, 5, and 2.5 cps/ml was prepared by Allplex, were also retested with AlinSARS (n=11). All samples were negative for the other three pathogens using Allplex. After routine testing, samples had been stored at -20°C for up to 48 weeks before retesting.

Since 

Spiked throat wash specimens were prepared by spiking 10 µl of leftover media from SARS-CoV-2 positive swab samples into 1.1 ml of either different residual negative patient throat wash specimens (n=10) or 0.9% NaCl solution as control (n=1), respectively. Samples with either high positive (about 6 log cps/ml) or low positive (around the detection limit) target concentrations, respectively, were tested with AlinSARS.

The detection rates of RT-SARS were assessed by testing multiple replicates of the RT-SARS dilution panel with target concentrations between 1000 and 2.5 cps/ml. RT-SARS exhibited 100% detection rate at a concentration of 50 cps/ml (Table 1) (Table S1) , confirming the high specificity of both assays. 

In February/March 2021, the prevalence of VOCs in our laboratory rapidly increased from 10% to 75%. While variant Beta only accounted for ≤1%, the vast majority (up to 74%) consisted of the variant Alpha. As expected, all 24 samples (23 Alpha and one Beta specimens) with Ct-values ranging from 16 to 39 by Allplex were also detected by AlinSARS, exhibiting comparable Ct values (data not shown). At 50 cps/ml, 100% detection rates were observed with RT-SARS and AlinSARS, exceeding the sensitivity claimed by the manufacturer. A high analytical sensitivity is essential to identify and contain outbreaks during the SARS-CoV-2 pandemic [14] and is fundamental in cases of low viral load due to poor swabbing technique of a potentially infectious patient [15] . The observed high sensitivity of RT-SARS and AlinSARS agrees well with previous studies evaluating their LOD and comparing the assays with multiple commercial SARS-CoV-2 assays [15, 16, 17, 18, 19, 20, 21] .

findings [16, 18, 21, 22] .

coronaviruses [20] .

Allplex and Inhouse, we found 100% PPA and 100% NPA in all comparisons between RT-SARS, AlinSARS, Alin4Plex, Allplex, and the E gene of Inhouse. Only with the RdRp target region of Inhouse, a lower PPA of 84% compared to all other assays was observed. High PPAs and NPAs were similarly described comparing RT-SARS with a CDC-based in-house assay and the ThermoFisher TaqPath RT-PCR COVID-19 EUA assay [20, 18] . Furthermore, excellent agreement was obtained between RT-SARS and AlinSARS [18] . Finally, in our study, high concordance was observed between Alin4Plex and Allplex for the detection of FluA, FluB, and RSV. The two

Previously, mutations in the S gene encoding the spike protein have been shown to affect the performance of some diagnostic PCR assays targeting the S gene [23] . [24] . The Alin4Plex assay is a multiplex PCR assay allowing differentiation of several respiratory pathogens in a single test, thus enabling laboratories to process more tests per week and saving precious testing materials. Information on the incidence 

The analytical and clinical performance of the Abbott RealTime SARS-CoV-2, the Alinity m SARS-CoV-2, and the Alinity m Resp-4-Plex assays qualify these tests as valuable automated PCR methods. The ability to rapidly test for SARS-CoV-2 on the random access Alinity m analyzer, either as single plex or as multiplex SARS-CoV-2/FluA/FluB/RSV assay, will expand options to further increase testing capacities with the objective to slow down the spread of viral respiratory infections. 

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 40.79 ± 0.35 0.9% cps = copies; n = number of samples; Ct = cycle threshold; SD = standard deviation; CV = coefficient of variation J o u r n a l P r e -p r o o f 

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Package insert Alinity m SARS-CoV-2 AMP Kit

Package insert Alinity m Resp-4-Plex AMP Kit

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Instructions for use: LightMix® SarbecoV E-gene plus EAV control

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Validation and verification of the Abbott RealTime SARS-CoV-2 assay analytical and clinical performance

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Improved molecular laboratory productivity by consolidation of testing on the new random-access analyzer Alinity m