key: cord-0754026-ivuieskw
authors: Lefeuvre, Caroline; Pivert, Adeline; Przyrowski, Emilie; Bouthry, Elise; Darviot, Estelle; Mahieu, Rafaël; Lunel‐Fabiani, Françoise; Ducancelle, Alexandra; Le Guillou‐Guillemette, Hélène
title: Comparison of performance between three SARS‐CoV‐2 molecular assays (Aptima™, Laboratory Developed Test‐Fusion, and R‐GENE®) with special attention to turnaround time, a key point in laboratory management
date: 2022-03-07
journal: J Med Virol
DOI: 10.1002/jmv.27675
sha: c0fceab282e897dd8d8b4c115dc03fc9e67ff023
doc_id: 754026
cord_uid: ivuieskw

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) highlights the importance of rapid diagnostic testing to identify individuals with SARS‐CoV‐2 infections and to limit the spread of the virus. Many molecular assays have become commercially available to cope with this surging demand for timely diagnosis of COVID‐19 cases, but identifying individuals requires accurate diagnostic tools. We compared the performance of three molecular SARS‐CoV‐2 assays: Aptima™ SARS‐CoV‐2 assay running on the Panther system (Hologic), an in‐house assay (Laboratory Developed Test, LDT) running on the Fusion module of the Panther Fusion system (LDT‐Fusion; Hologic), and the R‐GENE® SARS‐CoV‐2 assay (bioMérieux). In addition, we also evaluated the turnaround time. This parameter is crucial to managing the SARS‐CoV‐2 diagnosis and represents a key point in the quality management at the laboratory. Aptima™ and LDT‐Fusion assays exhibited an excellent positive percent agreement (PPA) (100.0%), while the R‐GENE® assay showed a slightly decreased PPA (98.2%). The Hologic assays have a higher throughput with less hands‐on time than the R‐GENE® assays (24–25 vs. 71 min). Both Hologic assays are used on a fully automated random‐access testing system with on‐demand testing capabilities that avoid run series, unlike the R‐GENE® assay. Automated random‐access testing systems should be preferred during periods of high SARS‐CoV‐2 prevalence.

the detection of viral agents compared to conventional diagnostic assays. 3 Many molecular assays have commercially available to cope with this surging demand for timely diagnosis of COVID-19 cases.

At our institution, three molecular assays are used interchangeably to cope with the testing demand: the Aptima™ SARS-CoV-2 assay running on the Panther system (Hologic), an in-house assay (Laboratory Developed Test, LDT) running on the Fusion module of the Panther-Fusion system (herein referred as LDT-Fusion;

Hologic) and the R-GENE ® SARS-CoV-2 assay (bioMérieux).

We have had concerns before the integration of R-GENE ® and Aptima™ assays in our laboratory. Aptima™ is a transcriptionmediated amplification (TMA) assay and does not provide a semiquantitative result with a threshold amplification cycle (C t ) value. R-GENE ® assay requires a second real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) in case of equivocal results.

Aptima™ assay was recently evaluated and compared with other assays for the detection of SARS-CoV-2 in clinical respiratory specimens. [4] [5] [6] [7] One study mentioned that the Fusion Open Access protocol allows the use of LDT SARS-CoV-2 8 and one study compared it to Aptima™ assay. 5 Nevertheless, this is the first study comparing the R-GENE ® assay to any other assays for SARS-CoV-2 testing. In this study, we compared, therefore, the performance of three assays (two reverse transcriptase-polymerase chain reaction [RT-PCR] assays and one TMA assay) for the detection of SARS-CoV-2 in clinical specimens. In addition, we also evaluated the TAT which is crucial to managing the SARS-CoV-2 diagnosis.

We assayed 85 frozen specimens (stored for 2 months at −80°C after initial collection) from the 2020 winter season in parallel on three assays. Among 85 specimens, 76 nasopharyngeal swabs, 6 nasopharyngeal aspirates, and 3 bronchoalveolar lavages were assayed. The median age of the cohort was 62 years old. The gender distribution of the cohort was 33 women and 52 men. These samples came from patients who consulted at the University Hospital of Angers.

The Aptima™ assay targets two virus sequences located on the ORF1ab gene. A virus transport medium (VTM) (500 µl) was manually placed in the appropriate specimen lysis tube containing 710 µl of lysis buffer. On the Panther system, 360 µl of this mix was used for the lysis and capture of nucleic acids.

The LDT-Fusion assay corresponds to an in-house RT-PCR assay running on the Fusion module of the Panther-Fusion system using primers (nCoV_IP2 and nCoV_IP4) targeting two virus sequences on the RNA-dependent RNA polymerase (RdRP) gene and uses primer/probe sets from the National Reference Center of respiratory viruses (Institut Pasteur). 9 Samples were prepared as described for the Aptima™ assay.

The R-GENE ® assay includes two multiplexes rRT-PCR: first targeting nucleocapsid and RdRp genes and performed for the screening of SARS-COV-2; and subsequently, a second targeting Sarbecovirus E gene and performed for diagnostic confirmation in cases of equivocal results. Nucleic acid extraction from 200 µl of VTM was performed on the NucliSENS ® easyMAG ® system (bioMérieux) and amplification on the Mx3005P QPCR System (Agilent technologies). 

The study was carried out in accordance with the Declaration of Helsinki. This study was a noninterventional study, with no alterations of the usual sampling procedures. Biological material data were obtained only for standard viral diagnosis following physicians' prescriptions (no specific sampling, no modification of the sampling protocol). Data analyses were carried out using an anonymized database. 

In summary, the three assays provided comparable qualitative results and confirmed previous evaluations results for LDT-Fusion 5 and Aptima™ assays. 5- 7 We reported the results of a first performance evaluation for the R-GENE ® assay not previously evaluated and compared. The negative percent agreement ranged from 96.7% to 100.0%, suggesting that each assay has good specificity.

Due to the high testing demand occurring in the pandemic context, it is essential to choose assays with reduced TAT to meet health requirements legacy regarding the timeliness of results for physicians and to lead to a significant improvement of patients' care. 10 Rigorous TAT analysis taking into account the human and technical resources is a key point in laboratory management; it is important to estimate it when setting up a new assay and then it is often used as a quality indicator. 11 A limitation of the R-GENE ® assay is the manual setup of the multiwell plate (not automated) and the longer TAT for the nucleic acid extraction step. 12 The TAT of R-GENE ® could be even longer in some cases because 9/85 samples in our study required further investigation to confirm equivocal results.

Both Hologic assays are used on a fully automated randomaccess testing system with on-demand testing capabilities that avoid run series, unlike the R-GENE ® assay. Random access offers the capability to simultaneously load a significant number of samples on a single instrument at any time and specimens can be continuously analyzed. 10 Our data show that implementing the Panther/Fusion platform in our laboratory was associated with a great reduction in TAT that was largely due to the difference in the HOT when compared to the R-GENE ® . The time of automation is longer with Aptima™ than LDT-Fusion. However, the Aptima™ assay has the advantage of continuous loading of reagents and specimens during the process, which offers time-saving. One of the limitations of the Aptima™ assay is that it does not provide a semiquantitative assessment of the amount of virus. Thus, an RLU value around 

The authors would like to thank Chris Milne for proofreading the English manuscript.

Hélène Le Guillou-Guillemette has received honoraria from being a consultant or speaker from Hologic and research grants from Hologic 

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Lefeuvre http://orcid.org/0000-0003-4934-5926

Potential preanalytical and analytical vulnerabilities in the laboratory diagnosis of coronavirus disease 2019 (COVID-19)

In vitro diagnostic assays for COVID-19: recent advances and emerging trends

Molecular diagnosis of respiratory virus infections

High-throughput transcription-mediated amplification on the Hologic Panther is a highly sensitive method of detection for SARS-CoV-2

The workflow of each assay represented on the graph corresponds to the time for six specimens per run. The NucliSENS ® easyMAG ® system (the automated process of extraction) can extract 24 samples per run. To process the 120 samples, 5 extraction runs of 24 samples and 2 PCR runs must be considered, that is, a turnaround time of 16h13. LDT, Laboratory Developed Test; PCR, polymerase chain reaction 5. Tremeaux P, Lhomme S, Abravanel F, et al. Evaluation of the Aptima TM transcription-mediated amplification assay (Hologic ® ) for detecting SARS-CoV-2 in clinical specimens

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Rapid random access detection of the novel SARS-coronavirus-2 (SARS-CoV-2, previously 2019-nCoV) using an open access protocol for the Panther Fusion

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