key: cord-321287-mh2j2koa
authors: Trémeaux, Pauline; Lhomme, Sébastien; Abravanel, Florence; Raymond, Stéphanie; Mengelle, Catherine; Mansuy, Jean-Michel; Izopet, Jacques
title: Evaluation of the Aptima™ Transcription-Mediated Amplification assay (Hologic®) for detecting SARS-CoV-2 in clinical specimens
date: 2020-07-06
journal: J Clin Virol
DOI: 10.1016/j.jcv.2020.104541
sha: 
doc_id: 321287
cord_uid: mh2j2koa

BACKGROUND: The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which appeared in late 2019, has been limited by isolating infected individuals. However, identifying such individuals requires accurate diagnostic tools. OBJECTIVE: This study evaluates the capacity of the Aptima™ Transcription-Mediated Amplification (TMA) assay (Hologic® Panther System) to detect the virus in clinical samples. STUDY DESIGN: We compared the Aptima™ assay to two in-house real-time RT-PCR techniques, one running on the Panther Fusion™ module and the other on the MagNA Pure 96 and Light-Cycler 480 instruments. We included a total of 200 respiratory specimens: 100 tested prospectively and 100 retrospectively (25 -ve/75 +ve). RESULTS: The final Cohen’s kappa coefficients were: κ = 0.978 between the Aptima™ and Panther Fusion™ assays, κ = 0.945 between the Aptima™ and MagNA/LC480 assays and κ = 0.956 between the MagNA/LC480 and Panther Fusion™ assays. CONCLUSION: These findings indicate that the Aptima™ SARS-CoV-2 TMA assay data agree well with those obtained with our routine methods and that this assay can be used to diagnose coronavirus disease 2019 (COVID-19).

This study evaluates the performance of the automated commercial Aptima™ SARS-CoV-2 assay (Hologic® Panther™ System) on clinical samples. This test, which uses transcription-mediated amplification (TMA) to detect SARS-CoV-2, was compared to two in-house reverse transcriptasepolymerase chain reaction (RT-PCR) assays we presently use to diagnose COVID-19. 

The Aptima™ SARS-CoV-2 assay was performed following the manufacturer's instructions. Virus transport medium (500 µL) was manually placed in the Panther™ tube containing 710 µL of lysis buffer.

The instrument used 360 µL of this mix for the lysis and capture of nucleic acids. The Aptima™ assay targets two virus sequences located on the ORF1ab gene. An internal control was included.

Our two in-house assays that both target virus sequences on the SARS-CoV-2 RNA-dependent RNApolymerase (RdRp) gene, use primers and probes (IP-2 and IP-4) and amplification programs from the Institut Pasteur, Paris (4). The first assay uses the MagNA Pure 96 instrument (Roche Diagnostics) to extract nucleic acids from 200 µL of virus transport medium eluted in 100 µL. RT-PCR is then J o u r n a l P r e -p r o o f performed on the Light-Cycler 480 instrument (LC480) (Roche Diagnostics) using the AccuStart™ Taq DNA polymerase (QuantaBio) and 2 µL of nucleic acids (total reaction volume: 10 µL). The second assay uses the same primers and probes on the Panther Fusion™ module (Hologic®) in open access. Samples were prepared as described above for the Aptima™ assay.

Because there is no reference standard method for the detection of SARS-CoV-2, we calculated the concordance between assays by using Cohen's kappa coefficient (κ), the positive percent agreement, the negative percent agreement and overall percent agreement, all with 95% confidence intervals (6, 7).

The prospective study provided 2 positive and 98 negative samples, and there was perfect concordance between results of the Aptima™ assay and those of the Panther Fusion™ assay and very good concordance between the Aptima™ assay and the MagNA/LC480 assay. One sample was faintly positive with the MagNA/LC480 assay (virus IP-4 target, Ct = 39.97 and IP-2 -ve) and negative with the other two assays (sample A2 0141 0134, cf. Table S1 in Supplementary materials).

All positive samples with Ct values < 35 (n = 50) with the Panther Fusion™ assay tested positive with the other two assays. The MagNA/LC480 assay detected only one virus target (IP-4) in 6 of these samples (initial Ct: 32-35) (cf. Table S2 in Supplementary materials).

The 25 (Ct > 35) positive samples detected by the Panther Fusion™ assay included 20 (80%) samples that were positive with The Aptima™ assay and 17 (68%) that were positive with the MagNA/LC480 J o u r n a l P r e -p r o o f assay. These latter included 13 (65%) in which only one virus target (IP-4) was detected. Five (20%) samples were negative with the Aptima™ assay and eight (32%) with the MagNA/LC480 assay, four of these were negative with both assays. These nine samples were tested again with the Panther Fusion™ assay since sample storage can result in the partial degradation of virus RNA, and thus to reduced assay sensitivity. Four were negative and five were positive or inconclusive (three with only IP-2, one with only IP-4 and one with both targets) (cf. Table S2 ). These discrepancies could also be due to selection bias -samples were chosen based on the Panther Fusion™ assay results.

All the Panther Fusion™ assay negative samples were also negative with the Aptima™ assay, except for one invalid sample. One sample was positive with the MagNA/LC480 assay, but displayed atypical PCR curves. A second RT-PCR on the same nucleic acid extract was negative (sample A2 0139 3655, cf. Table S2 ).

The invalid rate, all results included, with the Aptima™ assay was 0.5% (1/200 The Aptima™ SARS-CoV-2 TMA assay was given emergency use authorization by the United States Food and Drug Administration (8) and the CE mark for in vitro diagnostic use in Europe.

with those obtained with our in-house assays. It includes an internal control to detect inhibition of amplification. The Panther™ instrument also provides random access, which allows urgent samples prioritization to obtain results within 3.5 hours with the Aptima™ assay, and can deliver up to 60 results per hour. This can help laboratories provide rapid, high-throughput diagnosis during the COVID-19 pandemic crisis.

None declared.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

[4] World Heanlth Organisation. 24 January 2020 2020. Molecular assays to diagnose COVID-19: Summary  table  of  available  protocols. https://www.who.int/docs/defaultsource/coronaviruse/whoinhouseassays.pdf?sfvrsn=de3a76aa_2&download=true. Accessed 10 February 2020.

[5] Tang YW, Schmitz JE, Persing DH, Stratton CW. 2020. Laboratory Diagnosis of COVID-19: Current Issues and Challenges. J Clin Microbiol 58. Emergency Use Authorization Information -In Vitro Diagnostic Products https://www.fda.gov/emergencypreparedness-and-response/mcm-legal-regulatory-and-policy-framework/emergency-use-authorization covidinvitrodev. Accessed 05 June 2020. Ct: cycle threshold, RLU: relative light unit J o u r n a l P r e -p r o o f

China Novel Coronavirus I, Research T. 2020. A Novel Coronavirus from Patients with Pneumonia in China

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