key: cord-0837302-c14arapr
authors: Aupaix, Antoine; Lazarova, Elena; Chemais, Monia
title: A brief performance evaluation and literature review of Abbott ID Now COVID-19 rapid molecular-based test
date: 2021-09-20
journal: J Virol Methods
DOI: 10.1016/j.jviromet.2021.114293
sha: 6322c1845ec78dfce108713e7d34bb4485f349fc
doc_id: 837302
cord_uid: c14arapr

The qualitative ID Now COVID-19 assay combines claimed performance and ease of use that seem to position it as a reliable test for urgent patient management. However, the declared limit of detection (LOD) of 125 genome equivalents/mL is not confirmed by the published studies, which observed a range of LOD varying from 276 to 20.000 copies/mL. We decided to establish the LOD value on more robust basis using serial dilutions of a SARS-CoV-2 culture supernatant sample of defined concentration. Afterwards, we tested the analytical performances of the assay with 23 QCMD external quality control measurements. Hence, taking into consideration the additional dilution in the sample receiver cup, we found a lower 95% LOD of 64 copies/mL. For its intended use and with the new established LOD, ID Now COVID-19 proved to be a suitable test for the diagnosis of COVID-19 in contagious patients, as proposed by the latest Belgian recommendations.

In late 2019, an outbreak of pneumonia cases with flu-like symptoms of unknown origin began in Wuhan, China. Soon, several studies confirmed a human to human transmission and showed that some cases of this new respiratory disease could cause acute respiratory distress syndrome (ARDS), multiple organ failure (MOF) and death. [1, 2, 3] Early January 2020, a new virus was identified by genome sequencing [4] as a betacoronavirus family-related virus, named "Severe Acute Respiratory Syndrome Coronavirus 2" (SARS-CoV-2) by the International Committee on Taxonomy of Viruses. Rapidly, the new virus spread all J o u r n a l P r e -p r o o f over the world causing over 85.000 confirmed cases and nearly 3.000 deaths by the end of February 2020. [5] On March 11, WHO officially declared this new disease, called Coronavirus 2019 (COVID- 19) , as a global pandemic.

SARS-CoV-2 is the seventh coronavirus with human-to-human transmission (especially through small droplets from upper respiratory tract). It can cause a wide panel of symptoms, from mild forms (cough, temperature, flu-like symptoms) to severe ones, particularly in elderly subjects. The molecular characteristics of SARS-CoV-2 explaining its wide and rapid spread and the existence of healthy carriers and pre-symptomatic contagiousness cause a challenge for diagnostic strategy. Therefore, reliable and easy performing tests are needed.

The first validated test was Reverse Transcription-Polymerase Chain Reaction (RT-PCR) using nasal, nasopharyngeal and oropharyngeal swabs. [6] Various countries developed their own assays based on National Reference Center RT-PCRs. [7] Nowadays, RT-PCRs targeting multiple genes, such as nucleocapsid protein, RNA-polymerase and envelop protein genes, remain the most reliable diagnostic test for COVID-19 [8] and various platforms and commercial assays are largely used.

In our laboratory, we use the following diagnostic strategy: antigen-based tests for outpatients with symptoms (confirmed with RT-PCR in case of negative result), Cepheid Xpert Xpress SARS-CoV2 RT-PCR (on Cepheid GeneXpert, Cepheid, Sunnyvale, California, US) for patients from emergency department before hospitalization, and finally, we send non-urgent sample to a bigger laboratory which uses several RT-PCR platforms. Like most labs, we are regularly confronted with a limited supply of Cepheid reagents. To meet the increasing demand for diagnostic tests and to offset shortages and delays in delivery of reagents, we needed a rapid and reliable molecular biology test in addition to our diagnostic strategy.

We here evaluate the Abbott ID Now COVID-19 assay, recently available in Belgium only since January 2021, which uses nicking enzyme amplification reaction (NEAR) technology, targeting RdRp gene. This test was claimed with a LOD of 125 genome equivalents/mL. In fact, the objective of our study was to assess this expected performance. We used a reference viral material from the Belgian National Reference Center for Respiratory Pathogens (Katholieke Universiteit Leuven, Brussels, Belgium) and the external controls from all three challenges of 2020 and the first 2021 challenge of Quality Control for Molecular Diagnostics (QCMD, Glasgow, Scotland) assessment programs [9] . We also reviewed the published literature on the subject. Finally, we interpret our results in the light of the recent recommendations of the Belgian Scientific Public Health Institute.

The standards used in this study have two different origins:

(a) A sample of heat-inactivated SARS-CoV-2 supernatant, with a known viral load of 1.1

x 10 9 copies/mL, supplied by the Belgian National Reference Center for Respiratory Pathogens. We carried out serial dilutions in isotonic solution of sodium chloride so that we could dispose of concentrations between 1.1 x 10 2 and 1.1 x 10 8 copies/mL. 

This assay is a rapid molecular test based on the nicking enzyme amplification reaction (NEAR) technology, allowing to give a qualitative result (positive, negative, uninterpretable) for the detection of the RNA-dependent RNA polymerase (RdRp) gene segment of SARS-CoV-2 in 13 minutes reaction time. It also includes an internal control. After an isothermal nucleic acid amplification, this assay specifically identifies the amplified RNA targets with fluorescently labeled beacons. All testing was performed according to the manufacturer's instructions except the type of sample used. Indeed, the use of direct nasopharyngeal swabs is recommended by the test procedure without mentioning the possible use of liquid transport medium testing. However, based on the protocol of published studies [10] we transferred 200 µL of each viral medium directly to the sample receiver cup in order to perform the assay. Therefore, instead of depositing a swab in the sample receiver cup, that contains already 2.5 ml of elution buffer, we deposited 200 µl of viral medium in that 2.5 ml volume of elution buffer, thus adding into the procedure of this assay an additional 13.5-fold-dilution.

This is a molecular test based on real-time reverse transcription PCR (RT-PCR) amplification technology and allowing to give a semi-quantitative result for the combined detection of the nucleocapsid gene (N2) and the envelope gene (E) of SARS-CoV-2. The sample is deposited into single-use cartridges containing all reagents and controls, and housing the extraction and the RT-PCR process once the cartridge is placed in the GeneXpert instrument system. All testing was performed according to the manufacturer's instructions, i.e. direct analysis of 300 µl of viral transport media without additional dilution step in the reaction cup.

Serial dilutions of the reference material and all 23 QCMD samples were analyzed with Abbott ID Now COVID-19 assay and with Cepheid Xpert Xpress SARS-CoV-2 assay.

In order to assess the analytical sensitivity, serial dilutions were analyzed, starting from a concentration of 1.1 x 10 8 copies/ml up to 110 copies/ml. Near the published limits of detection, starting from 22,000 copies/mL, 8 replicates of each dilution were tested. When we reached the LOD, we ran tests in triplicates for the subsequent dilutions. All tests were performed by the same operator in a standardized manner in which each sample was vortexed 40 seconds prior to analysis. The 95% LOD was then determined using Probit regression, as recommended by CLSI guidelines (document EP17-A2) with MedCalc Software Version 20.009.

The results allowing analytical sensitivity assessment are presented in Table 1 . Additional to the declared viral load, the actual measured viral load, corrected with the 13.5-fold dilution in the reaction cup of ID Now, is presented for each dilution. 

The results of the QCMD quality controls are presented in Table 2 . It is to be noted that contrary to Cepheid Xpert Xpress SARS-CoV-2 assay which directly measures the declared viral load, ID Now COVID-19 assay presents always a dilution when liquid samples are used.

Consequently, we also present here the calculated concentration of SARS-CoV-2 viral load inside the ID Now receiver cup considering this additional 13.5-fold dilution. Seventeen QCMD samples were positive for SARS-CoV-2 with declared RNA concentration varying from 100 to 200.000 copies/mL. ID Now correctly detected 12 out of 17 resulting with a PPA of 70.6%. All 6 negative samples were negative on ID Now (NPA of 100%). Four of these samples contained other coronaviruses (coronavirus 229E, coronavirus NL63 and coronavirus OC43). The overall agreement was calculated as 78.3%. All positive and negative QCMD samples were correctly assessed by Cepheid Xpert Xpress SARS-CoV-2 assay.

Several studies have already compared ID Now COVID-19 assay with a reference method (RT-PCR) showing PPA value from 48 to 94% [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] and a LOD widely ranging from 262 copies/mL (Lephart et al., 2020) to 20,000 copies/mL and even higher [23] , data presented in Table 3 and   in Table 4 . Literature data on test comparisons, number of specimens included and ID Now performances are presented in Table 3 . Summary of published data of the analytical sensitivity of the assay are presented in Table 4 . These were obtained with serial dilutions of positive samples or external quality controls and are expressed in copies/mL when available. In both tables, an additional column informs if the dilution in the sample receiver cup was considered or not for ID Now analytical performances and sensitivity establishment. 

The qualitative ID Now COVID-19 test became available in Belgium at the same time as the recommendations of the Belgian authorities demanding to each laboratory to calibrate its RT-PCRs in order to provide and report results interpretation in terms of viral load [24] . It was therefore essential to know the precise LOD of the ID Now assay before integrating it into our diagnostic strategy. However, we were intrigued by the fact that ID Now was declared by the manufacturer with a LOD of 125 genome equivalents/mL which is even lower than the LOD of RT-PCR, e.g. the declared LOD for Cepheid Xpert Xpress SARS-CoV-2 is of 250 copies/mL. Moreover, the literature shows LOD values inconsistent with the manufacturer declared LOD, and in variable proportions ranging from two times up to 300 times greater than the claimed value. An additional question for us was to know what challenge we were facing with respect to the national recommendations by introducing a qualitative test probably less sensitive for the detection of SARS-CoV-2 in respiratory specimens than RT-PCR. Therefore, we decided to define ourselves the analytical sensitivity of ID Now COVID-19 using reference viral materials, CLSI guidelines for 95% LOD determination, and considering the 13.5-fold additional dilution in the reagent cup. Furthermore, our 95% LOD was challenged with precisely quantified external controls from QCMD assessment programs.

Our study found a 95% LOD of 64 copies/ml, which is even lower than the manufacturer product insert (Figure 1 and Table 1 ). This surprising finding was further sustained by the results of QCMD external controls (Table 2) These findings further confirm that comparing results of different quantitative PCR platforms should always consider an eventual pre-analytical dilution which is namely the case of ID Now COVID-19. Unfortunately, many published studies did not take into consideration this systematic pre-analytical bias, which partially explains the heterogeneity in the analytical performance evaluations (Table 3 and Table 4 ). Moreover, regarding the literature specifically on the analytical sensitivity, the 13.5-fold dilution factor was not considered in any of the study protocols using liquid samples, except by the one by Lephart et al. (Table 4 ). In that study, the determination of the LOD of ID Now COVID-19 assay implied different dilution factors in order to reach within the sample receiver cup the concentrations to which the other PCR platforms were subjected (Table 4 ). The LOD value determined by Lephart et al. is comparable to the one declared by the manufacturer and to the one determined in this study, despite the differences within the study protocols and the definition of LOD. Indeed, both studies considered, although in different ways, the existence of this dilution factor in the sample receiver cup when defining the analytical sensitivity of ID Now COVID-19 assay. However, if the 13.5-fold dilution factor is omitted, 95% LOD would be determined as 859 copies/ml instead of 64 copies/ml (Figure 1a and Figure 1b) , which is much closer to the published data.

Regarding QCMD external controls' expected results and compared to Cepheid Xpert Xpress assay, our determination of PPA of ID Now COVID-19 of 70.6% is comparable to the one of the various published studies (Table 3 ). In order to further assess the clinical significance of the discordant results, we followed the recommendations of the Belgian Risk Assessment Group (Sciensano, Scientific Public Health Institute, Brussel, Belgium) [24] which stated that samples J o u r n a l P r e -p r o o f can be considered "very strongly positive" when they present viral load higher or equal to 10 7 copies/mL, "strongly positive" higher or equal to 10 5 copies/mL and "positive" higher or equal to 10³ copies/mL. Below the threshold of 10³ copies/mL, samples are reported as "weakly positive" and patients may be considered no longer infectious if the clinical and serologic evidence supports an old infection or the absence of infection. Therefore, in a second analysis, we divided the positive samples into two groups: higher than 10³ copies/mL receiver cup viral load and lower. All 3 highly charged samples were correctly identified. A total of 14 positive QCMD samples presented a receiver cup viral load below 10³ copies/mL and 9 out of them were correctly detected by ID Now COVID-19 assay. These findings further underline the ample analytical performances of ID Now.

All of these data were sufficiently satisfactory in order to introduce the ID Now platform in our COVID-19 screening strategy, alongside with the GeneXpert platform. As acknowledged, ID Now being a qualitative test, does not allow evaluation of the viral load of samples positive for SARS-CoV-2, as recommended by the Belgian Public Health Institute (ref). However, the analytical sensitivity calculated in our study demonstrates that this assay can detect all ranges of viral loads reported by the 4 categories, including the one with the lowest concentrations (<10 3 RNA copies /ml).

In conclusion, we here defined the analytical sensitivity of ID Now COVID-19 assay using reference viral materials, CLSI guidelines for 95% LOD determination, and considering the additional dilution in the sample receiver. Surprisingly, ID Now COVID-19 assay presents molecular grade performance characteristics comparable to more complex and timeconsuming RT-PCR assays. Its analytical performances, combined with the very short 13 minutes reactional time and the friendly device-guided handling procedure, constitute an J o u r n a l P r e -p r o o f additional advantage of ID Now COVID-19 for setting up a rapid diagnosis within the clinical laboratories or in relocated forms of laboratories.

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.

The authors have nothing to disclose and there was no conflict of interest. J o u r n a l P r e -p r o o f Abbreviations: Ct, cycle threshold. a If not specified, viral load corresponds to RNA concentration for SARS-CoV-2. Values were initially given in log10 copies/mL. We converted those in copies/mL and rounded to the nearest whole number. When a control contained another coronavirus, we specified the viral load between parenthesis after the virus scientific name. 

A Novel Coronavirus from Patients with Pneumonia in China

Characteristics of and Important Lessons From the Coronavirus Disease 2019 (COVID-19) Outbreak in China: Summary of a Report of 72 314 Cases From the Chinese Center for Disease Control and Prevention

Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study

Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding

COVID-19) Dashboard. n

COVID-19)

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

International external quality assessment for SARS-CoV-2 molecular detection and survey on clinical laboratory preparedness during the COVID-19 pandemic

Clinical Evaluation of Three Sample-to-Answer Platforms for Detection of SARS-CoV-2

Comparison of Two Commercial Molecular Tests and a Laboratory-Developed Modification of the CDC 2019-nCoV Reverse Transcriptase PCR Assay for the Detection of SARS-CoV-2

Evaluation of the COVID19 ID NOW EUA assay

Performance of Abbott ID Now COVID-19 Rapid Nucleic Acid Amplification Test Using Nasopharyngeal Swabs Transported in Viral Transport Media and Dry Nasal Swabs in a New York City Academic Institution

Comparison of Cepheid Xpert Xpress and Abbott ID Now to Roche cobas for the Rapid Detection of SARS-CoV-2

Comparison of Abbott ID Now, DiaSorin Simplexa, and CDC FDA Emergency Use Authorization Methods for the Detection of SARS-CoV-2 from Nasopharyngeal and Nasal Swabs from Individuals Diagnosed with COVID-19

Commercial Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Molecular Assays: Superior Analytical Sensitivity of cobas SARS-CoV-2 Relative to NxTAG CoV Extended Panel and ID NOW COVID-19 Test

Comparison of Abbott ID Now and Abbott m2000 Methods for the Detection of SARS-CoV-2 from Nasopharyngeal and Nasal Swabs from Symptomatic Patients

How many are we missing with ID NOW COVID-19 assay using direct nasopharyngeal swabs? Findings from a mid-sized academic hospital clinical microbiology laboratory

Comparative study of four SARS-CoV-2 Nucleic Acid Amplification Test (NAAT) platforms demonstrates that ID NOW performance is impaired substantially by patient and specimen type

Clinical Evaluation and Utilization of Multiple Molecular In Vitro Diagnostic Assays for the Detection of SARS-CoV-2

Evaluation rapide multicentrique du test moléculaire rapide ID-NOW pour détection de l'ARN du SARS-CoV2, technologie de LAMP

Comparison of abbott ID NOW COVID-19 rapid molecular assay to cepheid xpert xpress SARS-CoV-2 assay in dry nasal swabs

Résultats de l'évaluation de la performance analytique pour la détection du SARS-CoV-2 dans le cadre de l'épidémie de COVID-19 comparaison avec la technique de référence du CNR IPP

Abbreviations: NP, nasopharyngeal; VTM, viral transport medium

PPA, positive percent agreement; NPA, negative percent agreement

Emergency Use Authorization; PPV, positive predictive value

FDA, Food and Drug Administration

Paired samples means that several specimen types were taken for each patient

In the studies cited, for samples in VTM, 200µL of liquid sample were added to the 2.5mL of elution buffer. We give here the information whether the authors used dry swabs for ID Now assay instead of swabs in VTM/UTM, in order to avoid a 13.5-fold additional dilution. a ID NOW

f Simplexa COVID-19 Direct Kit (Diasorin Molecular LLC

h Panther Fusion SARS-CoV-2

i RT-qPCR on SuperScript™ III Platinum® One-Step Quantitative RT-PCR System

Abbreviations: VTM, viral transport medium; BGM cells; Buffalo Green Monkey cells; NP, nasopharyngeal; UVT, Universal Viral Transport. 1 The results from this study were given in cycle threshold (respectively Ct30.1 for supernatant and Ct29 for the clinical sample) aligned with those of the Pasteur Institute [25] . We converted those results in copies/mL using data source from the reference method, the RT-PCR on SuperScript™ III Platinum® One-Step Quantitative RT-PCR System, with Kit Extraction NucleoSpin Dx Virus performed in Pasteur National Reference Center, Paris, France. a Zeptometrix Corporation, Buffalo, New York. b Exact Diagnostic, Fort Worth, Texas. c ThermoFisher, Waltham, Massachusetts. d Simplexa COVID-19 Direct Kit (Diasorin Molecular LLC, Cypress, California). e cobas SARS-CoV-2 Test (Roche Molecular Systems, Inc, Pleasanton, California).