key: cord-0910991-pduf6rom
authors: De Luca, Caterina; Gragnano, Gianluca; Conticelli, Floriana; Cennamo, Michele; Pisapia, Pasquale; Terracciano, Daniela; Malapelle, Umberto; Montella, Emma; Triassi, Maria; Troncone, Giancarlo; Portella, Giuseppe
title: Evaluation of a fully closed real time PCR platform for the detection of SARS-CoV-2 in nasopharyngeal swabs: a pilot study
date: 2021-04-09
journal: J Clin Pathol
DOI: 10.1136/jclinpath-2021-207516
sha: 86c716aeb662980320dfd706880926df9c2931d5
doc_id: 910991
cord_uid: pduf6rom

AIMS: To date, reverse transcriptase PCR (RT-PCR) on nasopharyngeal swabs is the ‘gold standard’ approach for the diagnosis of COVID-19. The need to develop easy to use, rapid, robust and with minimal hands-on time approaches are warranted. In this setting, the Idylla SARS-CoV-2 Test may be a valuable option. The aim of our study is to evaluate the analytical and clinical performance of this assay on previously tested SARS-CoV-2 people by conventional RT-PCR based approach in different settings, including initial diagnosis and clinical follow-up. METHODS: To evaluate the sensitivity and specificity of the Idylla SARS-CoV-2 Test, we retrieved 55 nasopharyngeal swabs, previously analysed by a fully validated assay, from symptomatic patients or from people who have been in close contact with COVID-19 positive cases. Discordant or high discrepant cases were further analysed by a third technique. In addition, a second subset of 14 nasopharyngeal swab samples with uncertain results (cycle threshold between 37 and 40), by using the fully validated assay, from patients with viral infection beyond day 21, were retrieved. RESULTS: Overall, Idylla showed a sensitivity of 93.9% and a specificity of 100.0%. In addition, in the additional 14 nasopharyngeal swab samples, only five (35.7%) featured a positive result by the Idylla SARS-CoV-2 Test. CONCLUSIONS: We demonstrated that the Idylla SARS-CoV-2 Test may represent a valid, fast, highly sensitive and specific RT-PCR test for the identification of SARS-CoV-2 infection.

The COVID-19 caused by the SARS-CoV-2 was first identified in Wuhan (Hubei region, China) at the end of 2019. 1 2 The infection has rapidly spread all over the world, and the pandemic status was determined by the WHO on 11 March 2020. 3 At the time of writing (12 February 2021), more than 107 million people have been infected by SARS-CoV-2 with more than 2 million deaths, worldwide. 4 Overall, SARS-CoV-2 is a betacoronavirus, enveloped, positive-sense, single stranded RNA virus from the Coronaviridae family. 5 6 The viral genome, composed by about 30 000 nucleotides, contains genes encoding for nucleocapsid (N), envelop (E), membrane (M), internal (I) and spike (S) structural proteins and two open reading frame genes (ORF1a and ORF1b) encoding for 16 nonstructural proteins including the RNA-dependent RNA polymerase. 7 The 'gold standard' for the diagnosis of COVID-19 is reverse transcriptase PCR (RT-PCR) on nasopharyngeal swabs. 8 9 However, despite the high sensitivity and specificity, several limitations affected the routinely performance of this diagnostic tool. In particular, highly trained personnel is required. In addition, a complex infrastructure is mandatory to manage hundreds or thousands of daily testing while contemporary reducing personnel biological risks exposure. Finally, it is fundamental to mediate between the necessities to batch a minimum number of testing samples while giving the results as quickly as possible (within 24 hours). In order to overcome these limitations, several efforts have been spent to develop novel, easy to use, rapid, robust and with minimal hands-on time requirements diagnostic tools. 10 In this setting, the fully automated RT-PCR Idylla platform (Biocartis NV, Mechelen, Belgium) may be a valuable solution. This technology enables a rapid, robust, sensitive and specific approach useful to reduce the risk of sample cross-contamination and personnel exposure to high risk specimens. 11 As previously reported, the Idylla platform has been successfully adopted by our molecular predictive laboratory at the Department of Public Health of the University of Naples Federico II for predictive purposes in advanced stage solid tumour patients during the healthcare emergency. 12 Besides the predictive role, a novel cartridge (see the Materials and methods section) to assess the infectious SARS-CoV-2 status has been developed by Biocartis NV. 13 In the present study, our aim is to evaluate the analytical and clinical performance of this novel disposable cartridge on previously tested SARS-CoV-2 people by conventional RT-PCR based approach in different settings, including initial diagnosis and clinical follow-up.

This study was designed to evaluate the efficiency of Idylla SARS-CoV-2 Test to identify SARS-CoV-2 viral RNA. To this end, we have retrieved from the archive of the Clinical Pathology Laboratory at the Department of Translational Medical Sciences,

University of Naples Federico II, 55 nasopharyngeal swabs that had been collected and preserved in sterile viral medium Universal Transport Medium (UTM, Copan Diagnostic, Brescia, Italy) from symptomatic patients or from people who have been in close contact with COVID-19 positive cases. These latter, had previously been tested by a fully validated assay (Real-Time SARS-CoV-2 kit, #09N77-095, Abbott Laboratories, Chicago, Illinois, USA) which detect the RdRP and the N gene of the SARS-CoV-2, that had been Food and Drug Administration authorised for the emergency use, featuring a complete analysis run of 6 hours on average. To assess sensitivity and specificity of the Idylla SARS-CoV-2 Test against the Abbott Real-Time SARS-CoV-2 kit, samples, including positive (n=35) and negative (n=20) nasopharyngeal swabs, were retested by the Idylla SARS-CoV-2 Test. Cases showing discrepant results and lack of agreement between Idylla SARS-CoV-2 Test and Abbott Real-Time SARS-CoV-2 kit, were further analysed by a third technique (RealStar SARS-CoV-2 RT-PCR Kit, Altona Diagnostics GmbH, Hamburg, Germany). In addition, with the aim to evaluate the adoption of the Idylla SARS-CoV-2 Test to end the quarantine, a second subset of 14 nasopharyngeal swab samples with uncertain results (cycle threshold (Ct) between 37 and 40), by using Abbott Real-Time SARS-CoV-2 assay, from patients with viral infection beyond day 21, were retrieved. In all these instances, a third methodology (RealStar SARS-CoV-2 RT-PCR Kit) was also adopted.

At the Clinical Pathology Laboratory at the Department of Translational Medical Sciences, University of Naples Federico II, nasopharyngeal swabs are routinely analysed by using Abbott Real-Time SARS-CoV-2 kit, as described in the instructions of the manufacturer, on the Alinity platform. Briefly, 800 µL of UTM were required. Results were considered as positive when the Ct value on N and/or on ORF1b genes was equal or less than 37. 14

The Idylla SARS-CoV-2 Test is a fully automated RT-PCR system that adopts disposable cartridges. Briefly, 200 µL of UTM were directly pipetted into the SARS-CoV-2 Test cartridge. Via microfluidic channels, RNA was extracted after cell lysis performed by a combination of HIFU, enzymatic/chemical digestion, and heat. The extracted RNA was converted in complementary DNA (cDNA) by the RT enzyme and it was then transported into five PCR chambers for amplification. Here, there are dried primers and probes designed to detect two N and three ORFb1 target regions. This is a fluorescent-based assay allows to detect two SARS-CoV-2 RNA targets such as N gene (nucleocapsid phosphoprotein gene) and ORF1b region, they are covered by five PCR targets (two N and three ORF1b targets). In addition, each amplification chamber the amplification of MS2 Bacteriophage is adopted as an internal control, to monitor that RNA extraction and amplification steps have been correctly carried out into the cartridge.

After 90 min run, the final report is displayed on the Idylla console as 'positive', 'negative' or 'not valid'. As a general rule, a positive result requires at least two N amplified targets (with a Ct value ≤41.9) and/or at least one or more ORF1b amplified targets (in this case a Ct value cut-off is not required, due to the very high specificity of this gene amplification). Negative results indicate the absence of SARS-CoV-2 target amplification.

RealStar SARS-CoV-2 RT-PCR Kit is a RT-PCR based technology that enabling the qualitative detection of target regions in SARS-Cov-2 E and S genes, has been adopted following manufacturer instructions.

Overall, Idylla assay generated valid results in almost all analysed samples (96.4%, 53/55). Interestingly, among successfully analysed cases, a 96.2% concordance rate (51/53), between the Idylla SARS-CoV-2 Test and the Real-Time SARS-CoV-2 kit, has been obtained. Of note no false positive results have been reported by the Idylla SARS-CoV-2 Test (specificity 100.0%) (table 1) (online supplemental  table 3 ). Interestingly, in the vast majority of analysed samples (11/14, 78 .6%) the adoption of a third methodology (RealStar SARS-CoV-2 RT-PCR Kit) confirmed the Idylla SARS-CoV-2 Test results (online supplemental table 3) . Conversely, three positive Idylla SARS-CoV-2 Test cases were classified as 'uncertain' (n=2) or 'negative' (n=1) with the third methodology (online  supplemental table 3) .

Our study demonstrated that the Idylla Test is a sensitive, specific, easy to use and rapid assay for SARS-CoV-2 detection in nasopharyngeal swabs. In particular, in our experience the Idylla SARS-CoV-2 Test was able to reach a specificity of 100.0% and a sensitivity of 93.9% by comparing the results with a reliable reference technology. Only two cases showed discrepancies with Idylla assay being negative. These cases may represent true false negative cases although is not possible to rule-out technical issues related to the thawing of archival frozen nasopharyngeal swabs.

In the difficult time of COVID-19 pandemic, there is an urgent need of rapid and automated tests for the detection of SARS-CoV-2 infection. In fact, RT-PCR requires several dozen or even hundreds of samples to be grouped in batches to be tested in parallel, with a delay in results (6-24 hours). In this setting, it is key the employment of rapid assays for urgent cases, such as the characterisation of the COVID-19 status before an emergency surgery or in a vaginal delivery. The possibility to Original research quickly process selected cases represents an advantage in clinical practice that has led the Idylla technology to be widespread for mutational testing in oncological patients. Indeed, this automated molecular technology is often present in molecular laboratories dealing with oncological procedures which represents also an opportunity to apply rapid assay to select the patients with COVID-19.

To date, several tests and technologies have been developed for the diagnosis of SARS-CoV-2 infection. [15] [16] [17] [18] However, in particular to reduce hands-on time, potential exposure to SARS-CoV-2 infected samples and to reduce the risk of sample cross-contamination, it is mandatory to draw the attention to fully automated technologies. In this setting, the fully automated Idylla platform may represent a valid diagnostic tool. During the COVID-19 pandemic, a technological shift towards fully automated platform due to laboratory personnel reduction to cope the spread of virus has been reported. In a single and a multicentre experience coordinated by the Molecular Predictive Pathology Laboratory at the Department of Public Health of the University of Naples Federico II, fully automated platforms, such as Idylla, may significant simplify predictive molecular testing, while contemporary ensuring safety and cost-effectively management of laboratory staff. 12 19 Recently, the novel Idylla SARS-CoV-2 Test has been developed. 13 Overall, the Idylla system adopted a console computer that can be associated with up to eight separately operating instruments. Each separately operating instruments can process disposable cartridges able to perform viral RNA extraction, retrotranscription, amplification and data analysis in about 90 min. 13 Interestingly, this assay has recently obtained the CE-IVD mark with a lower limit of detection for viral genomic of 500 copies/mL. 13 In our experience, among 53 successfully analysed cases, only two 'false negative' results have been reported by the Idylla SARS-CoV-2 Test. Overall, these cases were further analysed by another RT-PCR assay (Altona RealStar SARS-CoV-2 RT-PCR Kit). 20 This latter was able to analyse target regions in E and S genes. Interestingly, with this RT-PCR approach a negative and undetermined results have been reported (online supplemental table 1 ). In addition, in five cases (#1, #16, #25, #30 and #31) with a high discrepancy between the standard technology and the Idylla SARS-CoV-2 Test in terms of Ct and Cq, respectively, the third methodology (Altona assay) confirmed a positive result (online supplemental  table 2 ). However, it should be borne in mind that these discrepancies may related to adoption a of a fluorescence threshold by using Abbott technology, whereas the Idylla SARS-CoV-2 calculates the Cq based on normalised amplification curves. In this study, we have selected only positive cases within a Ct equal or less than 37. Interestingly, it has been reported that positive amplification with a Ct value >37 in patients with a viral infection beyond day 21, may be not able to spread the virus. 21 To this end, in a selection of 14 nasopharingeal swabs belonging to patients with viral infection beyond day 21 and tested as uncertain by Abbott Real-Time SARS-CoV-2 kit, only 5 (35.7%) out of 14 nasopharyngeal swabs featured a positive result by the Idylla SARS-CoV-2 Test. These data may arise interest for the adoption of the Idylla SARS-CoV-2 Test to end the quarantine. 

Of note, in the vast majority of these samples (11/14, 78 .6%) the adoption of a third methodology (RealStar SARS-CoV-2 RT-PCR Kit) confirmed the Idylla SARS-CoV-2 Test results (online supplemental table 3) . Conversely, only three positive Idylla SARS-CoV-2 Test cases were classified as 'uncertain' (n=2) or 'negative' (n=1) with the third methodology (online  supplemental table 3) .

However, despite the encouraging results, several limitations affected this pilot study. In particular, the number of analysed samples may be extremely low to better assess the sensitivity and specificity of this novel assay. Thus, further investigation is required to assess whether the Idylla SARS-CoV-2 Test may be useful to further analyse cases with undetermined and unclear results (Ct between 37 and 40) that requires a rapid evaluation. In these cases, the Idylla SARS-CoV-2 Test may be considered a component of a diagnostic algorithm that exploits both conventional and automated RT-PCR platforms.

In conclusion, we demonstrated that the Idylla SARS-CoV-2 Test may represent a valid, fast, highly sensitive and specific RT-PCR test for the identification of SARS-CoV-2 infection. Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.

Competing interests UM has received personal fees (as consultant and/or speaker bureau) from Boehringer Ingelheim, Roche, MSD, Amgen, Thermo Fisher Scientifics, Diaceutics, GSK, Merck and AstraZeneca, unrelated to the current work. GT reports personal fees (as speaker bureau or advisor) from Roche, MSD, Pfizer and Bayer, unrelated to the current work.

Ethics approval All information regarding human material was managed using anonymous numerical codes, and all samples were handled in compliance with the Declaration of Helsinki (http://www. wma. net/ en/ 30publications/ 10policies/ b3/).

Provenance and peer review Not commissioned; externally peer reviewed.

Data availability statement All data relevant to the study are included in the article or uploaded as supplemental information.

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Pasquale Pisapia http:// orcid. org/ 0000-0002-6429-0620 Umberto Malapelle http:// orcid. org/ 0000-0003-3211-9957 Giancarlo Troncone http:// orcid. org/ 0000-0003-1630-5805