key: cord-0854483-bfkuj6z0
authors: Giovacchini, Nicla; Coppi, Marco; Aiezza, Noemi; Baccani, Ilaria; Malentacchi, Francesca; Pollini, Simona; Antonelli, Alberto; Rossolini, Gian Maria
title: Rapid screening for SARS-CoV-2 VOC Alpha (202012/01, B.1.1.7) using the Allplex™ SARS-CoV-2 / FluA / FluB / RSV Assay
date: 2021-10-07
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2021.10.005
sha: c48ea4a60d71a15fb6a5769e43462b4a372ef5e6
doc_id: 854483
cord_uid: bfkuj6z0

Background The emergence of SARS-CoV-2 variants of concern (VOCs) for increased transmissibility and potentially capable of immune-escape, mandates for epidemiological surveillance. Genomic alterations present in VOCs can affect the results of RT-qPCR assays for routine diagnostic purposes, leading to peculiar profiles that can be used for rapid screening of variants. In this work, we report on a peculiar profile observed with the Allplex™ SARS-CoV-2/ FluA/ FluB/ RSV Assay and VOC Alpha (202012/01, lineage B.1.1.7, also named VOC-UK), which was the first identified SARS-CoV-2 VOC. Methods Samples were analyzed by two RT-qPCR assays: the Allplex™ SARS-CoV-2/ FluA/ FluB/ RSV Assay (ASFR, Seegene Technologies Inc; Seoul, South Korea), and the TaqPath COVID-19 RT-PCR (Thermo Fisher Scientifics, USA). Definition of SARS-CoV-2 variant was carried out by Sanger sequencing of relevant S-gene regions and, in some cases, by whole genome sequencing (WGS) using the ARTIC-nCoV workflow on a MiniION (Oxford Nanopore Technologies, Oxford, UK) or a MiSeq ILLUMINA platform (San Diego, California, US). Results Of 173 SARS-CoV-2-positive specimens, all those of lineage B.1.1.7 (N=71) showed an average Cq difference between the N and S gene of +11±2 (range, + 8/+15). None of the other specimens, including several different lineages (Wild-type for the analyzed regions, N=22; Gamma, N=63; Delta, N=9; B.1.258, N=3; B.1.160, N=3; B.1.177.7, N=1; B.1.1.420, N=1), exhibited a similar difference of Cq values. Conclusions The peculiar pattern of delayed N gene positivity could constitute a convenient method for screening of VOC Alpha, simultaneous to viral detection, when using the Allplex™ SARS-CoV-2/ FluA/ FluB/ RSV Assay.

1 HIGHLIGHTS  Rapid detection of SARS-CoV-2 VOCs might be of epidemiological importance  Cq difference between N and S gene was found only with VOC Alpha using the ASFR Assay  This peculiar positivity profile can be a convenient proxy for tracking of VOC Alpha

The emergence of SARS-CoV-2 variants of concern (VOCs) for increased transmissibility and potentially capable of immune-escape, mandates for epidemiological surveillance. Genomic alterations present in VOCs can affect the results of RT-qPCR assays for routine diagnostic purposes, leading to peculiar profiles that can be used for rapid screening of variants. In this work, we report on a peculiar profile observed with the Allplex™ SARS-CoV-2/ FluA/ FluB/ RSV Assay and VOC Alpha (202012/01, lineage B.1.1.7, also named VOC-UK), which was the first identified SARS-CoV-2 VOC. The emergence and spreading of SARS-CoV-2 variants has mandated for their epidemiological surveillance. Although sequencing the entire viral genome represents the reference approach for variants identification, it is expensive and time-consuming and requires specialized laboratories.

Therefore, there is considerable interest for rapid molecular methods able to screen for VOCs, which could be helpful to rapidly suspect their presence by the routine diagnostic laboratory and timely implement tailored contact tracing and restriction measures. For instance, the TaqPath COVID-19 RT-PCR (Thermo Fisher Scientifics, USA), which fails amplification of the S gene target in case of the 69-70 deletion typical of VOC-Alpha, has successfully been used for rapid screening of this VOC based on such a peculiar amplification profile (Kidd et al., 2021) .

In this study, we report on the observation that VOC-UK could easily and rapidly be suspected by (Table 1) In addition to the ASFR assay, all specimens were also analyzed with the TaqPath COVID-19 RT-PCR, which has been used for rapid screening of VOC-UK (Kidd et al., 2021) .

The analysis of results obtained with specimens of the B.1.1.7 lineage always revealed a notable difference between the Cq values for the N and S genes (mean, +112; range, +8 / +15), while such a difference was not observed with specimens of the other lineages ( Table 1 ). The TaqPath COVID-19 RT-PCR assay revealed a missing S gene target amplification with all specimens of VOC-UK, but also with B.1.258 (Table 1) , as expected from the presence of the HV69-70 Spike deletion also in members of this lineage (Brejová et al., 2021) .

The delayed detection of the N-gene target (i.e. higher Cq values) with the ASFR assay might be due to the presence of mutations 28280 GAT->CTA and C28977T in the N nucleocapsid gene, likely affecting the oligonucleotide/probe region and leading to a lower RT-qPCR efficiency.

In conclusion, our data suggest that, finding a considerably higher (>8) N-gene Cq value vs. S-gene Cq value, could be highly suggestive for the presence of VOC-UK at the same time as viral detection.

The delayed N-gene target amplification by ASFR assay may constitute a more specific proxy for VOC-UK compared with the TaqPath test, which presumptively identifies VOC-UK based on a missing S-gene target amplification due to the presence of the HV69-70 deletion which is also present in other lineages, such as B.1.258Δ. In fact, members of the latter lineage did not present a delayed N-gene amplification with the ASFR assay (Table 1) .

Further experiments, including additional variants with alterations in the N gene sequence will be required to confirm the specificity of the ASFR "N-late" method for rapid screening of VOC-UK.

A SARS-CoV-2 mutant from B.1.258 lineage with ∆H69/∆V70 deletion in the Spike protein circulating in Central Europe in the fall 2020

European Centre for Disease Prevention and Control. Assessing SARS-CoV-2 circulation, variants of concern, non-pharmaceutical interventions and vaccine rollout in the EU/EEA, 15th update -10

Impact of B.1.1.7 variant mutations on antibody recognition of linear SARS-CoV-2 epitopes

S-variant SARS-CoV-2 lineage B1.1.7 is associated with significantly higher viral loads in samples tested by ThermoFisher TaqPath RT-qPCR

Early transmissibility assessment of the N501Y mutant strains of SARS-CoV-2 in the United Kingdom

Improvements to the ARTIC multiplex PCR method for SARS-CoV-2 genome sequencing using nanopore

Results were obtained from the analysis of anonymized data derived from diagnostic routine testing.