key: cord-0711361-uiahguvc authors: Sánchez-Calvo, Juan Manuel; Arboledas, Juan Carlos Alados; Vidal, Luis Ros; de Francisco, José Luis; Prieto, María Dolores López title: Diagnostic pre-screening method based on N-gene dropout or delay to increase feasibility of SARS-CoV-2 VOC B.1.1.7 detection date: 2021-07-16 journal: Diagn Microbiol Infect Dis DOI: 10.1016/j.diagmicrobio.2021.115491 sha: e06b98381437ce93af542f7b8a7b2945af2b1626 doc_id: 711361 cord_uid: uiahguvc OBJECTIVES: : To compare the RT-PCR Allplex SARS-CoV-2/FluA/FluB/RSV Assay (Allplex assay) with other methods of detection of VOC B.1.1.7. METHODS: : Suspected and non-suspected cases of VOC B.1.1.7 were defined according to the VirSNiP assay, which detects N501Y and deletion H69-V70. For pre-screening, the Allplex™ and TaqPath assays were used. RESULTS: : One hundred and sixteen suspected and 113 non-suspected cases were included. In the suspected cases, the Allplex assay showed N-gene dropout, or delayed Ct values of 6.27±1.21 and 6.66±1.41 compared with those of the RdRP and S-gene target, respectively. Agreement between the Allplex and TaqPath assays was 100% when the RdRP and S-gene targets had Ct values <35. Agreement between the Allplex and VirSNiP assays was 100% with Ct value <30. CONCLUSIONS: : The Allplex assay showed excellent agreement with the current pre-screening method for VOC B.1.1.7. In addition, its automated processing enhances the feasibility of widespread use in laboratories. In the current pandemic, the rapid spread of new variants of SARS-CoV-2 poses a significant public health challenge. Some of them, like the new alpha variant, also known as Variant of Concern (VOC) B.1.1.7, spread faster than others (Leung et al., 2021) . Some emerging variants have also proved to be more contagious and can reduce the efficacy of vaccines (Ortuso et al., 2021) . VOC B.1.1.7 emerged in England in September 2020 and is currently the dominant circulating SARS-CoV-2 variant (Public Health England, 2021) . This new variant has been detected in over 60 countries, including Spain (Centro de Coordinación de Alertas y Emergencias Sanitarias, 2021). In the United States, according to the Centers for Disease Control and Prevention's (CDC) national genomic surveillance program, the estimated biweekly prevalence of B.1.1.7, the dominant variant in most of the states from February 27 to March 13, 2021, was 27.2% (CDC and NCIRD, 2021) . This variant is defined by 23 mutations. The most unusual and concerning single mutation in this cluster is N501Y in the spike protein, which has been associated with increasing binding affinity to human ACE2. It is possible that this mutation, alone or in combination with deletion H69-V70 in the N-terminal domain of the spike protein, enhances the transmissibility of the virus (Public Health England, 2021) . The N501Y mutation has been detected in other emerging variants, such as 20H/501Y.V2 and B.1.351, detected mainly in South Africa, and P.1, which is now spreading across Brazil and has been found in 24 other countries (Tegally et al., 2020; Faria et al., 2021) . The H69-V70 deletion has also been detected in the cluster 5 variant, identified in both minks and humans in Denmark (Lassaunière et al., 2020) . The European Centre for Disease Prevention and Control (ECDC) recommends that laboratories should consider implementing pre-screening reverse transcription PCR (RT-PCR) approaches in samples where SARS-CoV-2 has been detected to detect S-gene dropout (deletion H69-V70), or a screening method to detect the N501Y mutation in variant viruses. These methods should not be a substitute for sequencing, which is necessary to confirm specific variants (ECDC, 2021) . The CDC suggests that S-gene target failure results can help identify potential B.1.1.7 cases, although these results should be confirmed by sequencing (Galloway et al., 2021) . In Spain, the Ministry of Health recommends the use of techniques based on S-gene target failure, such as the Applied Biosystems TaqPath RT-PCR COVID-19 kit, to screen for VOC B.1.1.7 (Ministerio de Sanidad, accessed 11/04/2021). This method has been previously validated and the sequencing results were fully concordant with the RT-PCR profiles for VOC B.1.1.7. In the cluster 5 variant, the H69-V70 deletion also results in an S-gene dropout (Bal et al., 2021) . These strategies have been validated to screen only for this VOC. In order to detect other circulating variants, performance of a RT-PCR approach such as mutation detection is necessary. Although there are some mutations in VOC B.1.1.7, detection of the N501Y mutation and H69-V70 deletion could be a good strategy to screen for this variant and serve as a proxy to identify the B.1.1.7 lineage (Goncalves et al., 2021) . Leung et al. The diagnostic gold standard of this variant is whole-genome sequencing (WGS), which is the currently recommended method to confirm these variants (ECDC, 2021; Galloway et al., 2021; Ministerio de Sanidad, 2021) . However, sequencing is generally expensive and takes longer than targeted detection methods. Consequently, sequencing capacity is currently limited in most EU/EEA Member States (ECDC, 2021). As a result, most laboratories use methods that require manual screening processes to detect this variant, which is timeconsuming and presents an increased risk of contamination. There is a need therefore for methods involving automated processing systems. The aim of this study was to compare an automated technique, the real-time RT-PCR Allplex SARS-CoV-2/FluA/FluB/RSV Assay (Allplex assay) with current methods of detection of VOC B.1.1.7. This assay was used in our laboratory during flu seasonal period.The assay is applied on Seegene´s integrated All-in-One automated platform which enables detect and differentiate eight target genes, including three different target genes of SARS-CoV-2, influenza A virus (Flu A), influenza B virus (Flu B), human respiratory syncytial virus A/B, with two Internal Controls (IC) (Endogenous IC and Exogenous IC). A cross-sectional study was performed to analyze the agreement between two prescreening approaches based on RT-PCR and a diagnostic screening method that can be used as a surrogate marker for detection of VOC B.1.1.7. To conduct the study for SARS-CoV-2 VOC B.1.1.7 detection, nasopharyngeal swab samples were collected from symptomatic and asymptomatic child and adult outpatients. 2.2 First pre-screening method for detecting VOC B.1.1.7 In the first step, infection was confirmed using the Allplex TM SARS-CoV-2/FluA/FluB/RSV Assay (Seegene, Seoul, South Korea), performed in a real-time thermal cycler (CFX96 Bio-Rad, California, United States (US)). This assay is a multiplex real-time PCR assay designed to detect the nucleocapsid (N), spike (S) and RNA-dependent RNA polymerase (RdRP) gene targets for SARS-CoV-2, influenza A, influenza B and respiratory syncytial virus (RSV) A/B in a single tube. The kit use an endogenous IC to validate correct sampling for health professional collected specimens, which is an RNase human, and an exogenous IC. Viral RNA extraction was performed with the viral DNA/RNA extraction cartridge kit STARMag (Seegene, Seoul, South Korea), using automated nucleic acid extraction instrument (STARlet system). Extraction was performed according to the manufacturer's instructions. Viral RNA was eluted with 100 µL buffer and used for RT-PCR assay. Briefly, 10 μL of extracted RNA was added to 5 μL of EM8 and 5 μL of SARS-CoV-2/FluA/FluB/RSV MuDT Oligo Mix (SC2FabR-MOM). The amplification condition of RT-PCR consisted of 1 cycle of 20 minutes at 50°C, 15 minutes at 95ºC, followed by 2 cycles of 10 seconds at 95ºC, 40 seconds at 60ºC and 20 seconds at 72ºC, and followed by 41 cycles of 10 seconds at 95ºC, 15 seconds at 60ºC and 10 seconds at 72ºC. The results were interpreted with an automatic data analyzer and laboratory informatics system was interlocking with Seegene Viewer. The FAM channel was used to detect S-gene and RSV, the HEX channel to detect RdRPgene and Flu B, the Cal Red 610 channel to detect N-gene and Flu A, and Quasar 670 channel to detect endogenous and exogenous IC. In the second step, all samples were re-analyzed in the QuantStudio 5 real-time PCR system using the Applied Biosystems TaqPath RT-PCR COVID-19 kit (Thermo Fisher Scientific, Waltham, U.S.), which includes the N-, S-and open reading frame (ORF1ab) gene targets. Mutation detection was performed on a Roche LightCycler 480 system (Roche Diagnostics, San Cugat, Spain), using the reagents VirSNiP SARS-CoV-2 spike N501Y and VirSNiP SARS-CoV-2 spike deletion H69-V70 (TIB MOLBIOL, Eresburgstrasse, Berlin, Germany). This method was selected as a surrogate method of detection of VOC B.1.1.7 and a proxy for the diagnostic gold standard. Suspected cases were patients with confirmed SARS-CoV-2 infection and detection of the N501Y mutation and spike deletion H69-V70. Non-suspected cases were positive samples that did not contain either N501Y or the H69-V70 deletion. To further confirm this variant, WGS was performed on a random subsample (n=5) of SARS-CoV-2, using the NextSeq 500 system (Illumina, San Diego, U.S.). WGS was performed in the sequencing reference laboratory of the Hospital Universitario Virgen del Rocio. The protocol is an adaption of several circulating protocols on SARS-CoV-2 sequencing using the ARTIC protocol and Illumina's Nextera DNA Flex library prep kit. Consensus genome sequence was obtained using the nf-core/viralrecon pipeline (https://nf-co.re/viralrecon). Clade assignment and annotated mutations are generated through the nextclade tool (https://clades.nextstrain.org/). Using the pangolin tool (https://cov-lineages.org/) lineages are assigned and for those genomes where a lineage cannot be obtained, an imputed lineage is assigned using the in-house impuSARS tool (https://www.biorxiv.org/content/10.1101/2021.04.13.439668v1). These samples were selected from the TaqPath kit and showed an N-gene cycle threshold (Ct) value <30. The WGS study should be performed on samples with RNA concentrations of 100-1000 ng. All samples were suspected cases of VOC B.1.1.7. The results were analyzed with SPSS version 20. Parametric tests (Student's t-test) were used for the analysis of Ct values. The results were shown as means ± standard deviation. The agreement analysis was assessed with Cohen's kappa coefficient (κ). P-values <0.05 were considered significant. Samples were collected during the third wave of COVID-19 in Spain. The studies were carried out in accordance with General Data Protection Regulation (Regulation (EU) 2016/679 and Directive 95/46/EC) and the organic data protection law of Spain 3/2018. No additional samples were collected for the purpose of this study. During February 2021, 229 nasopharyngeal swab samples were selected to identify suspected and non-suspected cases. Cases were preliminarily selected according to the amplification profile of the Allplex assay. The VirSNiP assay was used to define cases and identified 116 suspected cases of VOC B.1.1.7 and 113 non-suspected cases. To determine whether the Allplex assay was comparable to the TaqPath kit, we compared cases with delayed or N-gene dropout in the Allplex assay with S-gene target failure in the TaqPath kit. In suspected cases, the diagnostic pre-screening method performed with Allplex yielded delayed N-gene Ct values of 6.27±1.21 and 6.66±1.41 in comparison with those of the RdRP and S-gene targets, respectively. The N gene was not detected in 28.5% (n=33) of cases (Figure 1) . The pre-screening method with the TaqPath kit did not detect the S gene in 99.1% (115/116) of the cases. Among the non-suspected cases, the Allplex assay detected the Ct values of the N gene earlier than the RdRP (Ct value -1.00±1.37) and S-(Ct value -1.41±1.31) gene targets in 93.8% (n=106) of cases (Figure 1) . The N-gene target was not detected in 6.2% (n=7) of the cases. The Ct values of the 3 targets using the TaqPath kit were similar. The ORF1ab gene and S-gene target were not detected in 2.60% (n=3) and1.76% (n=2) of cases, respectively. With respect to the agreement analysis between the Allplex and TaqPath assays, the total agreement (TA) was 99.1%, regardless of the Ct values of the RdRP and S-gene targets. In addition, using a cutoff Ct value of <35 in the RdPR and S-gene targets, the TA was 100% (Table 1) . To analyze agreement between the Allplex and VirSNiP assays, we selected cases with Ct values >35 in the RdRP and S-gene targets and N-gene target failure with the Allplex assay Early detection of new SARS-CoV-2 variants is one of the main strategies recommended by the ECDC and CDC to control the pandemic (ECDC, 2021; Galloway et al., 2021) . Our results show the utility of a widely available pre-screening method for detection of the VOC B.1.1.7. In the laboratories based on TaqPath multiplex assay, the possible detection of B.1.1.7 is simultaneous with detection of SARS-CoV-2. This technology are only available in some laboratories. The microbiology laboratories which do not use this assay for detection of VOC B.1.1.7, require the re-analysis of all positive cases for other systems. The Allplex assay can be performed on automated robotic systems, since it integrates all sample processing tools in one compact hands-free unit, improving workflow efficiency and reducing the cost of reanalyzing all samples positive by the currently recommended systems. In Spain, the currently recommended system for the screening of VOC B.1.1.7 is the TaqPath kit (Ministerio de Sanidad, 2021) , which is also a very time-consuming manual technique with more complex processing. In our study, the Allplex assay showed very high agreement with this method. Thus, the Allplex assay can be used in places where there is a high prevalence of the VOC B.1.1.7, with little or no co-circulation of other variants that cause N-gene dropout or delayed Ct values. In this context, it could be used as a proxy measure of the incidence of this variant. Another advantage of the Allplex assay is that it can also be used to detect Flu A, Flu B, and RSV in respiratory samples from patients with signs and symptoms of respiratory infection. In our series, the two cases positive for N501Y that were considered non-suspected cases by the VirSNiP assay could be a new or emerging variant, such as B.1.351 or P.1 (Tegally et al., 2020; Faria et al., 2021) . In these samples, the RNA concentration was low (Ct value >35) so that failure to detect the H69-V70 deletion cannot be ruled out, although these cases could also be VOC B.1.1.7. The main reasons for failure to detect these genes using real-time RT-PCR systems are mutations, deletions or insertions in the gene targets of SARS-CoV-2. In the Allplex assay, mutations in the VOC B.1.1.7 variant in the S-and RdRP-gene targets did not affect the amplification curve pattern compared with the N gene. A study comparing amplification curve patterns of the envelope (E), N, and RdRP/S genes of the Allplex SARS-CoV-2 assay (Seegene) found peculiar, non-sigmoidal amplification curves in the RdRP/S genes that were associated with B.1.1.7 variant strains (Ibba et al., 2021) . Although the Allplex assay used the S-gene target, the Allplex SARS-CoV-2 assay used a combination of two gene targets: RdRP and the S gene. Mutations in the genomic region of these genes probably affected amplification efficiency. Nevertheless, these mutations did not affect the amplification efficiency of the S-gene target in the Allplex assay. The mutations most frequently detected in the N protein of VOC B.1.1.7 are D3L and S235F (Rambaut et al., 2020) . These mutations were detected in all the sequenced samples. Other mutations detected in all samples analyzed, such as M1X, R203K and G204R, could affect the amplification efficiency of the N-gene target. Although the N-gene target of the Seegene assay is proprietary, these mutations are the most probable cause of reduced efficiency in the detection of this gene. In other variants of concern, such as the B 1.351 and P.1 variants, the main mutations in the N protein are non-synonymous ones, such as T205I and P80R, respectively (Gómez et al., 2021) . These mutations were not detected in our samples. The main limitation of this study is that the circulation of other variants with other mutations in the N protein could also result in N-gene dropout or delay, leading to false positives. Two studies found that the C29200A mutation may be responsible for failure of amplification of the N gene by Cepheid's Xpert assay (Ziegler et al., 2020; Hasan et al., 2021) . We do not know whether other VOCs, such as B.1.351, P.1, B.1.427 and B.1.429, are able to affect the sensitivity and specificity of the N gene (Deng et al., 2021) . These results should be confirmed by WGS whenever possible. In summary, the Allplex assay is the first method that can be used to screen for VOC B.1.1.7 by N-gene dropout or delay. Though WGS should be performed to confirm this variant, the Allplex assay can be used as an excellent screening method when the RdRp and S-gene targets have a Ct value <30. Furthermore, its automated processing enhances the feasibility of widespread use in laboratories. Juan Manuel Sánchez: Conceptualization, methodology, formal analysis, data curation, writing original draft, editing and reviewing. Juan Carlos Alados: Supervision, conceptualization, editing and reviewing manuscript. Luis Ros: Analysis, resources, editing and reviewing manuscript. Jose Luis de Francisco: Analysis and resources. María Dolores López: Conceptualization, methodology, formal analysis, editing and review manuscript. All authors approved the final manuscript. All authors declare no conflicts of interest regarding this study. 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London, United Kingdom: Public Health England Preliminary genomic characterisation of an emergent SARS-CoV-2 lineage in the UK defined by a novel set of spike mutations. nCoV-2019 Genomic Epidemiology-Virological Emergence and rapid spread of a new severe acute respiratory syndromerelated coronavirus 2 (SARS-CoV-2) lineage with multiple spike mutations in South Africa SARS-CoV-2 samples may escape detection because of a single point mutation in the N gene We would like to thank all the technicians on the Covid team at the Hospital Universitario de Jerez who have worked very hard during this pandemic, and also Dr. Jose Antonio Cordoba and Dr. Javier Pascual who helped us to improve the design and clarity of this manuscript.