Ninja Omicron:  BA.1 subvariant showing a BA.2-like pattern using a variant-specific PCR assay due to a single point mutation downstream the spike 69/70 deletion.

doi:10.21203/rs.3.rs-1730154/v1

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Case Report

Ninja Omicron: BA.1 subvariant showing a BA.2-like pattern using a variant-specific PCR assay due to a single point mutation downstream the spike 69/70 deletion.

https://doi.org/10.21203/rs.3.rs-1730154/v1

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Background:

SARS-CoV-2 variant tracking is key to the genomic surveillance of the pandemic. While next-generation sequencing (NGS) is commonly used for variant determination, it is expensive and time-consuming. Variant-specific PCR (vsPCR) is a faster, cheaper method that detects specific mutations that are considered variant-defining. These tests usually rely on specific amplification when a mutation is present or a specific melting temperature peak after amplification.

Case presentation:

A discrepant result between vsPCR and NGS was found in seventeen SARS-CoV-2 samples from Galicia, Spain. A cluster of BA.1 Omicron SARS-CoV-2 variant showed a BA.2-like melting temperature pattern due to a point mutation (C21772T) downstream the deletion of the spike aminoacids 69/70. As the 69/70 deletion is widely used for differentiation between BA.1 and BA.2 by vsPCR, C21772T can cause BA.1 samples to be misinterpreted as BA.2. Over a thousand BA.1 sequences in the EpiCoV database contain this mutation.

Conclusions:

To our knowledge, this is the first case of a point mutation causing a vsPCR algorithm to misclassify BA.1 samples as BA.2. This is an example of how mutations in the targets of vsPCR tests based on melting curve analysis can lead to variant misclassification. NGS confirmation of vsPCR results is relevant for the accuracy of the epidemiological surveillance. In order to overcome the possible impact of novel mutations, diagnostic tools must be constantly updated.

SARS-CoV-2

Omicron

Next Generation Sequencing

Variant-specific PCR

Surveillance

BA.1

BA.4

BA.5

During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, epidemiological surveillance teams have relied on Next-Generation-Sequencing (NGS) to track the spread of new variants and mutations. As a cheaper, faster alternative, commercial providers have developed assays in order to detect mutations that define a specific variant (variant-specific PCR, vsPCR) (1, 2). These methods rely on melting curve assays or multiplex quantitative RT-PCRs. Mutations commonly targeted in these assays include E484K (specific of VOCs Beta and Gamma), N501Y (specific of VOCs Alpha, Beta, Gamma and Omicron) or L452R (specific of VOCs Delta and Omicron BA.4/5). One noteworthy example is the case of the deletion of nucleotides 21765–21770, causing the deletion of spike aminoacids 69 and 70 (69/70del). First detected in Alpha variant, it causes a negative result in the S-gene of some RT-PCR detection kits (S-Gene Target Failure, SGTF). Detection of the 69/70del became relevant again as it appears in Omicron subvariant (PANGO lineage) BA.1 but not in BA.2, therefore being a perfect candidate for the determination of Omicron subvariant using a vsPCR assay (3).

Here, we report a case of false designation of a BA.1 SARS-CoV-2 strain as BA.2 due to a detection failure of the 69/70 deletion by one vsPCR assay. The objective was to establish the genomic basis of this finding and to know the potential implications for vsPCR-based SARS-CoV-2 surveillance.

In March 2022, 17 patients diagnosed with SARS CoV-2 infection at the Complexo Hospitalario Universitario de Vigo (Vigo, Galicia, Spain) showed discrepant SARS-CoV-2 variant results between an automated vsPCR assay and NGS. For the vsPCR assay, RNA extraction was performed with GXT 96 X3 Extraction kit v1.0 (Hain Lifescience GmbH, Nehren, Germany) on the GenoXtract ® fleXT instrument (Bruker) and PCR was performed with Fluorotype SARS-CoV-2 varID Q (Hain Lifescience GmbH, Nehren, Germany) on the FluoroCycler XT (Hain Lifescience) instrument according to manufacturer’s instructions. The mutation A67V (C21762T) upstream the 69/70del is usually present in BA.1 variants. In consequence, some tests like the Hain test used for these cases, updated their reagents to detect the specific combination of A67V + 69/70del (4, 5). A negative result in the A67V 69/70del-specific PCR was obtained and the presence of the BA.2 variant was determined by the interpretation algorithm.

For NGS, RNA extraction from the same samples was performed using QIASymphony DSP Virus Pathogen Midi kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. Whole-Genome Sequencing was performed using the COVIDSeq Assay Kit (Illumina Inc., San Diego, CA, USA). Some samples were sequenced by Miseq using MiSeq Reagents v3 (Illumina Inc.) and a Hamilton instrument for automated library preparation. The rest were sequenced on an iSeq 100 using iSeq 100 I1 Reagents (Illumina Inc.) after manual library preparation. Bioinformatic analysis was performed by a custom made pipeline and UShER genome was used for variant determination (Supplementary data 1). Upon NGS analysis, the samples were characterised as Omicron BA.1.1.14 with the synonymous mutation C21772T immediately downstream the 69/70del. We suggest that this mutation prevented the detection of the 69/70del. This deletion leads to the loss of the aminoacids histidine (H) and valine (V). Given that ATA, ATC and ATT all translate into isoleucine (I), mutation C21772T did not cause a substitution in the aminoacid sequence (Table 1).

Table 1

**Nucleotide changes associated with the 69/70 codon deletion of spike of SARS CoV-2 for BA.1 and BA.2 variants and for BA.1 variant with C21772T mutation**.
Variant	Nucleotide sequence (21761–21775) (codons 67–71)	Aminoacid sequence (spike 67–71)
Wuhan-Hu-1	GCTATACATGTCTCT	AIHVS
BA.1	GTTA‒‒‒‒‒‒TCTCT	VI‒‒S
BA.1 + C21772T	GTTA‒‒‒‒‒‒TTTCT	VI‒‒S
BA.2	GCTATACATGTCTCT	AIHVS
Mutation C21762T (characteristic of BA.1 subvariant) and mutation C21772T (described in this paper) are highlighted.

The discrepant samples belonged to a cluster of BA.1.1.14 with G9049A which accounts for 96% of the BA.1.1.14 samples in Spain (9). We identified 19 samples in the cluster presenting mutation C21772T, all of them detected in Galicia, Spain. Seventeen out of 19 samples were detected in our laboratory. A multiple alignment was performed to confirm that the samples were monophyletic (Supplementary figure 1). The multiple alignments and phylogenetic trees were performed and constructed on R (version 4.1.1, https://cran.r-project.org/) using the packages ggtree (6), ggplot (7), msa (8), and custom-made code.

Contact tracing showed that 10 sequences pertained to high school students, and 4 of them were epidemiologically related. All GISAID accession IDs for these 17 sequences are listed in Supplementary table 1. It is noteworthy that, upon alignment against the Wuhan-Hu-1 reference, some alignments misplace the 69/70del, and thus, the mutation is numbered as A21766T instead of C21772T (Supplementary table 2). That is the case of databases such as CoVSpectrum and Nextclade (9,10).

The 17 BA.1 + C21772T samples were re-tested using the Hain assay with the QIAsymphony nucleic acid extract, giving the same negative result and BA.2 interpretation (Figure 1, Supplementary table 3). To support this result, we tested the samples with a second vsPCR assay. A negative result with a shift in melting temperature was observed with VirSnip SARS-CoV-2 A67V 69/70del and LightCycler 1-step RT-polymerase (TibMolBiol, Berlin, Germany) on the cobas z480 instrument (Roche Diagnostics, Basel, Switzerland). (Figure 2, Supplementary table 4).

The spike deletion 69/70 has been used for Omicron detection since its emergence because it was not characteristic of the Delta variant circulating at that time. Afterwards, it was also useful to differentiate between BA.1 (69/70del positive) and BA.2 (69/70del negative) subvariants. Instead of relying on the negative result on S-gene to detect BA.1 (SGTF) (3), some commercial tests such as the ones used in this paper offer a specific RT-PCR kit to detect the A67V-69/70del. In the same manner 69/70del causes SGTF, new mutations can cause a failure in these PCR-based assays.

We report the appearance of a cluster of 17 samples of BA.1.1.14 identified as BA.2 by a commercial kit based on melting temperature curve analysis due to a point mutation two bases downstream from the deletion (C21772T). As of May 16th, there are 1315 sequences from BA.1 with the 69/70del and C21772T mutation, belonging to different BA.1 subvariants (9) suggesting that this mutation could spontaneously appear at different times.

If over a thousand BA.1 variants with the mutation C21772T have been detected since the appearance of the Omicron variant, it could be possible that there was an underestimation of the frequency of BA.1 in surveillance services that relied mainly on melting curve based vsPCR assays for variant tracking. In addition, while this paper is focused on commercial tests used for genomic surveillance of SARS-CoV-2, the same issue can arise for in-house methods developed for wastewater samples (11). This is, to the best of our knowledge, the first report of C21772T causing a negative result in 69/70del-targeted RT-PCR assays. It is highly likely that other mutations can cause similar effects in SARS-CoV-2 variant tracking in the future.

New challenges arise for variant tracking with PCR-based subvariant surveillance at the Omicron wave of SARS-CoV-2: as total cases increase, so do the chances of diagnostic PCR tests affected by a novel mutation such as the one previously reported (12) or the one we report today. In addition, the Omicron variant shows more than 30 non-synonymous mutations in the spike gene, causing changes in common diagnostic tests. For example, some pre-Omicron commercial melting curve assays targeting spike mutation N501Y give a negative result for all Omicron samples, likely due to the mutations surrounding aminoacid 501 (13, 14). As an added challenge, the recently discovered BA.4 and BA.5 Omicron subvariants show a specific mutational pattern not expected by the interpretation software, making variant determination with vsPCR increasingly complex. For example, mutation A67V makes possible the differentiation between BA.1 and BA.4/5. However, BA.4 and BA.5 are genetically identical in the area surrounding 69/70del, so other targets are needed for lineage determination.

Finally, recombinant SARS-CoV-2 variants such as PANGO lineage XD or XE, which combine AY.4/BA.1 and BA.1/BA.2 respectively, increase the complexity for taxonomic classification of the virus using methods based in the detection of combinations of single point mutations. As Omicron subvariants show a high number of spike mutations, and given all the complexities mentioned, we recommend commercial kit providers to increase their targets in order to obtain a more specific differentiation between Omicron clusters with vsPCR assays.

In the current phase of the pandemic, where testing numbers have been decreasing, the odds of missing relevant novel mutations is higher. It is therefore essential to keep adequate surveillance with the resources available. As mutations in the target area of vsPCR tests based on melting curve analysis can lead to variant misclassification, NGS confirmation of PCR results is relevant for the accuracy of the epidemiological surveillance. Update of diagnostic tools could help overcome mutations that can impact their result.

NGS

Next-Generation Sequencing

vsPCR

variant-specific PCR

SARS-CoV-2

Severe Acute Respiratory Syndrome CoronaVirus 2

VOCs

Variants of Concern

69/70del

spike gene aminoacid 69–70 deletion

SGTF

Spike Gene Target failure

Ethics approval and consent to participate:

No ethical approval was required for this study as samples were collected for routine surveillance.

Consent for publication:

Not applicable (see Ethics approval and consent to participate).

Availability of data and materials:

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

The genetic sequences from the samples analysed are available on GISAID and all ID accession numbers can be found in Supplementary table 1.

Competing interests:

The authors declare that they have no competing interests.

Funding:

This work received funding from the ECDC/HERA incubator grant 2021/PHF/23776 and Consellería de Sanidade, Galicia, Spain.

Author’s contributions:

Carlos Daviña-Núñez, Sonia Perez-Castro: Bioinformatic analysis, data interpretation, article writing

Lucía Martínez-Lamas: Hain Lifescience variant-specific PCR, data interpretation

Jorge Julio Cabrera-Alvargonzalez, Sonia Rey-Cao, Raquel Carballo-Fernandez: Data interpretation, article writing

Montse Godoy-Diz: VirSnip variant-specific PCR

Leticia López-Bóveda: Library preparation for Next-Generation Sequencing

Víctor del Campo-Pérez, Silvia Suárez-Luque: Contact tracing and epidemiological surveillance

Benito Regueiro-García: Study design and article writing

Acknowledgements:

We would like to acknowledge the staff from the Microbiology service of the Complexo Hospitalario Universitario de Vigo (CHUVI), for their contribution to epidemiological surveillance, their work and dedication.

Authors’ information:

Carlos Daviña-Núñez and Sonia Pérez-Castro contributed equally to this work.

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No competing interests reported.

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You are reading this latest preprint version

Ninja Omicron: BA.1 subvariant showing a BA.2-like pattern using a variant-specific PCR assay due to a single point mutation downstream the spike 69/70 deletion.

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