key: cord-0259521-vyajnog5 authors: Harankhedkar, S.; Chatterjee, G.; Rajpal, S.; Khan, A.; Srivastava, T.; Mirgh, S.; Gokarn, A.; Punatar, S.; Shetty, N.; Joshi, A.; Nair, S.; Murthy, V.; Khattry, N.; Tembhare, P.; More, A.; Kamtalwar, S.; Chavan, P.; Bhat, V.; Patil, A.; Dhumal, S.; Bhat, P.; Subramanian, P.; Tripathi, R.; Munipally, S.; Gujral, S.; Gupta, S.; Patkar, N. title: N Gene Target Failure (NGTF) for detection of Omicron: a way out for the "stealth" too? date: 2022-01-30 journal: nan DOI: 10.1101/2022.01.28.22269801 sha: 305958dc72459aa0f9d4c859c2c94a03a8f5162c doc_id: 259521 cord_uid: vyajnog5 S-gene target failure (SGTF) is neither specific nor accurate for identification of Omicron lineage of SARS-CoV-2. We observed N-gene target failure (NGTF) in 402 out of 412 SARS-CoV2 positive cases from December to mid-January 2022 using a commercially available assay. This phenomenon was not observed with more than 15,000 cases tested previously. We sequenced the genome of five samples with NGTF and compared these results with six cases where NGTF was not seen. We confirm that cases with NGTF were the Omicron lineage while cases with preserved N-gene amplification belonged to Delta lineage. We discovered that the ERS31-33 deletion (nucleotide 28362-28370del) overlaps with N gene probe used, explaining NGTF. As the 'stealth' Omicron variant also harbors ERS31-33 deletion, this approach will work for the detection of 'stealth' Omicron variant as well. We suggest that NGTF can be used as a low cost, rapid screening strategy for detection of Omicron. The ongoing COVID19 pandemic has witnessed the emergence of novel strains due to the accumulation of mutations in the SARS-CoV-2 genome [1] . Since December 2020, variants of concern (VOC) have been discovered, namely, Alpha (B. 1 [3] . As compared to the original Wuhan strain of SARS-CoV-2, the Omicron variant distinctively harbors a higher number of mutations and is highly transmissible resulting in a higher infectivity rate [4] [5] . This has resulted in a sharp spike in the number of cases all over the globe. This Omicron variant is characterized by more than thirty mutations (including single nucleotide variants as well as insertions and deletions) in the spike protein-coding regions which has raised concerns regarding higher transmissibility and efficacy of vaccines. Furthermore, recent evidence has also emerged that therapeutic monoclonal antibodies have a minimal response against the Omicron variant, presumably due to mutations in the spike protein-encoding regions [5] . This creates the possibility of a clinical need for diagnosis of Omicron variant and distinguishing this from other SARS-CoV-2 variants. Although the definitive evidence of strain evolution can be obtained by genome sequencing this technique is time-consuming and is available to only a fraction of the laboratories due to the high expertise required to perform this assay. Real-time polymerase chain reaction (RT-PCR) based testing remains the mainstay for relatively rapid testing and community diagnosis. In India (and presumably all over the world), there is a paucity of commercially available kits that can specifically detect the Omicron variant. It has been suggested that S-gene amplification failure on RT-PCR is a surrogate method to suspect Omicron. However, S-gene amplification failure has also been observed (when using the TaqPath COVID-19 Combo Kit, Thermo Fisher) . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted January 30, 2022. ; https://doi.org/10.1101/2022.01.28.22269801 doi: medRxiv preprint with the alpha variant of SARS-CoV-2 [6] . In this manuscript, we describe a commercially available RT-PCR assay that can be used as a rapid screening tool to detect the Omicron variant as a result of N-gene amplification failure. We demonstrate sequence level evidence that this approach detects the Omicron variant and reliably distinguishes it from the Delta variant of SARS-CoV-2. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted January 30, 2022. ; https://doi.org/10.1101/2022.01.28.22269801 doi: medRxiv preprint Nasopharyngeal and oropharyngeal swabs were received in either viral transport media (VTM) or molecular transport media (MTM) from the fever clinic at the discretion of the treating physician. The testing cohort included hospital staff as well as cancer patients. Real-time polymerase chain reaction (RT-PCR) was performed using a commercially available SARS- SAM files were processed further using Samtools (v.1.9) and variants were called using iVar (v.1.2) at a minimum alternative frequency of 0.01. Lastly, Nextclade web application was used to process the fasta sequences for identification of clade and mutation calling. The Phylogenetic Assignment of the named Global Outbreak LINeages (PANGOLIN) method of lineage classification was followed [8] . . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 30, 2022. ; https://doi.org/10.1101/2022.01.28.22269801 doi: medRxiv preprint We have performed over 15000 tests for detection of SARS-CoV-2 RNA, using a commercially available RT-PCR kit (Huwel Life Sciences, Hyderabad, India), which involves the detection of E (Envelope) and N (Nucleocapsid) genes of the virus (Figure 2) whereas all six cases with both N and E gene amplification belonged to Delta. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 30, 2022. ; https://doi.org/10.1101/2022.01.28.22269801 doi: medRxiv preprint The S gene target failure (SGTF) resulting from the HV69-70del is predominantly considered for identifying Omicron variant by most commercial kits [9] . However, mutations also affect other The second Omicron variant of the BA.2 lineage, termed 'stealth' Omicron, is genetically distinct from the BA.1 lineage and reported to escape detection on routinely used PCR kits as it does not harbour HV69-70del, therefore not resulting in S gene dropout [6] [11]. We did not encounter Omicron BA.2 lineage in our sequenced cases, however, we are confident that the NTGF by our RT-PCR method can be utilized to detect the BA.2 lineage as well, as the ERS31-33del in N gene is observed in both BA.1 and BA.2 lineages of Omicron variant [3] . We also emphasize that preservation of N gene or its dropout would depend on further viral genetic mutations and the probe design used for the gene in individual kits and that these findings concern the NTGF in the present kit used in the current clinical scenario only. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 30, 2022. We describe a readily available RT-PCR strategy for rapid diagnosis of Omicron variant based on N-gene amplification failure. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Zenodo. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 30, 2022. 11. https://github.com/cov-lineages/pango-designation/issues/361 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 30, 2022. ; https://doi.org/10.1101/2022.01.28.22269801 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted January 30, 2022. ; https://doi.org/10.1101/2022.01.28.22269801 doi: medRxiv preprint Features, evaluation, and treatment of coronavirus (COVID-19). StatPearls Omicron mutations enhance infectivity and reduce antibody neutralization of SARS-CoV-2 virus-like particles. medRxiv Omicron mutations enhance infectivity and reduce antibody neutralization of SARS-CoV-2 virus-like particles. medRxiv SARS-CoV-2 and variant diagnostic testing approaches in the United States Disentangling primer interactions improves SARS-CoV-2 genome sequencing by multiplex tiling PCR. PloS one CoV2 infection in Mumbai. Correlation of Viral Genomic Sequencing Data and Clinical Outcomes Silico Design of Specific Primer Sets for