Detection of Spike Gene Target Failure As a marker of Variant B 1.1.7 among SARS CoV-2 positive cases in No (1) DSGH

AbstractThe appearance of the SARS-CoV-2 lineage B.1.1.7 in the UK in late 2020, associated with faster transmission, sparked the need to find effective ways to monitor its spread. The set of mutations that characterise this lineage include a deletion in position 69 and 70 of the spike protein, which is known to be associated with Spike Gene Target Failure (SGTF) in a commonly used three gene diagnostic qPCR assay. The lower cost and faster turnaround times compared to whole genome sequencing make the use of qPCR for monitoring of the variant spread an attractive proposition. However, there are several potential issues with this approach. Here we use 826 SARS-CoV-2 samples collected in a hospital setting as part of the Hospital Onset COVID Infection (HOCI) study where qPCR was used for viral detection, followed by whole genome sequencing (WGS), to identify the factors to consider when using SGTF to infer lineage B.1.1.7 prevalence in a hospital setting, with potential implications for locations where this variant has recently been introduced.

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Discrepancy between PCR based SARS-CoV-2 tests suggests the need to re-evaluate diagnostic assays

BMC Research Notes ◽

10.1186/s13104-021-05722-5 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Muhammad Zain Mushtaq ◽

Sadia Shakoor ◽

Akbar Kanji ◽

Najma Shaheen ◽

Asghar Nasir ◽

...

Keyword(s):

Binding Sites ◽

Case Detection ◽

Gene Target ◽

Missed Opportunities ◽

Spike Gene ◽

Diagnostic Assays ◽

Additional Testing ◽

Pcr Assays

Abstract Objective We investigated the discrepancy between clinical and PCR-based diagnosis of COVID-19. We compared results of ten patients with mild to severe COVID-19. Respiratory samples from all cases were tested on the Roche SARS-CoV-2 (Cobas) assay, Filmarray RP2.1 (bioMereiux) and TaqPath™ COVID19 (Thermofisher) PCR assays. Results Laboratory records of ten patients with mild to severe COVID-19 were examined. Initially, respiratory samples from the patients were tested as negative on the SARS-CoV-2 Roche® assay. Further investigation using the BIOFIRE® Filmarray RP2.1 assay identified SARS-CoV-2 as the pathogen in all ten cases. To investigate possible discrepancies between PCR assays, additional testing was conducted using the TaqPath™ COVID19 PCR. Eight of ten samples were positive for SARS-CoV-2 on the TaqPath assay. Further, Spike gene target failures (SGTF) were identified in three of these eight cases. Discrepancy between the three PCR assays could be due to variation in PCR efficiencies of the amplification reactions or, variation at primer binding sites. Strains with SGTF indicate the presence of new SARS-CoV-2 variant strains. Regular modification of gene targets in diagnostic assays may be necessary to maintain robustness and accuracy of SARS-CoV-2 diagnostic assays to avoid reduced case detection, under-surveillance, and missed opportunities for control.

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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

Virus Genes ◽

10.1007/s11262-021-01866-5 ◽

2021 ◽

Cited By ~ 1

Author(s):

Broňa Brejová ◽

Kristína Boršová ◽

Viktória Hodorová ◽

Viktória Čabanová ◽

Lenka Reizigová ◽

...

Keyword(s):

Central Europe ◽

Drop Out ◽

Spike Protein ◽

Substantial Fraction ◽

Gene Target ◽

The Czech Republic ◽

Spike Gene ◽

Amino Terminal ◽

Terminal Domain ◽

Amino Terminal Domain

AbstractSARS-CoV-2 mutants carrying the ∆H69/∆V70 deletion in the amino-terminal domain of the Spike protein emerged independently in at least six lineages of the virus (namely, B.1.1.7, B.1.1.298, B.1.160, B.1.177, B.1.258, B.1.375). We analyzed SARS-CoV-2 samples collected from various regions of Slovakia between November and December 2020 that were presumed to contain B.1.1.7 variant due to drop-out of the Spike gene target in an RT-qPCR test caused by this deletion. Sequencing of these samples revealed that although in some cases the samples were indeed confirmed as B.1.1.7, a substantial fraction of samples contained another ∆H69/∆V70 carrying mutant belonging to the lineage B.1.258, which has been circulating in Central Europe since August 2020, long before the import of B.1.1.7. Phylogenetic analysis shows that the early sublineage of B.1.258 acquired the N439K substitution in the receptor-binding domain (RBD) of the Spike protein and, later on, also the deletion ∆H69/∆V70 in the Spike N-terminal domain (NTD). This variant was particularly common in several European countries including the Czech Republic and Slovakia but has been quickly replaced by B.1.1.7 early in 2021.

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Discrepancy between PCR based SARS-CoV-2 tests suggests the need to re-evaluate diagnostic assays

10.21203/rs.3.rs-551208/v1 ◽

2021 ◽

Author(s):

Zain Mushtaq ◽

Sadia Shakoor ◽

Akbar Kanji ◽

Najma Shaheen ◽

Asghar Nasir ◽

...

Keyword(s):

Binding Sites ◽

Rt Pcr ◽

Pcr Assay ◽

Gene Target ◽

Missed Opportunities ◽

Spike Gene ◽

Diagnostic Assays ◽

Additional Testing ◽

Pcr Assays ◽

Possible Cause

Abstract Objective: To investigate the possible cause of discrepancy between clinical and RT-PCR based diagnosis of patients with COVID-19.Results: Laboratory records of ten patients with mild to severe COVID-19 were examined. Initially, respiratory samples from the patients were tested using the Roche SARS-CoV-2 Cobas assay and were found to be negative. Further investigation for other viral causes of pneumonia was conducted using the Filmarray RP2.1 assay, which identified SARS-CoV-2 as the pathogen in all ten cases. To investigate whether this could be due to discrepancies between PCR assay, additional testing was conducted using the TaqPath TM COVID19 PCR. Eight of ten samples were positive for SARS-CoV-2 on the TaqPath assay. Further, Spike gene target failures (SGTF) were identified in three of these eight cases. Discrepancy between the three PCR assays could be due to variation in PCR efficiencies of the amplification reactions or, could be due to variation in the primer binding sites. The identification of strains with SGTF identify the possible presence of new SARS-CoV-2 variant strains. Regular modification of gene targets in diagnostic assays may be necessary to maintain robustness and accuracy of SARS-CoV-2 diagnostic assays to avoid reduced case detection, under-surveillance, and missed opportunities for control.

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Multiplex qPCR discriminates variants of concern to enhance global surveillance of SARS-CoV-2

PLoS Biology ◽

10.1371/journal.pbio.3001236 ◽

2021 ◽

Vol 19 (5) ◽

pp. e3001236

Author(s):

Chantal B. F. Vogels ◽

Mallery I. Breban ◽

Isabel M. Ott ◽

Tara Alpert ◽

Mary E. Petrone ◽

...

Keyword(s):

South Africa ◽

United Kingdom ◽

Severe Acute Respiratory Syndrome ◽

Immune Responses ◽

Primary Target ◽

Pcr Assay ◽

Gene Target ◽

Spike Gene ◽

The United Kingdom ◽

The World

With the emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants that may increase transmissibility and/or cause escape from immune responses, there is an urgent need for the targeted surveillance of circulating lineages. It was found that the B.1.1.7 (also 501Y.V1) variant, first detected in the United Kingdom, could be serendipitously detected by the Thermo Fisher TaqPath Coronavirus Disease 2019 (COVID-19) PCR assay because a key deletion in these viruses, spike Δ69–70, would cause a “spike gene target failure” (SGTF) result. However, a SGTF result is not definitive for B.1.1.7, and this assay cannot detect other variants of concern (VOC) that lack spike Δ69–70, such as B.1.351 (also 501Y.V2), detected in South Africa, and P.1 (also 501Y.V3), recently detected in Brazil. We identified a deletion in the ORF1a gene (ORF1a Δ3675–3677) in all 3 variants, which has not yet been widely detected in other SARS-CoV-2 lineages. Using ORF1a Δ3675–3677 as the primary target and spike Δ69–70 to differentiate, we designed and validated an open-source PCR assay to detect SARS-CoV-2 VOC. Our assay can be rapidly deployed in laboratories around the world to enhance surveillance for the local emergence and spread of B.1.1.7, B.1.351, and P.1.

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Monitoring the rise of the SARS-CoV-2 lineage B.1.1.7 in Tenerife (Spain) since mid-December 2020

10.1101/2021.03.14.21253535 ◽

2021 ◽

Author(s):

Julia Alcoba-Florez ◽

Jose M. Lorenzo-Salazar ◽

Helena Gil-Campesino ◽

Antonio Íñigo-Campos ◽

Diego García-Martínez de Artola ◽

...

Keyword(s):

Genome Sequencing ◽

Canary Islands ◽

Viral Genome ◽

Sharp Increase ◽

Nasopharyngeal Swab ◽

Gene Target ◽

Spike Gene ◽

Rapid Transmission ◽

The Uk ◽

Local Transmission

AbstractStarting in December 2020, a sharp increase of COVID-19 cases occurred in Tenerife compared to the rest of the Canary Islands (Spain). Because of the direct touristic connections between Tenerife and the UK, and the rapid transmission and dominance of the SARS-CoV-2 B.1.1.7 variant of concern (VOC-202012/01) by the end of November 2020 in South England, here we measured the proportion of B.1.1.7 cases occurring between the 18th of December 2020 and the 25th of February 2021. Out of the 2,091 COVID-19 positive nasopharyngeal swab samples assessed, 226 showed a spike gene target failure (SGTF). Subsequent viral genome sequencing further confirmed that 93.2% of them corresponded to the B.1.1.7 lineage. Furthermore, a rapid increase in the proportion of SGTF variants was detected in up to 10.7% of positive cases during the Christmas season despite stricter measures for containing the transmission were imposed in Tenerife in the period. These results support the local transmission of SARS-CoV-2 B.1.1.7 lineage in Tenerife since late December 2020 although it is not yet dominant.

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PCR assay to enhance global surveillance for SARS-CoV-2 variants of concern

10.1101/2021.01.28.21250486 ◽

2021 ◽

Author(s):

Chantal B.F. Vogels ◽

Mallery Breban ◽

Tara Alpert ◽

Mary E. Petrone ◽

Anne E. Watkins ◽

...

Keyword(s):

South Africa ◽

Immune Responses ◽

Open Source ◽

Primary Target ◽

Pcr Assay ◽

Gene Target ◽

Spike Gene ◽

The World ◽

The Uk

AbstractWith the emergence of SARS-CoV-2 variants that may increase transmissibility and/or cause escape from immune responses1–3, there is an urgent need for the targeted surveillance of circulating lineages. It was found that the B.1.1.7 (also 501Y.V1) variant first detected in the UK4,5 could be serendipitously detected by the ThermoFisher TaqPath COVID-19 PCR assay because a key deletion in these viruses, spike Δ69-70, would cause a “spike gene target failure” (SGTF) result. However, a SGTF result is not definitive for B.1.1.7, and this assay cannot detect other variants of concern that lack spike Δ69-70, such as B.1.351 (also 501Y.V2) detected in South Africa6 and P.1 (also 501Y.V3) recently detected in Brazil7. We identified a deletion in the ORF1a gene (ORF1a Δ3675-3677) in all three variants, which has not yet been widely detected in other SARS-CoV-2 lineages. Using ORF1a Δ3675-3677 as the primary target and spike Δ69-70 to differentiate, we designed and validated an open source PCR assay to detect SARS-CoV-2 variants of concern8. Our assay can be rapidly deployed in laboratories around the world to enhance surveillance for the local emergence spread of B.1.1.7, B.1.351, and P.1.

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Tracking SARS-CoV-2 lineage B.1.1.7 dissemination: insights from nationwide spike gene target failure (SGTF) and spike gene late detection (SGTL) data, Portugal, week 49 2020 to week 3 2021

Eurosurveillance ◽

10.2807/1560-7917.es.2021.26.10.2100130 ◽

2021 ◽

Vol 26 (10) ◽

Author(s):

Vítor Borges ◽

Carlos Sousa ◽

Luís Menezes ◽

António Maia Gonçalves ◽

Miguel Picão ◽

...

Keyword(s):

Confidence Interval ◽

Age Distribution ◽

Rt Pcr ◽

Gene Target ◽

Spike Gene ◽

Viral Loads ◽

Late Detection

We show that the SARS-CoV-2 B.1.1.7 lineage is highly disseminated in Portugal, with the odds of B.1.1.7 proportion increasing at an estimated 89% (95% confidence interval: 83–95%) per week until week 3 2021. RT-PCR spike gene target late detection (SGTL) can constitute a useful surrogate to track B.1.1.7 spread, besides the spike gene target failure (SGTF) proxy. SGTL/SGTF samples were associated with statistically significant higher viral loads, but not with substantial shift in age distribution compared to non-SGTF/SGTL cases.

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Increased hazard of death in community-tested cases of SARS-CoV-2 Variant of Concern 202012/01

10.1101/2021.02.01.21250959 ◽

2021 ◽

Cited By ~ 4

Author(s):

Nicholas G. Davies ◽

Christopher I. Jarvis ◽

W. John Edmunds ◽

Nicholas P. Jewell ◽

Karla Diaz-Ordaz ◽

...

Keyword(s):

Absolute Risk ◽

Care Home ◽

Pcr Amplification ◽

Deprivation Level ◽

Severe Illness ◽

Gene Target ◽

Spike Gene ◽

Risk Of Death ◽

The United Kingdom ◽

Novel Variant

VOC 202012/01, a SARS-CoV-2 variant first detected in the United Kingdom in September 2020, has spread to multiple countries worldwide. Several studies have established that this novel variant is more transmissible than preexisting variants, but have not identified whether it leads to any change in disease severity. We analyse a large database of SARS-CoV-2 community test results and COVID-19 deaths, representing 52% of all SARS-CoV-2 community tests in England from 1 September 2020 to 5 February 2021. This subset of SARS-CoV-2 tests can identify VOC 202012/01 because mutations in this lineage prevent PCR amplification of the spike gene target (S gene target failure, SGTF). We estimate that the hazard of death among SGTF cases is 58% (95% CI 40–79%) higher than among non-SGTF cases after adjustment for age, sex, ethnicity, deprivation level, care home residence, local authority of residence and test date. This corresponds to the absolute risk of death for a male aged 55–69 increasing from 0.6% to 0.9% (95% CI 0.8–1.0%) over the 28 days following a positive test in the community. Correcting for misclassification of SGTF and missingness in SGTF status, we estimate a 71% (48–97%) higher hazard of death associated with VOC 202012/01. Our analysis suggests that VOC 202012/01 is not only more transmissible than preexisting SARS-CoV-2 variants but may also cause more severe illness.

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Proliferation of SARS-CoV-2 B.1.1.7 Variant in Pakistan-A Short Surveillance Account

Frontiers in Public Health ◽

10.3389/fpubh.2021.683378 ◽

2021 ◽

Vol 9 ◽

Author(s):

Massab Umair ◽

Muhammad Salman ◽

Zaira Rehman ◽

Nazish Badar ◽

Qasim Ali ◽

...

Keyword(s):

Large Scale ◽

Low Frequency ◽

Control Measures ◽

Initial Screening ◽

Rt Pcr ◽

Gene Target ◽

Pakistani Population ◽

Spike Gene ◽

Thermofisher Scientific ◽

The United Kingdom

The emergence of a more transmissible variant of SARS-CoV-2 (B1. 1.7) in the United Kingdom (UK) during late 2020 has raised major public health concerns. Several mutations have been reported in the genome of the B.1.1.7 variant including the N501Y and 69-70deletion in the Spike region that has implications on virus transmissibility and diagnostics. Although the B.1.1.7 variant has been reported by several countries, only three cases have been reported in Pakistan through whole-genome sequencing. Therefore, the objective of the study was to investigate the circulation of B.1.1.7 variant of concern (VOC) in Pakistani population. We used a two-step strategy for the detection of B.1.1.7 with initial screening through TaqPathTM COVID-19 CE-IVD RT-PCR kit (ThermoFisher Scientific, Waltham, US) followed by partial spike (S) gene sequencing of a subset of samples having the spike gene target failure (SGTF). From January 01, 2021, to February 21, 2021, a total of 2,650 samples were tested for SARS-CoV-2 and 70.4% (n = 1,867) showed amplification of all the 3 genes (ORF, N, and S). Notably, 29.6% (n=783) samples have been SGTF that represented numbers from all the four provinces and suggest a rather low frequency during the first 3 weeks of January (n = 10, n = 13, and n = 1, respectively). However, the numbers have started to increase in the last week of January, 2021. During February, 726 (93%) cases of SGTF were reported with a peak (n = 345) found during the 3rd week. Based on the partial sequencing of SGTF samples 93.5% (n = 29/31) showed the characteristic N501Y, A570D, P681H, and T716I mutations found in the B.1.1.7 variant. In conclusion, our findings showed an upsurge of B.1.1.7 cases in Pakistan during February, 2021 affecting 15 districts and warranting large scale genomic surveillance, strengthening of laboratory network and implementation of appropriate control measures in the country.

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