key: cord-0880935-ddvmbknt authors: Pinkhover, N. P.; Sanchez, E.; Pontbriand, K. M.; Okello, K.; Garvey, L. M.; Fletcher, K. P.; Pum, A.; Li, K.; DeOliveira, G.; Proctor, T. B.; Feenstra, J. D. M.; Sorel, O.; Gandhi, M.; Auclair, J. title: SARS-CoV-2 Variant Surveillance Using Tandem Targeted RT-PCR-based Genotyping Assays and Whole Genome Sequencing date: 2022-02-01 journal: nan DOI: 10.1101/2022.01.31.22270226 sha: 793a063525669d21c35372615ccb9ac59bbf0f55 doc_id: 880935 cord_uid: ddvmbknt Genomic surveillance is critical for tracking SARS-CoV-2 Variants of Concern (VOC) and for rapid detection of emerging variants. Whole genome sequencing (WGS) is the predominant method for genomic surveillance; but it is a laborious process for large-scale testing. The aim of this study was to assess the performance of a PCR-based mutation panel for the discrimination of 5 known VOC; Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2) and Omicron (B.1.1.529). Genotyping analysis was performed on 128 SARS-CoV-2 positive samples collected at the Life Science Testing Center at Northeastern University from April-December 2021. RNA extraction was performed using MagMax Viral/Pathogen II Nucleic Acid Isolation Kit. SARS-CoV-2 detection was confirmed using the TaqPath COVID-19 Combo Kit. Variant determination was conducted using a panel of TaqMan SARS-CoV-2 single nucleotide polymorphism (SNP) assays. On November 25, 2021, the emerging VOC (omicron) was reported by South Africa and the panel was quickly modified to detect omicron by substituting P681H and K417N assays. Based on the SNP panel analysis, variant identification in 128 samples were as follows: Alpha (N=34), Beta (N=1), Gamma (N=7), Delta (N=41) and Omicron (N=21). The genotyping panel accurately assigned lineages to all samples, confirmed by Ion Torrent GeneStudio S5 WGS. VOC discrimination using RT-PCR genotyping is a rapid, versatile method for detecting known and emerging SARS-CoV-2 variants. The versatility of SNP panels allows monitoring of emerging strains by simple layout adaptations. RT-PCR genotyping assays can expedite variant identification, enable high-throughput variant surveillance, and support WGS prioritization for detection of new variants. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19) was discovered in Wuhan, China in December of 2019. From poor disease monitoring programs to inconsistent public health precautions, numerous factors impacted the evolution of the COVID-19 pandemic. Vaccination coverage, non-pharmaceutical interventions and surveillance testing strategies are critical for mitigating the impact of COVID-19 but as these protective measures vary across countries SARS-CoV-2 continues to spread worldwide. CoV-2 cases in the United States. 3 Sequence analysis of VOC revealed the S-gene as a site bearing numerous mutations, of which some are shared between different VOC. [3] [4] [5] [6] 8 This finding highlights the important need of a genotyping approach for tracking mutations of concern (MOC) expressed in SARS-CoV-2 variants. Indeed, disease monitoring programs should exhibit high sensitivity, quick turn-aroundtimes and workflows should remain largely unaffected by varying positivity rates. Here, we describe an economic, convenient, and multimodal method for SARS-CoV-2 genomic surveillance using targeted RT-PCR-based genotyping assays ( Table 1) . The Life Science Testing Center (LSTC) at Northeastern University (NU) is a CAP and CLIA certified clinical laboratory established in August 2020 for routine COVID-19 testing for NU's student, faculty, and staff. As of January 22, 2022, 1,411,171 samples have been analyzed using RT-PCR of which, 7328 samples have been tested positive, mirroring the Greater Boston positivity rates and trends 9 ( Figure 1A ). We developed and implemented a RT-PCR-based genotyping approach to conduct extensive VOC screening. We analyzed N=128 confirmed SARS-CoV-2 positive samples collected between April and December 2021 thus covering the period when Alpha, Beta, Gamma, Delta, and Omicron VOC were circulating. 3, 8, 10 In addition, vaccine breakthrough cases (N=226) were tested between July and December 2021 using this targeted RT-PCRbased genotyping approach. [11] [12] [13] [14] [15] In this study we evaluated the analytical performance and the economic impact of genotyping approaches compared to whole genome sequencing (WGS) for large-scale screening of SARS-CoV-2 VOC. 5, 6, 8 A total of 128 SARS-CoV-2 positive samples that were initially collected between April and December 2021, were tested using our targeted RT-PCR-based genotyping assay. This RT-PCR approach included either, an 8-assay (N=44 samples tested) or a 12-assay (N=84 samples tested) MOC panel (Table 1 ) to detect SARS-CoV-2 VOC and VUM. Using the interpretation criteria described in Table 1 Figure 1B ). Overall, 24 SARS-CoV-2 positive samples were not assigned to any VOC lineage, however, it should be noted that 39 samples of the 128 tested were pooled-swab surveillance samples. 8 of the 24 samples with undetermined VOC status were pooled samples, which infringes on the intended application of the mutation panel (see SI). The Alpha VOC was most pronounced in April-May 2021 while the Delta variant was detected over the entire screening period with a peak in cases in August ( Figure 1B ). In April 2021, we detected a spike in non-VOC positive samples which could be attributed to unstable viral recombination prior to Delta's stabilization ( Figure 1B) . 10, 12, 13 Finally, the omicron variant became the most frequently detected VOC in December 2021 ( Figure 1B ). 3 No significant difference in mean Ct values for any of the three gene targets reported by the TaqPath TM COVID-19 Combo Kit used for SARS-CoV-2 detection were observed between the 5 VOC (see SI). 14 In addition, we monitored VOC trends in vaccine breakthrough COVID-19 cases from July-December 2021, where breakthrough cases (N=226) in fully vaccinated individuals were recorded, respectively ( Figure 1D ). The higher frequency of breakthrough cases coincided first with the broad wave of Delta cases (N=151) from July to December 2021 10,12-15 , followed by a rapid spike of Omicron cases (N=75) December 01 to December 27, 2021, at Northeastern University, as identified by the MOC panel genotyping approach ( Figure 1D) . A side-by-side cost analysis was performed by comparing the mutation panel genotyping approach for VOC surveillance to the WGS, with respect to cost of instrumentation, consumables, staff, computing capacity and data storage and other variables ( Figure 1C ). [4] [5] [6] 8 The average cost per sample for the genotyping approach using a 12-assay mutation panel was calculated to be over 8 times lower than WGS, without factoring in the longer turn-around-time for obtaining the WGS results when compared to RT-PCR. 5, 6, 8 Surveillance of SARS-CoV-2 variants is essential for timely implementation of public health measures aimed at limiting the viral spread and effects of the ongoing COVID-19 pandemic. 3,11-15 While WGS has proven to be an invaluable tool for identification of new variants, the cost as well as the turnaround times for sample processing, data analysis remain challenging for wide implementation. [4] [5] [6] 8 In this study we demonstrated that genotyping assays offer a fast, reliable, and cost-effective method for monitoring of known SARS-CoV-2 variants within a population. Our results showed that our mutation panels were able to accurately assign lineages to VOC as confirmed with WGS. Although genotyping assays can only assign samples to lineages that are already defined in the database, a key feature of genotyping assays is that they can be rapidly updated as new VOC exhibiting new combinations of mutations arise 4-6 , which can be powerful given the rapid emergence and spread of previous VOC. 3, [10] [11] [12] 14 This study showed that when comparing surveillance technical costs, the 12-Assay Mutation panels afforded 98.88% VOC determination at >8 times cheaper than WGS testing per sample, and costs can be further reduced by targeting selected VOC. Importantly, this targeted RT-PCR-based genotyping approach enables high-throughput variant surveillance and can be easily implemented in a routine testing laboratory including in developing regions with limited access to WGS. With minimal automation, this genotyping approach generated VOC data for 96 samples with a turnaround of ~3.5 hours whereas WGS required 5-7 days to confirm VOC data on 96 samples. Thus, expanding the use of a RT-PCR-based mutation panel approach would complement WGS for tracking emergence and surveillance of VOC. WGS could be prioritized to target samples that have not been assigned to a VOC lineage using mutations panels or that are consistently inconclusive in a routine SARS-CoV-2 RT-PCR test. The use of mutation panels can further be valuable to measure the impact of VOC on parameters such as vaccine effectiveness in preventing SARS-CoV-2 infections. Our data showed a strong association between the emergence of both Delta and Omicron variants and an increase in the number of breakthrough infections during the same periods. Rapid tracking of variants with immune escape or higher transmissibility is key as COVID-19 might transition into an endemic state. Taken together these results showed that VOC genotyping using mutations panels is a compelling and versatile approach to monitor variant prevalence in real-time and that should be leveraged to adjust public health strategy. This study included anterior nasal swabs specimens collected between April and December 2021 at NU. Viral RNA extraction and purification was performed using the MagMax™ Viral/Pathogen II Nucleic Acid Isolation Kit and RT-PCR with TaqPath™ COVID-19 Combo Kit (both Thermo Fisher Scientific) on Applied All rights reserved. No reuse allowed without permission. (which was not certified by peer review) 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 February 1, 2022. ; Biosystems™ 7500 Fast Dx RT-PCR thermocyclers. 128 SARS-CoV-2 positive samples representing pooled (N=39) samples, asymptomatic (N=43) and symptomatic (N=26) individuals were subjected to variant of concern identification through target RT-PCR-based genotyping. SARS-CoV-2 VOC were detected using TaqMan™ SARS-CoV-2 Mutation Panel (Thermo Fisher Scientific) in an 8-assay or 12-assay format as defined in Table 1 . Data analysis was performed using Applied Biosystems™ Design and Analysis Software Version 2.5.1. For details refer to SI. Figure 1. A. SARS-CoV-2 test positivity rate in LSTC population between August 2020 and December 2021. Samples that were tested using the mutation panel are highlighted in blue whereas samples that were tested before implementation of our VOC surveillance assay are depicted in red. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) 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 February 1, 2022. ; Cost benefit analysis between Mutant Panel and WGS for variants of concern surveillance testing. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) 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 February 1, 2022. ; https://doi.org/10.1101/2022.01.31.22270226 doi: medRxiv preprint 1D. Breakthrough SARS-CoV-2 positive incidence (N=226) in fully vaccinated (14-days after second dose of either the Moderna or Pfizer COVID-19 vaccine) patient samples tested by the LSTC after Northeastern began its COVID-19 vaccination program on January 05, 2021. A distinct spike in breakthrough infection in December 2021 was notably correlated with the emergence of the Omicron VOC on NU's campuses, as Delta (N=73) and Omicron (N=75) accounted for all 148 of December breakthrough cases. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) 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 February 1, 2022. ; https://doi.org/10.1101/2022.01.31.22270226 doi: medRxiv preprint A Novel Coronavirus from Patients with Pneumonia in China Precision Health Diagnostic and Surveillance Network uses S Gene Target Failure (SGTF) combined with sequencing technologies to identify emerging SARS-CoV-2 variants Specific allelic discrimination of N501Y and other SARS-CoV-2 mutations by ddPCR detects B.1.1.7 lineage in Washington State Genotyping coronavirus SARS-CoV-2: methods and implications Manual for the surveillance of vaccine-preventable diseases20 Evaluation of NGS-based approaches for SARS-CoV-2 whole genome characterisation Springfield COVID-19 Vaccine Tracker Effectiveness of Covid-19 vaccines against the B.1.617.2 (delta) variant Furin Cleavage Site Is Key to SARS-CoV-2 Pathogenesis The Impact of Mutations in SARS-CoV-2 Spike on Viral Infectivity and Antigenicity Effectiveness of Pfizer-BioNTech and Moderna Vaccines in Preventing SARS-CoV-2 Infection Among Nursing Home Residents Before and During Widespread Circulation of the SARS-CoV-2 B.1.617.2 (Delta) Variant -National Healthcare Safety Network Ct threshold values, a proxy for viral load in community SARS-CoV-2 cases Vaccine Breakthrough Infections with SARS-CoV-2 Variants Table 1. Panel of mutations in the SARS-CoV-2 S-protein selected for genotyping and combinations of mutations used for lineage assignment to five variants of concern: Alpha, Beta, Gamma, Delta, and Omicron (in red) and other variants of interest/under monitoring Thank you to all involved in making this research possible. We are grateful for the incredible dedication, time, and energy provided by everyone at the LSTC to complete this impactful project.