key: cord-0266429-6o8ihee6 authors: N M, V.; A, K.; V, K.; A, R. K.; Subramanian, G.; Rathinasamy, A. S.; Ramakrishnan, U.; Potdar, V. A.; Sivadoss, R.; P, S.; T S, S. title: S Gene Target Failure (SGTF) in Commercial Multiplex RT-PCR assay as indicator to detect SARS-CoV-2 VOC B.1.1.7 lineage in Tamil Nadu, India date: 2021-12-16 journal: nan DOI: 10.1101/2021.12.14.21251883 sha: 5558714e79439dd7e1c780afad7323be2b6053bf doc_id: 266429 cord_uid: 6o8ihee6 Emergence of Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2) Variants of Concern (VOC) possessing improved virulence, transmissibility and/or immune-escape capabilities has raised significant public health concerns. In order to identify VOCs, WHO recommends Whole-Genome Sequencing approach, which is costly and involves longer completion time. Hence, potential role of commercial multiplex RT-PCR kit to screen variants rapidly is being attempted in this study. A total of 1200 suspected COVID samples from different districts of Tamil Nadu State (India) were screened with Thermo TaqPath RT-PCR kit and Altona Realstar RT-PCR Assay kit. Among 1200 screened, S-gene target failure (SGTF) phenomenon were identified in 112 samples while testing with TaqPath RT-PCR Kit. 100% concordant results were observed between SGTF phenomenon and whole-genome sequencing (WGS) results in detecting SARS-CoV-2 VOC B.1.1.7. TaqPath RT-PCR assay testing can be utilized by laboratories to screen rapidly the VOC B.1.1.7 variants, thus enabling early detection of B.1.1.7 variant infection and transmission in population. This in turn will pave way to implement suitable preventive measures by appropriate authorities to control the transmission of the viral variant. The current coronavirus disease 2019 pandemic caused by novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected more than 200 countries globally (Huang et al., 2020; Li et al., 2020; Wu et al., 2020) . As on 26 th July 2021, 19.5 crore confirmed cases of COVID-19 have been reported worldwide and 41.8 lakh deaths were recorded. At the same time, India recorded 3.14 crore confirmed cases and 4.2 lakh deaths due to (Https://Www.Worldometers.Info/Coronavirus/). In India, a medical student travelling from China (Wuhan) on 30th January 2020 was the first reported case of COVID-19 (Vaman et al., 2020) . Over the couple of years, SARS-CoV-2 have evolved to form new variants possessing improved transmissibility, virulence and/or immune-escape capabilities (CMMID COVID-19 Working Group et al., 2021; Davies et al., 2021; Rambaut et al., 2020) . Rapid spread of these new SARS-CoV-2 variants poses notable public health challenges (Sánchez-Calvo et al., 2021) . As several molecular diagnostics only target small regions in the viral genome, genetic changes in SARS-CoV-2 will significantly affect their diagnosis too (Wollschläger et al., 2021) . Among the several SARS-CoV-2 major variants of concern (VOCs) reported till date, B.1.1.7 (UK) is a dominant circulating variant that emerged in England during September 2020 (Public Health England, 2021) . The new variant has been detected in over 93 countries, including India (O'Toole Á et al., 2021) . This VOC has been reported to possess increased mortality and transmissibility (CMMID COVID-19 Working Group et al., 2021; Davies et al., 2021; Leung et al., 2021; Rambaut et al., 2020) . Hence, early identification of this variant in patients may help reduce further spread of infection. Identifying potential B.1.1.7 cases using S-gene target failure (SGTF) results has been recommended by CDC (Galloway et al., 2021) . European Centre for Disease Prevention and Control (ECDC) has also suggested laboratories to implement screening RT-PCR tests to detect SGTF among SARS-CoV-2 samples (ECDC 2021). However, both CDC and ECDC have emphasized the need to confirm specific variants by sequencing only (ECDC 2021; Galloway et al., 2021) . Multiplex PCR is the gold standard for SARS-CoV-2 diagnosis (Kevadiya et al., 2021) . Dual or triple targets detection is recommended (Corman et al., 2020; Kevadiya et al., 2021) . However, detecting viral variants is not intended with regular PCR kits. Performance of any SARS-CoV-2 assay will be significantly affected by SARS-CoV-2 variants present in samples. Several researchers have recorded gene dropouts using SARS-CoV-2 multiplex PCR approaches targeting S or N or E genes (Artesi et al., 2020; Hasan et al., 2021; Kidd et al., 2021; Umair et al., 2021; Washington et al., 2020; Ziegler et al., 2020) . Variant-specific PCR or Whole Genome Sequencing (WGS) techniques are either costly and/or involve longer completion time (Wang et al., 2021; Wollschläger et al., 2021) . Availability of a rapid screening approach will enable usage of WGS on samples of interest as well as maximize the use of WGS and expand scope of laboratories to actively participate in VOC identification process. . 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) preprint The copyright holder for this this version posted December 16, 2021. ; Thus, the current study focusses on SARS-CoV-2 S gene target failure (SGTF) detection as an indicator for the presence of VOC B.1.1.7 lineage using commercial multiplex PCR assay, further confirmed by whole-genome sequencing. The study was conducted from December 2020 to May 2021 (6 months) involving individuals presenting with ILI symptoms throughout the Tamil Nadu State. Samples from the individuals were classified into one of the following categories viz. International Travelers and their close contacts, Community Clusters, Family Clusters, Vaccine Breakthrough Cases, Deceased with no comorbidities, Children and Young Adults with Severe Lung Involvement. A total of 1200 samples (both oropharyngeal and nasal swabs) were collected in Viral Transport Media from suspected ILI symptoms presenting cases as per the protocol of WHO for sample collection and transport (WHO 2021). Care was taken to transport the samples in cold chain to State Public Health Laboratory (SPHL), Directorate of Public Health and Preventive Medicine, Chennai, Tamil Nadu, India as mandated by WHO (WHO 2021). The sample processing was carried out in a Type II Biological Safety Cabinet with HEPA filters, at SPHL, Chennai. RNA was extracted using KingFisher Flex Automated Nucleic Acid Extractor (MVP-II Protocol Kit) as per manufacturer instructions. All the samples were subjected to RT-PCR analysis using TaqPath COVID-19 Combo Kit (ThermoFisher Scientific, USA) that detects 3 genes viz. S, N and ORF-1ab (ThermoFisher Scientific TaqPath Kit, 2021). Samples that failed to amplify S-gene in TaqPath COVID-19 Combo kit were subjected to RealStar SARS-CoV-2 RT-PCR Kit (Altona Diagnostics, Germany) testing that identifies E and S genes (RealStar SARS-CoV-2 RT-PCR Kit, 2020). The results were analyzed using Quant Studio Design and Analysis Software (v1.5.0). Samples with Cycle Threshold (Ct) value of <25 in RT-PCR were alone selected and sent for sequencing studies. RNA extracts of the samples were sent either to National Institute of Virology (NIV), Pune or to InSTEM, Bengaluru for sequencing studies for confirmation of the variant form 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) preprint The copyright holder for this this version posted December 16, 2021. ; https://doi.org/10.1101/2021.12.14.21251883 doi: medRxiv preprint VTM and excess RNA extracts of the samples were stored at -80˚C after RT-PCR analysis. Figure 1 outlines the overview of the study. Ethical Approval was obtained for this study from Institutional Ethics Committee (IEC), Madras Medical College, Chennai-3 (ECR/270/Inst./TN/2013/RR-16/No.32092021). A total of 1200 respiratory specimens with suspected cases of SARS-CoV-2 were tested by TaqPath Distribution of SARS-CoV-2 Alpha Variant in the samples analyzed is displayed in Table 2 . A majority of the Alpha variants (62.6%) were reported from community clusters (40%) followed by family clusters (23%). In addition, Alpha variants were also reported in 17.3% of the International travelers and their close contact. Alpha variants in the remaining categories were reported to be not more than 8%. In age-wise distribution, 50.6% of samples among the alpha variants belonged to 19 to 45 age group. 17.3% samples of the alpha variants were reported in two age groups viz. 10-18 and 46-60. Individuals aged above 60 represented 8% of the alpha variants identified, whereas less than 10 years age group formed only 6%. Among the gender-wise distribution, 54.6% and 45.3% of women and men were found to be infected with SARS-CoV-2 alpha variant, respectively. Table 3 lists month-wise SARS-CoV-2 alpha and delta variants reported during the study period. In April 2021, a maximum of 32 alpha variants (out of 75) and 8 delta variants were recorded (Figure 3 ). . 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 this version posted December 16, 2021. ; Step 1 Step 2 Step 3 Step 4 Step 5 . 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 this version posted December 16, 2021. ; . 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 this version posted December 16, 2021. ; . 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) preprint The copyright holder for this this version posted December 16, 2021. ; https://doi.org/10.1101/2021.12.14.21251883 doi: medRxiv preprint In the present study, a Multiplex RT-PCR based strategy is demonstrated to reliably screen for VOCs . 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) preprint The copyright holder for this this version posted December 16, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 multiplex real-time RT-PCR Kit of Altona Diagnostics, Germany (RealStar SARS-CoV-2 RT-PCR Kit, 2020) . According to CDC and WHO recommendations, S-gene drop could act as an indicator for investigation of new SARS-CoV-2 variant and for further sequencing (Galloway et al., 2021; WHO 2021 WHO , 2021 . Our study primarily focused on SGTF in SARS-CoV-2 using TaqPath Assay Kit. Kidd et al. (2021) , Umair et al. (2021) and Washington et al., (2020) have recorded similar S-gene dropout pattern in SARS-CoV-2. Failure of the S gene target amplification using TaqPath COVID-19 RT-PCR Kit is attributed to H69-V70 deletion typically present in SARS-CoV-2 variant VOC B.1.1.7. Interestingly, other investigators also reported gene target failure phenomenon associated with N and E genes of SARS-CoV-2 (Artesi et al., 2020; Hasan et al., 2021; Sánchez-Calvo et al., 2021; Ziegler et al., 2020) . Primary reasons for failure to detect these genes in real-time RT-PCR systems are mutations, deletions or insertions in the gene targets. From our study, it is clearly evident that Community clusters and Family Clusters in Tamil Nadu were predominantly affected with VOC B.1.1.7 (Table 2) . Thus, substantiating increased transmissibility of this variant among the population (Leung et al., 2021) . Alpha variant has accounted for higher infections among the 19-45 age group individuals and only 5 cases below age 10 have been reported. Similar results have been reported elsewhere (Davies et al., 2020) . The investigators suggest school closure as prime reason for less SARS-CoV-2infection among the children. Our results reveal peak number of alpha variants reported during April 2021 and also emergence of delta variant infected cases started to be reported at around the same time. Our study predominantly reports identification of SGTF as B.1.1.7 variant. However, less sample size availability to detect the other two variants during our study is a limitation. Further studies with more samples from diverse population would aid in understanding the newly evolved SARS-CoV-2 infections in our region and its possible selection using a commercially available RT-PCR Assay. The comparison of Multiplex RT-PCR results by using two different kits targeting different genes allow us to differentiate partial identification of the new variant of SARS-CoV-2 suspicions that is further confirmed by whole genome sequencing results obtained. Multiplex RT-PCR could be used as a potential molecular tool for the rapid identification of SARS-CoV-2 variant so as to ensure efficient screening for timely and accurate diagnosis of SARS-CoV-2 variants. The study findings may aid in risk assessment and will be immediately reviewed to re-assess the overall level of risk to human health and potential implications of SARS-CoV-2 diagnosis, treatment and vaccine development. This . 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. 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The authors also will help us to take immediate preventive measures to control the transmission of new variant of SARS-CoV-2 infections in Tamil Nadu state.