Clinical Evaluation of a Fully-Automated High-Throughput Multiplex Screening-Assay to Detect and Differentiate the SARS-CoV-2 B.1.1.529 (Omicron) and B.1.617.2 (Delta) Lineage Variants.

BACKGROUND: The recently emerged SARS-CoV-2 B.1.1.529 lineage and its sublineages (Omicron variant) pose a new challenge to healthcare systems worldwide due to its ability to efficiently spread in immunized populations and its resistance to currently available monoclonal antibody therapies. RT-PCR-based variant tests can be used to screen large sample-sets rapidly and accurately for relevant variants of concern (VOC). The aim of this study was to establish and validate a multiplex assay on the cobas 6800/8800 systems to allow discrimination between the two currently circulating VOCs, Omicron and Delta, in clinical samples. METHODS: Primers and probes were evaluated for multiplex compatibility. Analytic performance was assessed using cell culture supernatant of an Omicron variant isolate and a clinical Delta variant sample, normalized to WHO-Standard. Clinical performance of the multiplex assay was benchmarked against NGS results. RESULTS: In silico testing of all oligos showed no interactions with a high risk of primer-dimer formation or amplification of human DNA/RNA. Over 99.9% of all currently available Omicron variant sequences are a perfect match for at least one of the three Omicron targets included in the multiplex. Analytic sensitivity was determined as 19.0 IU/mL (CI95%: 12.9-132.2 IU/mL) for the A67V + del-HV69-70 target, 193.9 IU/mL (CI95%: 144.7-334.7 IU/mL) for the E484A target, 35.5 IU/mL (CI95%: 23.3-158.0 IU/mL) for the N679K + P681H target and 105.0 IU/mL (CI95%: 80.7-129.3 IU/mL) for the P681R target. All sequence variances were correctly detected in the clinical sample set (225/225 Targets). CONCLUSION: RT-PCR-based variant screening compared to whole genome sequencing is both rapid and reliable in detecting relevant sequence variations in SARS-CoV-2 positive samples to exclude or verify relevant VOCs. This allows short-term decision-making, e.g., for patient treatment or public health measures.

Rapid Automated Screening for SARS-CoV-2 B.1.617 Lineage Variants (Delta/Kappa) through a Versatile Toolset of qPCR-Based SNP Detection.

BACKGROUND: The recent emergence of distinct and highly successful SARS-CoV-2 lineages has substantial implications for individual patients and public health measures. While next-generation-sequencing is routinely performed for surveillance purposes, RT-qPCR can be used to rapidly rule-in or rule-out relevant variants, e.g., in outbreak scenarios. The objective of this study was to create an adaptable and comprehensive toolset for multiplexed Spike-gene SNP detection, which was applied to screen for SARS-CoV-2 B.1.617 lineage variants. METHODS: We created a broad set of single nucleotide polymorphism (SNP)-assays including del-Y144/145, E484K, E484Q, P681H, P681R, L452R, and V1176F based on a highly specific multi-LNA (locked nucleic acid)-probe design to maximize mismatch discrimination. As proof-of-concept, a multiplex-test was compiled and validated (SCOV2-617VOC-UCT) including SNP-detection for L452R, P681R, E484K, and E484Q to provide rapid screening capabilities for the novel B.1.617 lineages. RESULTS: For the multiplex-test (SCOV2-617VOC-UCT), the analytic lower limit of detection was determined as 182 IU/mL for L452R, 144 IU/mL for P681R, and 79 IU/mL for E484Q. A total of 233 clinical samples were tested with the assay, including various on-target and off-target sequences. All SNPs (179/179 positive) were correctly identified as determined by SARS-CoV-2 whole genome sequencing. CONCLUSION: The recurrence of SNP locations and flexibility of methodology presented in this study allows for rapid adaptation to current and future variants. Furthermore, the ability to multiplex various SNP-assays into screening panels improves speed and efficiency for variant testing. We show 100% concordance with whole genome sequencing for a B.1.617.2 screening assay on the cobas6800 high-throughput system.

Clinical performance and accuracy of a qPCR-based SARS-CoV-2 mass-screening workflow for healthcare-worker surveillance using pooled self-sampled gargling solutions: A cross-sectional study.

INTRODUCTION: The large number of asymptomatic SARS-CoV-2 infections necessitates general screening of employees. We evaluate the performance of a SARS-CoV-2 screening program in asymptomatic healthcare-workers (HCW), utilizing self-sampled gargling-solution and sample pooling for RT-qPCR. METHODS: We conducted a cross-sectional retrospective study to collect real-life data on the performance of a screening-workflow based on automated-pooling and high-throughput qPCR testing over a 3-month-period at the University Hospital Hamburg. RESULTS: Matrix validation reveals that lower limit of detection for SARS-CoV-2 RNA in gargling-solution was 180 copies/mL (5-sample-pool). A total of 55,122 self-collected gargle samples (= 7513 HCWs) was analyzed. The median time to result was 8.5 hours (IQR 7.2-10.8). Of 11,192 pools analyzed, 11,041 (98.7%) were negative, 69 (0.6%) were positive and 82 (0.7%) were invalid. Individual testing of pool participants revealed 57 SARS-CoV-2 previously unrecognized infections. All 57 HCWs were either pre-symptomatic or asymptomatic (prevalence 0.76%,CI95%0.58-0.98%). Accuracy based on HCWs with gargle-solution and NP-swab available within 3-day-interval (N = 521) was 99.5% (CI95%98.3-99.9%), sensitivity 88.9% (CI95%65.3-98.6%) while specificity 99.8% (CI95%98.9-99.9). CONCLUSION: This workflow was highly effective in identifying SARS-CoV-2 positive HCWs, thereby lowering the potential of inter-HCW and HCW-patient transmissions. Automated-sample-pooling helped to conserve qPCR reagents and represents a promising alternative strategy to antigen testing in mass-screening programs.

Evaluation of a fully automated high-throughput SARS-CoV-2 multiplex qPCR assay with built-in screening functionality for del-HV69/70- and N501Y variants such as B.1.1.7.

BACKGROUND: New SARS-CoV-2 variants with increased transmissibility, like B.1.1.7, first detected in England or B.1.351, first detected in South Africa, have caused considerable concern worldwide. In order to contain the spread of these lineages, it is of utmost importance to have rapid, sensitive and high-throughput detection methods at hand. METHODS: A set of RT-qPCR assays was modified for a diagnostic SARS-CoV-2 multiplex assay including detection of the del-HV69/70 and N501Y mutations on the cobas6800 platform. Analytical sensitivity was assessed for both wild-type SARS-CoV-2 and B.1.1.7 lineage by serial dilution. For clinical performance, a total of 176 clinical samples were subjected to the test and results compared to a commercial manual typing-PCR assay and next generation sequencing as gold standard. RESULTS: The multiplex assay was highly sensitive for detection of SARS-CoV-2 RNA in clinical samples, with an LoD of 6.16 cp/ml (CI: 4.00-8.31). LoDs were slightly higher for detection of the HV69/70 deletion (85.92, CI: 61-194.41) and the N501Y SNP (105.99 cp/ml, CI: 81.59 - 183.66). A total of 176 clinical samples were tested with the assay, including 50 samples containing SARS-CoV-2 of the B.1.1.7 lineage, one containing B.1.351 and 85 non-B.1.1.7/B.1.351 lineage, of which three also harbored a HV69/70 deletion. All were correctly identified by the multiplex assay. CONCLUSION: We describe here a highly sensitive, fully automated multiplex PCR assay for the simultaneous detection of the del-HV69/70 and N501Y mutations that can distinguish between B.1.1.7 and other lineages. The assay allows for high-throughput screening for currently relevant variants in clinical samples prior to sequencing.