Genomic surveillance of SARS-CoV-2 using long-range PCR primers.

Introduction: Whole Genome Sequencing (WGS) of the SARS-CoV-2 virus is crucial in the surveillance of the COVID-19 pandemic. Several primer schemes have been developed to sequence nearly all of the ~30,000 nucleotide SARS-CoV-2 genome, using a multiplex PCR approach to amplify cDNA copies of the viral genomic RNA. Midnight primers and ARTIC V4.1 primers are the most popular primer schemes that can amplify segments of SARS-CoV-2 (400 bp and 1200 bp, respectively) tiled across the viral RNA genome. Mutations within primer binding sites and primer-primer interactions can result in amplicon dropouts and coverage bias, yielding low-quality genomes with 'Ns' inserted in the missing amplicon regions, causing inaccurate lineage assignments, and making it challenging to monitor lineage-specific mutations in Variants of Concern (VoCs). Methods: In this study we used a set of seven long-range PCR primer pairs to sequence clinical isolates of SARS-CoV-2 on Oxford Nanopore sequencer. These long-range primers generate seven amplicons approximately 4500 bp that covered whole genome of SARS-CoV-2. One of these regions includes the full-length S-gene by using a set of flanking primers. We also evaluated the performance of these long-range primers with Midnight primers by sequencing 94 clinical isolates in a Nanopore flow cell. Results and discussion: Using a small set of long-range primers to sequence SARS-CoV-2 genomes reduces the possibility of amplicon dropout and coverage bias. The key finding of this study is that long range primers can be used in single-molecule sequencing of RNA viruses in surveillance of emerging variants. We also show that by designing primers flanking the S-gene, we can obtain reliable identification of SARS-CoV-2 variants.

Genomic Surveillance of SARS-CoV-2 Using Long-Range PCR Primers.

Whole Genome Sequencing (WGS) of the SARS-CoV-2 virus is crucial in the surveillance of the COVID-19 pandemic. Several primer schemes have been developed to sequence the ~30,000 nucleotide SARS-CoV-2 genome that use a multiplex PCR approach to amplify cDNA copies of the viral genomic RNA. Midnight primers and ARTIC V4.1 primers are the most popular primer schemes that can amplify segments of SARS-CoV-2 (400 bp and 1200 bp, respectively) tiled across the viral RNA genome. Mutations within primer binding sites and primer-primer interactions can result in amplicon dropouts and coverage bias, yielding low-quality genomes with 'Ns' inserted in the missing amplicon regions, causing inaccurate lineage assignments, and making it challenging to monitor lineage-specific mutations in Variants of Concern (VoCs). This study uses seven long-range PCR primers with an amplicon size of ~4500 bp to tile across the complete SARS-CoV-2 genome. One of these regions includes the full-length S-gene by using a set of flanking primers. Using a small set of long-range primers to sequence SARS-CoV-2 genomes reduces the possibility of amplicon dropout and coverage bias.

Younger and Rural Children are More Likely to be Hospitalized for SARS-CoV-2 Infections.

Purpose: To identify characteristics of SARS-CoV-2 infection that are associated with hospitalization in children initially evaluated in a Pediatric Emergency Department (ED). Methods: We identified cases of SARS-CoV-2 positive patients seen in the Arkansas Children's Hospital (ACH) ED or hospitalized between May 27, 2020, and April 28, 2022 using ICD-10 codes within the Pediatric Hospital Information System (PHIS) Database. We compared infection waves for differences in patient characteristics, and used logistic regressions to examine which characteristics led to a higher chance of hospitalization. Findings: We included 681 pre-Delta cases, 673 Delta cases, and 970 Omicron cases. Almost 17% of patients were admitted to the hospital. Compared to Omicron infected children, pre-Delta and Delta infected children were twice as likely to be hospitalized (OR=2.2 and 2.0, respectively; p<0.0001). Infants less than 1 year of age were >3 times as likely to be hospitalized than children ages 5-14 years regardless of wave (OR=3.42; 95%CI=2.36-4.94). Rural children were almost 3 times as likely than urban children to be hospitalized across all waves (OR=2.73; 95%CI=1.97-3.78). Finally, those with a complex condition had nearly a 15-fold increase in odds of admission (OR=14.6; 95%CI=10.6-20.0). Conclusions: Children diagnosed during the pre-Delta or Delta waves were more likely to be hospitalized than those diagnosed during the Omicron wave. Younger and rural patients were more likely to be hospitalized regardless of wave. We suspect lower vaccination rates and larger distances from medical care influenced higher hospitalization rates.