Inactivation and Recovery of High Quality RNA From Positive SARS-CoV-2 Rapid Antigen Tests Suitable for Whole Virus Genome Sequencing.

The diagnostic protocol currently used globally to identify Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is RT-qPCR. The spread of these infections and the epidemiological imperative to describe variation across the virus genome have highlighted the importance of sequencing. SARS-CoV-2 rapid antigen diagnostic tests (RADTs) are designed to detect viral nucleocapsid protein with positive results suggestive of the presence of replicating virus and potential infectivity. In this study, we developed a protocol for recovering SARS-CoV-2 RNA from "spent" RADT devices of sufficient quality that can be used directly for whole virus genome sequencing. The experimental protocol included the spiking of RADTs at different concentrations with viable SARS-CoV-2 variant Alpha (lineage B.1.1.7), lysis for direct use or storage. The lysed suspensions were used for RNA extraction and RT-qPCR. In parallel, we also tested the stability of the viral RNA in the RADTs and the RNA extracted from the RADTs was used as a template for tiling-PCR and whole virus genome sequencing. RNA recovered from RADTs spiked with SARS-CoV-2 was detected through RT-qPCR with Ct values suitable for sequencing and the recovery from RADTs was confirmed after 7 days of storage at both 4 and 20°C. The genomic sequences obtained at each time-point aligned to the strain used for the spiking, demonstrating that sufficient SARS-CoV-2 viral genome can be readily recovered from positive-RADT devices in which the virus has been safely inactivated and genomically conserved. This protocol was applied to obtain whole virus genome sequence from RADTs ran in the field where the omicron variant was detected. The study demonstrated that viral particles of SARS-CoV-2 suitable for whole virus genome sequencing can be recovered from positive spent RADTs, extending their diagnostic utility, as a risk management tool and for epidemiology studies. In large deployment of the RADTs, positive devices could be safely stored and used as a template for sequencing allowing the rapid identification of circulating variants and to trace the source and spread of outbreaks within communities and guaranteeing public health.

SARS-CoV2 Reinfections in a University Teaching Hospital

Background The consequences of SARS-CoV2 reinfections for patients, healthcare workers and society are unclear. We reviewed the clinical, laboratory, and epidemiological characteristics of patients re-infected with genetically distinct strains of SARS-CoV2 identified by Whole Virus Genome Sequencing (WvGS). Methods Cases were selected based on a positive SARS-CoV-2 Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) test, clinical resolution, a negative interim test and a subsequent positive nasopharyngeal swab. Positive samples were prepared for sequencing by cDNA synthesis, tiled-PCR following the ARTIC protocol and amplicon sequencing using Illumina MiSeq platform. Raw reads were mapped to the reference sequence using bowtie and Samtools was used for variants calling and to generate the consensus sequences. Comparative sequence analysis was conducted by phylogenetic inference maximum likelihood method with RAxML using the multiple sequence aligned by MAFFT. Clades and variants were assigned respectively using Nextstrain and Pangolin COVID-19 lineage assigner (Figure 1). The clinical, radiological and laboratory data were collected from patient medical notes and laboratory information system. Results Two cases of SARS-CoV-2 reinfection were detected by RT-PCR (patient 1 and 2). CT values and strain variants are presented in Table 1. The time between detection of the first and second infection was 67 and 270 days respectively. WvGS confirmed that the second episodes were due to a genetically distinct strain of SARS CoV2. These reflected the dominant contemporaneous variants in circulation. Both patients were immunocompromised from co-morbidities and medications. First and subsequent infections were minimally symptomatic. Both cases were associated with known hospital outbreaks. They passed away within 2 weeks of the second infection of unrelated causes. Conclusion Two patients in this study were diagnosed with a SARS-CoV-2 reinfection confirmed by WvGS. A common factor in these cases was immunocompromise. Where a previously infected patient test shows a new positive or an unexpected reduction in CT value is observed, we recommend individual risk assessment to determine the timing of discontinuation of isolation and infection control precautions. Disclosures Jérôme Fennell, MB BCh BAO MSc PhD FRCPath FRCPI, Roche Diagnostics (Advisor or Review Panel member)

Genomic Evolution of SARS-CoV-2 Virus in Immunocompromised Patient, Ireland.

We examined virus genomic evolution in an immunocompromised patient with prolonged severe acute respiratory syndrome coronavirus 2 infection. Genomic sequencing revealed genetic variation during infection: 3 intrahost mutations and possible superinfection with a second strain of the virus. Prolonged infection in immunocompromised patients may lead to emergence of new virus variants.

Whole-genome Sequencing to Track Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Transmission in Nosocomial Outbreaks.

BACKGROUND: During the first wave of the coronavirus disease 2019 (COVID-19) pandemic, outbreaks of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in healthcare institutions posed a significant problem. Due to limited evidence, guidance on appropriate infection prevention and control (IPC) measures such as the wearing of face masks varied. Here, we applied whole virus genome sequencing (WvGS) to analyze transmission routes of SARS-CoV-2 in hospital-acquired (HA) COVID-19. METHODS: An investigation was undertaken for all HA cases of COVID-19 from March to April 2020. Fifty SARS-CoV-2 samples were analysed by WvGS and their phylogenetic relationship established. RESULTS: WvGS identified transmission events previously undetected by epidemiological analysis and provided evidence for SARS-CoV-2 transmission between healthcare workers (HCW) and patients and among HCW themselves. The majority of HA COVID-19 cases occurred in patients highly dependent on nursing care, suggesting the likely route of transmission was by close contact or droplet, rather than aerosol, transmission. Mortality among HA COVID-19 infections was recorded as 33%. CONCLUSIONS: This study provides evidence that SARS-CoV-2 transmission occurs from symptomatic and asymptomatic HCWs to patients. Interventions including comprehensive screening of HCWs for COVID-19 symptoms, PCR testing of asymptomatic HCWs upon identification of HA cases and implementation of universal use of surgical masks for all clinical care is indicated to prevent viral transmission. Our study highlights the importance of close collaboration between guidance bodies and frontline IPC experts for developing control measures in an emergency pandemic situation caused by a virus with undefined transmission modus.