Rapid, high-throughput, cost-effective whole-genome sequencing of SARS-CoV-2 using a condensed library preparation of the Illumina DNA Prep kit.

The ongoing COVID-19 pandemic necessitates cost-effective, high-throughput, and timely whole-genome sequencing (WGS) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses for outbreak investigations, identifying variants of concern (VoC), characterizing vaccine breakthrough infections, and public health surveillance. In addition, the enormous demand for WGS on supply chains and the resulting shortages of laboratory supplies necessitated the use of low-reagent and low-consumable methods. Here, we report an optimized library preparation method (the BCCDC cutdown method) that can be used in a high-throughput scenario, where one technologist can perform 576 library preparations (6 plates of 96 samples) over the course of one 8-hour shift. The same protocol can also be used in a rapid turnaround time scenario, from primary samples (up to 96 samples) to loading on a sequencer in an 8-hour shift. This new method uses Freed et al.'s 1,200 bp primer sets (Biol Methods Protoc 5:bpaa014, 2020, https://doi.org/10.1093/biomethods/bpaa014) and a modified and condensed Illumina DNA Prep workflow (Illumina, CA, USA). Compared to the original protocol, the application of this new method using hundreds of clinical specimens demonstrated equivalent results to the full-length DNA Prep workflow at 45% of the cost, 15% of consumables required (such as pipet tips), 25% of manual hands-on time, and 15% of on-instrument time if performing on a liquid handler, with no compromise in sequence quality. Results demonstrate that this new method is a rapid, simple, cost-effective, and high-quality SARS-CoV-2 WGS protocol. IMPORTANCE: Sequencing has played an invaluable role in the response to the COVID-19 pandemic. Ongoing work in this area, however, demands optimization of laboratory workflow to increase sequencing capacity, improve turnaround time, and reduce cost without compromising sequence quality. This report describes an optimized DNA library preparation method for improved whole-genome sequencing of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogen. The workflow advantages summarized here include significant time, cost, and consumable savings, which suggest that this new method is an efficient, scalable, and pragmatic alternative for SARS-CoV-2 whole-genome sequencing.

Prospective, clinical comparison of self-collected throat-bilateral nares swabs and saline gargle compared to health care provider collected nasopharyngeal swabs among symptomatic outpatients with potential SARS-CoV-2 infection.

Background: In British Columbia (BC), self-collected saline gargle (SG) is the only alternative to health care provider (HCP)-collected nasopharyngeal (NP) swabs to detect SARS-CoV-2 in an outpatient setting by polymerase chain reaction (PCR). However, some individuals cannot perform a SG. Our study aimed to assess combined throat-bilateral nares (TN) swabbing as a swab-based alternative. Methods: Symptomatic individuals greater than 12 years of age seeking a COVID-19 PCR test at one of two COVID-19 collection centres in Metro Vancouver were asked to participate in this study. Participants provided a HCP-collected NP sample and a self-collected SG and TN sample for PCR testing, which were either HCP observed or unobserved. Results: Three-hundred and eleven individuals underwent all three collections. Compared against HCP-NP, SG was 99% sensitive and 98% specific (kappa 0.97) and TN was 99% sensitive and 99% specific (kappa 0.98). Using the final clinical test interpretation as the reference standard, NP was 98% sensitive and 100% specific (kappa 0.98), and both SG and TN were 99% sensitive and 100% specific (both kappa 0.99). Mean cycle threshold values for each viral target were higher in SG specimens compared to the other sample types; however, this did not significantly impact the clinical performance, because the positivity rates were similar. The clinical performance of all specimen types was comparable within the first 7 days of symptom onset, regardless of the observation method. SG self-collections were rated the most acceptable, followed by TN. Conclusions: TN provides another less invasive self-collection modality for symptomatic outpatient SARS-CoV-2 PCR testing.

Historique: En Colombie-Britannique (C.-B.), l'autoprélèvement de gargarisme d'eau saline (GS) est la seule alternative aux écouvillons nasopharyngés (NP) prélevés par un professionnel de la santé (PdS) pour déceler le SRAS-CoV-2 par test PCR en milieu ambulatoire. Cependant, certaines personnes ne peuvent pas effectuer de GS. La présente étude visait évaluer l'écouvillonnage de la gorge et des deux narines (GN) pour remplacer le GS. Méthodologie: Les personnes symptomatiques de plus de 12 ans qui demandaient un test PCR de la COVID-19 à l'un des deux centres de dépistage de la COVID-19 du Grand Vancouver ont été invitées à participer à la présente étude. Les participants ont fourni un prélèvement NP recueilli par un PdS ainsi qu'un autoprélèvement de GS et GN en vue d'un test PCR, observés ou non par un PdS. Résultats: Au total, 311 personnes ont participé aux trois prélèvements. Par rapport au prélèvement NP-PdS, le GS avait une sensibilité de 99 % et une spécificité de 98 % (kappa 0,97) et le prélèvement GN, une sensibilité de 99 % et une spécificité de 99 % (kappa 0, 98). À l'aide de l'interprétation définitive du test clinique comme norme de référence, le prélèvement NP avait une sensibilité de 98 % et une spécificité de 100 % (kappa 0,98) et tant le GS que le prélèvement GN avaient une sensibilité de 99 % et une spécificité de 100 % (deux kappa 0,99). Les valeurs seuils du cycle moyen de chaque cible virale étaient plus élevées dans les échantillons de GS quand dans les autres types d'échantillons, mais n'avaient pas d'effet significatif sur le rendement clinique, puisque les taux de positivité étaient semblables. Le rendement clinique de tous les types d'échantillons était comparable dans les sept premiers jours suivant l'apparition de la maladie, quel que soit le mode d'observation. L'autoprélèvement de GS a été classé comme le plus acceptable, suivi du prélèvement GN. Conclusions: Le prélèvement GN est un autre mode d'autoprélèvement moins invasif chez les patients ambulatoires symptomatiques qui effectuent un test PCR du SRAS-CoV-2.

Optimization of magnetic bead-based nucleic acid extraction for SARS-CoV-2 testing using readily available reagents.

The COVID-19 pandemic has highlighted the need for generic reagents and flexible systems in diagnostic testing. Magnetic bead-based nucleic acid extraction protocols using 96-well plates on open liquid handlers are readily amenable to meet this need. Here, one such approach is rigorously optimized to minimize cross-well contamination while maintaining sensitivity.

Design and preliminary experience of a transportable equine operating theatre.

BACKGROUND: Equine surgical facilities are not always available, even in situations where high-value horses are involved in relatively high-risk activities. OBJECTIVES: To develop a self-contained equine surgical unit that can be relocated and transported by road. STUDY DESIGN: Proof of concept. METHODS: A modular operating suite for horses, which included a theatre, scrub room, instrument preparation and storage room, and an anaesthetic induction/recovery box: Transportable Equine Operating Theatre (TEOT) was designed, constructed and tested. A second, larger unit was then built following the same design criteria. RESULTS: Both units were based on modifications of standard 6.06 × 2.35 m (20 × 8 ft), steel shipping containers that were fitted out to provide anaesthetic and surgical facilities in a functional and safe working environment. The two TEOTs consisted of either two or three containers that had selected sections of their sides removed so that they could be joined together to create a single large internal space. This space was subdivided to create separate working areas. A dedicated anaesthetic induction/recovery box was fabricated from a modular steel frame that could be disassembled for transport. The component parts could be loaded onto the bed of a standard lorry for road transport. The unit was designed to incorporate equipment that is found in any high-quality equine operating theatre. Surgical procedures on adult Thoroughbred horses have been successfully completed in both units. MAIN LIMITATIONS: The two units have had minimal use to date, and there is little data on their performance. CONCLUSIONS: The equipment described here is a way to provide equine surgical facilities that can be assembled and disassembled to allow their relocation to different sites.

Evaluation of a high volume antigen test for detection of SARS-CoV-2.

Increasing transmission of SARS-CoV-2 infection in successive waves may strain the capacity of laboratories performing molecular diagnostic testing. Alternative testing approaches may offer additional diagnostic capacity. A high throughput chemiluminescent antigen assay (Ortho VITROS SARS-CoV-2 antigen test) was evaluated using both an inactivated virus preparation and prospective clinical samples (nasopharyngeal swabs in virus transport medium). The limit of detection of the assay was approximately 0.5 TCID50/ml, equivalent to a Ct value of 33. The assay was linear over a wide range. When 528 clinical samples were tested with the antigen assay, the sensitivity was 84.2% and the specificity was 100% (positive predictive value 100% and negative predictive value 97.7%). High volume antigen tests might be used to supplement molecular diagnostic testing capacity.

Detailed neuropathologic report of COVID-19 complicated by large intracerebral hemorrhage and periventricular lesions with macrophagic infiltrates.

Infection with the SARS-CoV-2 virus affects a wide range of systems. Significant involvement of the central nervous system has been described, including ischemic and hemorrhagic strokes. Thus far, neuropathologic reports of patients who passed away from COVID-19 have generally described non-specific findings, such as variable reactive gliosis and meningeal chronic inflammatory infiltrates, as well as the consequences of the infection's systemic complications on the brain, including ischemic infarcts and hypoxic/ischemic encephalopathy. The neuropathological changes in patients with COVID-19 and large hemorrhagic strokes have not been described in detail. We report the case of an elderly male who had a long course of COVID-19 and ultimately passed away from a large intracerebral hemorrhage. In addition to acute hemorrhage, neuropathologic examination demonstrated non-specific reactive changes and chronic periventricular lesions with macrophagic and perivascular lymphocytic infiltrates without evidence of demyelination or presence of SARS-CoV-2 by PCR test. This manuscript expands the spectrum of reported neuropathological changes in patients with COVID-19.