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.

SARS-CoV-2 Antiviral Prescribing Gaps Among Non-Hospitalized High-Risk Adults.

Within a multi-state clinical cohort, SARS-CoV-2 antiviral prescribing patterns were evaluated from April 2022-June 2023 among non-hospitalized SARS-CoV-2-infected patients with risk factors for severe COVID-19. Among 3,247 adults, only 31.9% were prescribed an antiviral agent (87.6% nirmatrelvir/ritonavir, 11.9% molnupiravir, 0.5% remdesivir), highlighting the need to identify and address treatment barriers.

Engineering synthetic phosphorylation signaling networks in human cells.

Protein phosphorylation signaling networks play a central role in how cells sense and respond to their environment. Here, we describe the engineering of artificial phosphorylation networks in which "push-pull" motifs-reversible enzymatic phosphorylation cycles consisting of opposing kinase and phosphatase activities-are assembled from modular protein domain parts and then wired together to create synthetic phosphorylation circuits in human cells. We demonstrate that the composability of our design scheme enables model-guided tuning of circuit function and the ability to make diverse network connections; synthetic phosphorylation circuits can be coupled to upstream cell surface receptors to enable fast-timescale sensing of extracellular ligands, while downstream connections can regulate gene expression. We leverage these capabilities to engineer cell-based cytokine controllers that dynamically sense and suppress activated T cells. Our work introduces a generalizable approach for designing and building phosphorylation signaling circuits that enable user-defined sense-and-respond function for diverse biosensing and therapeutic applications.

Estimating vaccine coverage in conflict settings using geospatial methods: a case study in Borno state, Nigeria.

Reliable estimates of subnational vaccination coverage are critical to track progress towards global immunisation targets and ensure equitable health outcomes for all children. However, conflict can limit the reliability of coverage estimates from traditional household-based surveys due to an inability to sample in unsafe and insecure areas and increased uncertainty in underlying population estimates. In these situations, model-based geostatistical (MBG) approaches offer alternative coverage estimates for administrative units affected by conflict. We estimated first- and third-dose diphtheria-tetanus-pertussis vaccine coverage in Borno state, Nigeria, using a spatiotemporal MBG modelling approach, then compared these to estimates from recent conflict-affected, household-based surveys. We compared sampling cluster locations from recent household-based surveys to geolocated data on conflict locations and modelled spatial coverage estimates, while also investigating the importance of reliable population estimates when assessing coverage in conflict settings. These results demonstrate that geospatially-modelled coverage estimates can be a valuable additional tool to understand coverage in locations where conflict prevents representative sampling.

Common features of cartilage maturation are not conserved in an amphibian model.

BACKGROUND: Mouse, chick, and zebrafish undergo a highly conserved program of cartilage maturation during endochondral ossification (bone formation via a cartilage template). Standard histological and molecular features of cartilage maturation are chondrocyte hypertrophy, downregulation of the chondrogenic markers Sox9 and Col2a1, and upregulation of Col10a1. We tested whether cartilage maturation is conserved in an amphibian, the western clawed frog Xenopus tropicalis, using in situ hybridization for standard markers and a novel laser-capture microdissection RNAseq data set. We also functionally tested whether thyroid hormone drives cartilage maturation in X tropicalis, as it does in other vertebrates. RESULTS: The developing frog humerus mostly followed the standard progression of cartilage maturation. Chondrocytes gradually became hypertrophic as col2a1 and sox9 were eventually down-regulated, but col10a1 was not up-regulated. However, the expression levels of several genes associated with the early formation of cartilage, such as acan, sox5, and col9a2, remained highly expressed even as humeral chondrocytes matured. Greater deviances were observed in head cartilages, including the ceratohyal, which underwent hypertrophy within hours of becoming cartilaginous, maintained relatively high levels of col2a1 and sox9, and lacked col10a1 expression. Interestingly, treating frog larvae with thyroid hormone antagonists did not specifically reduce head cartilage hypertrophy, resulting rather in a global developmental delay. CONCLUSION: These data reveal that basic cartilage maturation features in the head, and to a lesser extent in the limb, are not conserved in X tropicalis. Future work revealing how frogs deviate from the standard cartilage maturation program might shed light on both evolutionary and health studies.

Assessing Geographic Overlap between Zero-Dose Diphtheria-Tetanus-Pertussis Vaccination Prevalence and Other Health Indicators.

The integration of immunization with other essential health services is among the strategic priorities of the Immunization Agenda 2030 and has the potential to improve the effectiveness, efficiency, and equity of health service delivery. This study aims to evaluate the degree of spatial overlap between the prevalence of children who have never received a dose of the diphtheria-tetanus-pertussis-containing vaccine (no-DTP) and other health-related indicators, to provide insight into the potential for joint geographic targeting of integrated service delivery efforts. Using geospatially modeled estimates of vaccine coverage and comparator indicators, we develop a framework to delineate and compare areas of high overlap across indicators, both within and between countries, and based upon both counts and prevalence. We derive summary metrics of spatial overlap to facilitate comparison between countries and indicators and over time. As an example, we apply this suite of analyses to five countries-Nigeria, Democratic Republic of the Congo (DRC), Indonesia, Ethiopia, and Angola-and five comparator indicators-children with stunting, under-5 mortality, children missing doses of oral rehydration therapy, prevalence of lymphatic filariasis, and insecticide-treated bed net coverage. Our results demonstrate substantial heterogeneity in the geographic overlap both within and between countries. These results provide a framework to assess the potential for joint geographic targeting of interventions, supporting efforts to ensure that all people, regardless of location, can benefit from vaccines and other essential health services.

Evidence for SARS-CoV-2 Delta and Omicron co-infections and recombination.

BACKGROUND: Between November 2021 and February 2022, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta and Omicron variants co-circulated in the United States, allowing for co-infections and possible recombination events. METHODS: We sequenced 29,719 positive samples during this period and analyzed the presence and fraction of reads supporting mutations specific to either the Delta or Omicron variant. FINDINGS: We identified 18 co-infections, one of which displayed evidence of a low Delta-Omicron recombinant viral population. We also identified two independent cases of infection by a Delta-Omicron recombinant virus, where 100% of the viral RNA came from one clonal recombinant. In the three cases, the 5' end of the viral genome was from the Delta genome and the 3' end from Omicron, including the majority of the spike protein gene, though the breakpoints were different. CONCLUSIONS: Delta-Omicron recombinant viruses were rare, and there is currently no evidence that Delta-Omicron recombinant viruses are more transmissible between hosts compared with the circulating Omicron lineages. FUNDING: This research was supported by the NIH RADx initiative and by the Centers for Disease Control Contract 75D30121C12730 (Helix).

A Method for Variant Agnostic Detection of SARS-CoV-2, Rapid Monitoring of Circulating Variants, and Early Detection of Emergent Variants Such as Omicron.

The rapid emergence of SARS-CoV-2 variants raised public health questions concerning the capability of diagnostic tests to detect new strains, the efficacy of vaccines, and how to map the geographical distribution of variants to understand transmission patterns and loads on healthcare resources. Next-generation sequencing (NGS) is the primary method for detecting and tracing new variants, but it is expensive, and it can take weeks before sequence data are available in public repositories. This article describes a customizable reverse transcription PCR (RT-PCR)-based genotyping approach which is significantly less expensive, accelerates reporting, and can be implemented in any lab that performs RT-PCR. Specific single-nucleotide polymorphisms (SNPs) and indels were identified which had high positive-percent agreement (PPA) and negative-percent agreement (NPA) compared to NGS for the major genotypes that circulated through September 11, 2021. Using a 48-marker panel, testing on 1,031 retrospective SARS-CoV-2 positive samples yielded a PPA and NPA ranging from 96.3 to 100% and 99.2 to 100%, respectively, for the top 10 most prevalent World Health Organization (WHO) lineages during that time. The effect of reducing the quantity of panel markers was explored, and a 16-marker panel was determined to be nearly as effective as the 48-marker panel at lineage assignment. Responding to the emergence of Omicron, a genotyping panel was developed which distinguishes Delta and Omicron using four highly specific SNPs. The results demonstrate the utility of the condensed panel to rapidly track the growing prevalence of Omicron across the US in December 2021 and January 2022.

SARS-CoV-2 variant Delta rapidly displaced variant Alpha in the United States and led to higher viral loads.

We report on the sequencing of 74,348 SARS-CoV-2 positive samples collected across the United States and show that the Delta variant, first detected in the United States in March 2021, made up the majority of SARS-CoV-2 infections by July 1, 2021 and accounted for >99.9% of the infections by September 2021. Not only did Delta displace variant Alpha, which was the dominant variant at the time, it also displaced the Gamma, Iota, and Mu variants. Through an analysis of quantification cycle (Cq) values, we demonstrate that Delta infections tend to have a 1.7× higher viral load compared to Alpha infections (a decrease of 0.8 Cq) on average. Our results are consistent with the hypothesis that the increased transmissibility of the Delta variant could be due to the ability of the Delta variant to establish a higher viral load earlier in the infection as compared to the Alpha variant.

Mutations in emerging variant of concern lineages disrupt genomic sequencing of SARS-CoV-2 clinical specimens.

Mutations in emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages can interfere with laboratory methods used to generate viral genome sequences for public health surveillance. We identified 20 mutations that are widespread in variant of concern lineages and affect widely used sequencing protocols by the ARTIC network and Freed et al. Three of these mutations disrupted sequencing of P.1 lineage specimens during a recent outbreak in British Columbia, Canada. We provide laboratory validation of protocol modifications that restored sequencing performance. The study findings indicate that genomic sequencing protocols require immediate updating to address emerging mutations. This work also suggests that routine monitoring and protocol updates will be necessary as SARS-CoV-2 continues to evolve. The bioinformatic and laboratory approaches used here provide guidance for this kind of assay maintenance.

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.

Emergence and rapid transmission of SARS-CoV-2 B.1.1.7 in the United States.

The highly transmissible B.1.1.7 variant of SARS-CoV-2, first identified in the United Kingdom, has gained a foothold across the world. Using S gene target failure (SGTF) and SARS-CoV-2 genomic sequencing, we investigated the prevalence and dynamics of this variant in the United States (US), tracking it back to its early emergence. We found that, while the fraction of B.1.1.7 varied by state, the variant increased at a logistic rate with a roughly weekly doubling rate and an increased transmission of 40%-50%. We revealed several independent introductions of B.1.1.7 into the US as early as late November 2020, with community transmission spreading it to most states within months. We show that the US is on a similar trajectory as other countries where B.1.1.7 became dominant, requiring immediate and decisive action to minimize COVID-19 morbidity and mortality.

Passive Tick Surveillance and Detection of Borrelia Species in Ticks from British Columbia, Canada: 2002-2018.

Lyme disease, caused by Borrelia burgdorferi sensu lato (s.l.) complex, is the most common vector-borne disease in North America. This disease has a much lower incidence in western compared with eastern North America. Passive tick surveillance data submitted over 17 years from 2002 to 2018 were analyzed to determine the occurrence of tick species and the prevalence of Borrelia spp. in ticks in British Columbia (BC), Canada. The BC Centre for Disease Control Public Health Laboratory received tick submissions from physicians, veterinarians, and BC residents. Ticks were identified to species, and all ticks, except Dermacentor andersoni, were tested using generic B. burgdorferi s.l. primer sets and species-specific PCR primer sets for B. burgdorferi sensu stricto (s.s.). Tick submission data were analyzed to assess temporal and geographical trends, tick life stages, and tick species. Poisson regression was used to assess temporal trends in annual tick submissions. A total of 15,464 ticks were submitted. Among these, 0.29% (n = 10,235) of Ixodes spp. ticks and 5.3% (n = 434) of Rhipicephalus sanguineus ticks were found carrying B. burgdorferi s.s. B. burgdorferi s.s. was primarily detected in Ixodes pacificus (52%; n = 16) and Ixodes angustus ticks (19%; n = 6) retrieved from humans (n = 5) and animals (n = 26). B. burgdorferi was found in ticks submitted throughout the year. Ixodes spp. ticks were primarily submitted from the coastal regions of southwestern BC, and D. andersoni ticks were primarily submitted from southern interior BC. The number of human tick submissions increased significantly (p < 0.001) between 2013 and 2018. The annual prevalence of B. burgdorferi in ticks remained stable during the study period. These findings correspond to those observed in US Pacific Northwestern states. Passive tick surveillance is an efficient tool to monitor long-term trends in tick distribution and B. burgdorferi prevalence in a low endemicity region.

Genomic epidemiology identifies emergence and rapid transmission of SARS-CoV-2 B.1.1.7 in the United States.

As of January of 2021, the highly transmissible B.1.1.7 variant of SARS-CoV-2, which was first identified in the United Kingdom (U.K.), has gained a strong foothold across the world. Because of the sudden and rapid rise of B.1.1.7, we investigated the prevalence and growth dynamics of this variant in the United States (U.S.), tracking it back to its early emergence and onward local transmission. We found that the RT-qPCR testing anomaly of S gene target failure (SGTF), first observed in the U.K., was a reliable proxy for B.1.1.7 detection. We sequenced 212 B.1.1.7 SARS-CoV-2 genomes collected from testing facilities in the U.S. from December 2020 to January 2021. We found that while the fraction of B.1.1.7 among SGTF samples varied by state, detection of the variant increased at a logistic rate similar to those observed elsewhere, with a doubling rate of a little over a week and an increased transmission rate of 35-45%. By performing time-aware Bayesian phylodynamic analyses, we revealed several independent introductions of B.1.1.7 into the U.S. as early as late November 2020, with onward community transmission enabling the variant to spread to at least 30 states as of January 2021. Our study shows that the U.S. is on a similar trajectory as other countries where B.1.1.7 rapidly became the dominant SARS-CoV-2 variant, requiring immediate and decisive action to minimize COVID-19 morbidity and mortality.

Methods for preparing quantum gases of lithium.

Lithium is an important element in atomic quantum gas experiments because its interactions are highly tunable due to broad Feshbach resonances and zero-crossings and because it has two stable isotopes: 6Li, a fermion, and 7Li, a boson. Although lithium has special value for these reasons, it also presents experimental challenges. In this article, we review some of the methods that have been developed or adapted to confront these challenges, including beam and vapor sources, Zeeman slowers, sub-Doppler laser cooling, laser sources at 671 nm, and all-optical methods for trapping and cooling. Additionally, we provide spectral diagrams of both 6Li and 7Li and present plots of Feshbach resonances for both isotopes.

Evolutionary repression of chondrogenic genes in the vertebrate osteoblast.

Gene expression in extant animals might reveal how skeletal cells have evolved over the past 500 million years. The cells that make up cartilage (chondrocytes) and bone (osteoblasts) express many of the same genes, but they also have important molecular differences that allow us to distinguish them as separate cell types. For example, traditional studies of later-diverged vertebrates, such as mouse and chick, defined the genes Col2a1 and sex-determining region Y-box 9 as cartilage-specific. However, recent studies have shown that osteoblasts of earlier-diverged vertebrates, such as frog, gar, and zebrafish, express these 'chondrogenic' markers. In this review, we examine the resulting hypothesis that chondrogenic gene expression became repressed in osteoblasts over evolutionary time. The amphibian is an underexplored skeletal model that is uniquely positioned to address this hypothesis, especially given that it diverged when life transitioned from water to land. Given the relationship between phylogeny and ontogeny, a novel discovery for skeletal cell evolution might bolster our understanding of skeletal cell development.

Clinical signs of meibomian gland dysfunction (MGD) are associated with changes in meibum sphingolipid composition.

PURPOSE: Sphingolipids (SPL) play roles in cell signaling, inflammation, and apoptosis. Changes in SPL composition have been reported in individuals with MGD, but associations between clinical signs of MGD and compositional changes in meibum SPLs have not been examined. METHODS: Forty-three individuals underwent a tear film assessment. Groups were split into those with good or poor quality meibum. Meibum was collected then analyzed with liquid chromatography-mass spectroscopy to quantify SPL classes. Relative composition of SPL and major classes, Ceramide (Cer), Hexosyl-Ceramide (Hex-Cer), Sphingomyelin (SM), Sphingosine (Sph) and Sphingosine 1-phosphate (S1P) was calculated via mole percent. RESULTS: 22 and 21 individuals were characterized with good and poor quality meibum, respectively. Individuals with poor quality were older (60 ±â€¯8 vs 51 ±â€¯16 years) and more likely to be male (90% vs 64%). Relative composition analysis revealed that individuals with poor meibum quality had SPL composed of less Cer (33.36% vs 49.49%, p < 0.01), Hex-Cer (4.88% vs 9.15%, p < 0.01), and S1P (0.16% vs 0.31%, p = 0.05), and more SM (58.67% vs 38.18%, p < 0.01) and Sph (2.92% vs 2.87%, p = 0.97) compared to individuals with good quality meibum. Assessment of the ratio of Cer (pro-apoptotic) to S1P (pro-survival) showed that individuals with poor meibum quality had a relative increase in Cer (495.23 vs 282.69, p = 0.07). CONCLUSION: Meibum quality, a clinically graded marker of MGD, is associated with compositional changes in meibum sphingolipids. Further investigation of the structural and bioactive roles of sphingolipids in MGD may provide future targets for therapy.

High myopic photorefractive keratectomy outcomes with the Alcon Wavelight® EX500 excimer laser.

PURPOSE: To present refractive outcomes from consecutive cases with the Alcon Wavelight® EX500 excimer laser using photorefractive keratectomy (PRK) in patients with high myopia. METHODS: A retrospective chart review of consecutive cases of high myopic eyes (≥6.0 Diopters [D]) undergoing PRK with the Alcon Wavelight EX500 excimer laser (Alcon Laboratories, Fort Worth, TX, USA) was done. Moderately high myopic eyes (6.0 to <8.0 D [6 D]) were compared with high myopic eyes (8.0 D or greater [8 D]). Outcomes measured included pre- and postoperative refractive error, uncorrected distance visual acuity (UDVA), corrected distance visual acuity, spherical equivalent correction (SEQ), haze incidence, and intraocular pressure (IOP). RESULTS: One hundred eighteen eyes of 63 patients were evaluated, with 59 eyes having 12 months of follow-up. Thirty-one eyes of 19 patients had 8.0 D or more of myopia. Twelvemonth average LogMAR UDVA was -0.06 (20/17) for the 6 D group and -0.08 (20/16) for the 8 D group. Average 12-month SEQ was -0.18 D and preoperatively was -7.52 D for the 6 D group and -0.09 and -9.02 in the 8 D group. Sixty-five eyes (86%) and 24 eyes (96%) had an SEQ within 0.50 D of emmetropia at 3 months in the 6 and 8 D groups, respectively. One eye had visually significant haze developed at 8 months. Three eyes had IOP elevation that resolved with addition of short-term topical IOP-lowering medication. CONCLUSION: High myopic PRK with the Alcon Wavelight EX500 excimer laser yields excellent refractive outcomes with a low incidence of complications.

Long-term capsule clarity with a disk-shaped intraocular lens.

PURPOSE: To evaluate long-term capsular clarity with a modified disk-shaped 1-piece hydrophilic acrylic monofocal intraocular lens (IOL) (Zephyr) suspended between 2 complete haptic rings connected by a pillar of the haptic material. SETTING: John A. Moran Eye Center, University of Utah, Salt Lake City, Utah, USA. DESIGN: Experimental study. METHODS: Study and control (1-piece hydrophobic acrylic) IOLs were implanted into the right and left eyes, respectively, of 8 New Zealand rabbits. Eyes were examined at the slitlamp at set intervals for 6 months. At the end of the clinical follow-up, the globes were enucleated and capsular clarity was scored from the posterior view (Miyake-Apple technique). Then, all the eyes were processed for a complete histopathological evaluation. RESULTS: At 6 months, the slitlamp evaluation showed a posterior capsule opacification score of 0.28 ± 0.26 (SD) in the study group and 4 ± 0 in the control (P < .0001, paired t test). The anterior capsule was generally clear in the study group. This parameter was difficult to analyze in the control group because of synechiae formation and poor pupil dilation. CONCLUSIONS: The degree of capsular bag clarity observed at 6 months postoperatively in the study eyes in this rabbit model was exceptional. It was likely because of the IOL design, keeping the capsular bag open and expanded, and minimizing contact between the IOL and the anterior capsule.

Laboratory and Benchtop Performance of a Mealtime Insulin-Delivery System.

BACKGROUND: A basal bolus insulin regimen requires multiple daily insulin injections, which might discourage patient adherence. As a potential solution, a mealtime insulin-delivery system-a 3-day wearable bolus-only patch-was designed to manually administer mealtime insulin discreetly by actuating buttons through clothing, without the need for multiple needle sticks. METHOD: Extensive functional testing of the patch included dose accuracy (from initial fill of the device to empty), pressure-vacuum leak testing, last-dose lockout and occlusion detection (safety alert features that lock the dosing buttons when no insulin is delivered), assessments of insulin drug stability, toxicological risk (including chemical testing), and system biocompatibility. RESULTS: Dosing accuracy was 2 units ±10% (with U-100 insulin) over a range of environmental conditions, with ≥95% reliability and confidence. The fluid seal performance and the safety alert features performed with ≥95% reliability and ≥95% confidence. The system met acceptable standards for insulin (U-100 lispro and aspart) stability for its intended 3-day use, in addition to the operational requirements. The toxicological risk assessment and demonstrated biocompatibility suggested that the patch is safe for human use. CONCLUSIONS: Benchtop performance showed that the bolus-only patch is a safe, accurate, and reliable device for mealtime insulin delivery.