Targeted Amplification and Genetic Sequencing of the Severe Acute Respiratory Syndrome Coronavirus 2 Surface Glycoprotein (preprint)

The SARS-CoV-2 spike protein is a highly immunogenic and mutable protein that is the target of vaccine prevention and antibody therapeutics. This makes the encoding S-gene an important sequencing target. The SARS-CoV-2 sequencing community overwhelmingly adopted tiling amplicon-based strategies for sequencing the entire genome. As the virus evolved, primer mismatches inevitably led to amplicon drop-out. Given the exposure of the spike protein to host antibodies, mutation occurred here most rapidly, leading to amplicon failure over the most insightful region of the genome. To mitigate this, we developed SpikeSeq, a targeted method to amplify and sequence the S-gene. We evaluated 20 distinct primer designs through iterative in silico and in vitro testing to select the optimal primer pairs and run conditions. Once selected, periodic in silico analysis monitor primer conservation as SARS-CoV-2 evolves. Despite being designed during the Beta wave, the selected primers remain > 99% conserved through Omicron as of 2023-04-14. To validate the final design, we compared SpikeSeq data and National SARS-CoV-2 Strain Surveillance whole-genome data for 321 matching samples. Consensus sequences for the two methods were highly identical (99.998%) across the S-gene. SpikeSeq can serve as a complement to whole-genome surveillance or be leveraged where only S-gene sequencing is of interest. While SpikeSeq is adaptable to other sequencing platforms, the Nanopore platform validated here is compatible with low to moderate throughputs, and its simplicity better enables users to achieve accurate results, even in low resource settings.

Establishing SARS-CoV-2 Membrane protein-specific antibodies as a valuable serological target via high-content microscopy

The prevalence and strength of serological responses mounted towards SARS-CoV-2 proteins other than nucleocapsid (N) and spike (S), which may be of use as additional serological markers, remains underexplored. Using high content microscopy to assess antibody responses against full length StrepTagged SARS-CoV-2 proteins, we found that 85% (166/196) of unvaccinated individuals with RT-PCR confirmed SARS-CoV-2 infections and 74% (31/42) of individuals infected after being vaccinated developed detectable IgG against the structural protein M, which is higher than previous estimates. Compared with N antibodies, M IgG displayed a shallower time-dependent decay and greater specificity. Sensitivity for SARS-CoV-2 seroprevalence was enhanced when N and M IgG detection was combined. These findings indicate that screening for M seroconversion may be a good approach for detecting additional vaccine breakthrough infections and highlight the potential to use HCM as a rapidly deployable method to identify the most immunogenic targets of newly emergent pathogens. Graphical

Managing systemic risk in emergency management, organizational resilience and climate change adaptation

PurposeThis paper applies the theory of cascading, interconnected and compound risk to the practice of preparing for, managing, and responding to threats and hazards. Our goal is to propose a consistent approach for managing major risk in urban systems by bringing together emergency management, organisational resilience, and climate change adaptation.Design/methodology/approachWe develop a theory-building process using an example from the work of the Greater London Authority in the United Kingdom. First, we explore how emergency management approaches systemic risk, including examples from of exercises, contingency plans and responses to complex incidents. Secondly, we analyse how systemic risk is integrated into strategies and practices of climate change adaptation. Thirdly, we consider organisational resilience as a cross cutting element between the approaches.FindingsLondon has long been a champion of resilience strategies for dealing with systemic risk. However, this paper highlights a potential for integrating better the understanding of common points of failure in society and organisations, especially where they relate to interconnected domains and where they are driven by climate change.Originality/valueThe paper suggests shifting toward the concept of operational continuity to address systemic risk and gaps between Emergency Management, Organizational Resilience and Climate Change Adaptation.

Immune Reconstitution and Vaccinations in Multiple Myeloma: A Report From the 19th International Myeloma Society Annual Workshop.

Given the significance of the immune system and the important role of therapies within the context of the immune system in plasma cell disorders, the International Myeloma Society annual workshop convened a session dedicated to this topic. A panel of experts covered various aspects of immune reconstitution and vaccination. The top oral presentations were highlighted and discussed. This is a report of the proceedings.

When Characteristics of Clinical Trials Require Per-Protocol as Well as Intention-to-Treat Outcomes to Draw Reliable Conclusions: Three Examples.

Under exceptional circumstances, including high rates of protocol non-compliance, per-protocol (PP) analysis can better indicate the real-world benefits of a medical intervention than intention-to-treat (ITT) analysis. Exemplifying this, the first randomized clinical trial (RCT) considered found that colonoscopy screenings were marginally beneficial, based upon ITT analysis, with only 42% of the intervention group actually undergoing the procedure. However, the study authors themselves concluded that the medical efficacy of that screening was a 50% reduction in colorectal cancer deaths among that 42% PP group. The second RCT found a ten-fold reduction in mortality for a COVID-19 treatment drug vs. placebo by PP analysis, but only a minor benefit by ITT analysis. The third RCT, conducted as an arm of the same platform trial as the second RCT, tested another COVID-19 treatment drug and reported no significant benefit by ITT analysis. Inconsistencies and irregularities in the reporting of protocol compliance for this study required consideration of PP outcomes for deaths and hospitalizations, yet the study coauthors refused to disclose them, instead directing inquiring scientists to a data repository which never held the study's data. These three RCTs illustrate conditions under which PP outcomes may differ significantly from ITT outcomes and the need for data transparency when these reported or indicated discrepancies arise.

Prevalence of SARS-CoV-2 Viremia in Presymptomatic Blood Donors in the Delta and Omicron Variant Eras.

Presymptomatic plasma samples from 1596 donors reporting coronavirus disease 2019 infection or symptoms after blood donation were tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and anti-S and anti-N antibodies. Prior infection and vaccination both protected from developing SARS-CoV-2 RNAemia and from symptomatic infection. RNAemia rates did not differ in the Delta and Omicron variant eras.

Protocol for studying co-infection between SARS-CoV-2 and Staphylococcus aureus in vitro.

Bacterial co-infection is one of the most common complications of SARS CoV-2 infection. Here, we present a protocol for the in vitro study of co-infection between SARS CoV-2 and Staphylococcus aureus. We describe steps for quantifying viral and bacterial replication kinetics in the same sample, with the optional extraction of host RNA and proteins. This protocol is applicable to many viral and bacterial strains and can be performed in different cell types. For complete details on the use and execution of this protocol, please refer to Goncheva et al.1.

Approaches to Continuing COVID-19-Related Clinical Research Practices After the Pandemic-Must Cinderella Leave the Ball?

This Viewpoint reviews relevant components of federal regulation and guidance followed during the COVID-19 pandemic that may still be feasible and beneficial after the public health emergency ends.

Tele-Urology During COVID-19: Rapid Implementation of Remote Video Visits.

INTRODUCTION: COVID-19 has brought unprecedented challenges to the delivery of urological care. Following rapid implementation of remote video visits at our tertiary academic medical center serving a large rural population we describe and assess our experience with planned video visits and ongoing scheduling efforts. METHODS: Patients scheduled for video visits between April 14 and April 27, 2020 were included. Prospective and retrospective data were collected on patient and clinical characteristics as well as telemedicine outcomes. Multivariable logistic regression was performed to evaluate factors influencing video visit success. Concurrently scheduling data were collected from a separate cohort regarding patient access to technology and willingness to participate in video visits. RESULTS: A total of 209 patients were included with an overall video visit success rate of 67%. Of video visits that failed (69) reasons included no-show (35%), inability to connect to the telemedicine platform (23%) and lack of Internet access (10%). Nearly half of failed video visits (46.4%) were completed as phone visits. After adjustment for patient demographics commercial insurance was significantly associated with video visit success. In assessment of scheduling outcomes 179 patients were contacted to offer video visits. Of these patients 6.7% reported not having Internet access. Of those with Internet access 87% agreed to proceed with a video visit in lieu of visiting in person. CONCLUSIONS: Our experience indicates that rapid implementation of video telemedicine is feasible and highly accepted by patients. Efforts focused on standardized pre-visit patient education may further optimize successful telemedicine visits.

AI-guided pipeline for protein-protein interaction drug discovery identifies a SARS-CoV-2 inhibitor (preprint)

Protein-protein interactions (PPIs) offer great opportunities to expand the druggable proteome and therapeutically tackle various diseases, but remain challenging targets for drug discovery. Here, we provide a comprehensive pipeline that combines experimental and computational tools to identify and validate PPI targets and perform early-stage drug discovery. We have developed a machine learning approach that prioritizes interactions by analyzing quantitative data from binary PPI assays and AlphaFold-Multimer predictions. Using the quantitative assay LuTHy together with our machine learning algorithm, we identified high-confidence interactions among SARS-CoV-2 proteins for which we predicted three-dimensional structures using AlphaFold Multimer. We employed VirtualFlow to target the contact interface of the NSP10-NSP16 SARS-CoV-2 methyltransferase complex by ultra-large virtual drug screening. Thereby, we identified a compound that binds to NSP10 and inhibits its interaction with NSP16, while also disrupting the methyltransferase activity of the complex, and SARS-CoV-2 replication. Overall, this pipeline will help to prioritize PPI targets to accelerate the discovery of early-stage drug candidates targeting protein complexes and pathways.

Pressure injuries in COVID-19 patients: A tertiary center experience.

Pressure ulcers continue to severely impact patient outcomes and increase health care costs. We aimed to examine the incidence and risk factors related to pressure ulcers among COVID-19 patients. A retrospective was conducted between March 2020-April 2021. Baseline differences were examined using chi-square and Fischer's exact test. Logistic regression was employed to examine the association of the collected variables to development of new pressure ulcers. 4608 patients were included, of which eighty-three acquired new pressure ulcers. Risk factors were increased age, peripheral artery disease, abnormal albumin levels, but not prone position.

Multidisciplinary teams, efficient communication, procedure services, and telehealth improve cirrhosis care: A qualitative study.

BACKGROUND: Cirrhosis care and outcomes are improved with access to subspecialty gastroenterology and hepatology care. In qualitative interviews, we investigated clinicians' perceptions of factors that optimize or impede cirrhosis care. METHODS: We conducted 24 telephone interviews with subspecialty clinicians at 7 Veterans Affairs medical centers with high- and low-complexity services. Purposive sampling stratified Veterans Affairs medical centers on timely post-hospitalization follow-up, a quality measure. We asked open-ended questions about facilitators and barriers of care coordination, access to appointments, procedures, transplantation, management of complications, keeping up to date with medical knowledge, and telehealth use. RESULTS: Key themes that facilitated care were structural: multidisciplinary teams, clinical dashboards, mechanisms for appointment tracking and reminders, and local or virtual access to transplant and liver cancer specialists through the "specialty care access network extension for community health care outcomes" program. Coordination and efficient communication between transplant and non-transplant specialists and between transplant and primary care facilitated timely care. Same-day access to laboratory, procedural, and clinical services is an indicator of high-quality care. Barriers included lack of on-site procedural services, clinician turnover, patient social needs related to transportation, costs, and patient forgetfulness due to HE. Telehealth enabled lower complexity sites to obtain recommendations for complex patient cases. Barriers to telehealth included lack of credit (eg, VA billing equivalent), inadequate staff, lack of audiovisual technology support, and patient and staff discomfort with technology. Telehealth was optimal for return visits, cases where physical examination was nonessential, and where distance and transportation precluded in-person care. Rapid telehealth uptake during the COVID-19 pandemic was a positive disruptor and facilitated use. CONCLUSIONS: We identify multi-level factors related to structure, staffing, technology, and care organization to optimize cirrhosis care delivery.

Impact Of The Vaccination Against Covid-19 On Frontline Health Workers.

INTRODUCTION: Brazil has been facing the pandemic of COVID-19 since march 2020. To date, more than 540,000 people have died from this disease in the country. There are estimates that indicate that the population exposed to SARS-CoV-2 represents 1 to 20%, however, these data are questionable due to the number of asymptomatic and untested individuals. As a result, vaccination for COVID-19 has become the main means of achieving herd immunity. OBJECTIVES: To demonstrate, through local sampling, that broad and rapid vaccination may decrease the rate of COVID-19 detection in individuals potentially exposed to the SARS-CoV-2 virus. RESULTS: A total of 1,128 individuals were studied, including students and health professionals from Centro Universitário FMABC, who received the two doses of the vaccine for COVID-19 (Oxford/Astrazeneca ® and CoronaVac®). There was a 41% reduction in the demand for RT-PCR test after vaccination, in the studied period. And a 78.3% reduction in positive results after vaccination started Conclusion: The results of this study showed that, even vaccinating a population with higher exposure to the risk of contamination, there was a significant reduction in test positivity and in the demand to perform these tests. Emphasizing that vaccination is the best strategy to achieve herd immunity and to reduce the spread of the disease.

Researching COVID to enhance recovery (RECOVER) adult study protocol: Rationale, objectives and design (preprint)

Importance: SARS-CoV-2 infection can result in ongoing, relapsing, or new symptoms or other health effects after the acute phase of infection; termed post-acute sequelae of SARS-CoV-2 infection (PASC), or long COVID. The characteristics, prevalence, trajectory and mechanisms of PASC are ill-defined. The objectives of the Researching COVID to Enhance Recovery (RECOVER) Multi-site Observational Study of PASC in Adults (RECOVER-Adult) are to: (1) characterize PASC prevalence; (2) characterize the symptoms, organ dysfunction, natural history, and distinct phenotypes of PASC; (3) identify demographic, social and clinical risk factors for PASC onset and recovery; and (4) define the biological mechanisms underlying PASC pathogenesis. Methods: RECOVER-Adult is a combined prospective/retrospective cohort currently planned to enroll 14,880 adults aged [≥]18 years. Eligible participants either must meet WHO criteria for suspected, probable, or confirmed infection; or must have evidence of no prior infection. Recruitment occurs at 86 sites in 33 U.S. states, Washington, DC and Puerto Rico, via facility- and community-based outreach. Participants complete quarterly questionnaires about symptoms, social determinants, vaccination status, and interim SARS-CoV-2 infections. In addition, participants contribute biospecimens and undergo physical and laboratory examinations at approximately 0, 90 and 180 days from infection or negative test date, and yearly thereafter. Some participants undergo additional testing based on specific criteria or random sampling. Patient representatives provide input on all study processes. The primary study outcome is onset of PASC, measured by signs and symptoms. A paradigm for identifying PASC cases will be defined and updated using supervised and unsupervised learning approaches with cross-validation. Logistic regression and proportional hazards regression will be conducted to investigate associations between risk factors, onset, and resolution of PASC symptoms. Discussion: RECOVER-Adult is the first national, prospective, longitudinal cohort of PASC among US adults. Results of this study are intended to inform public health, spur clinical trials, and expand treatment options.

IMPLEMENTATION INSIGHTS from SITE CHAMPIONS and STATE LEADS POST-ROLLOUT of the SAFER BABY BUNDLE

Background: National rollout of the Safer Baby Bundle (SBB) is recommended by the National Stillbirth Action and Implementation Plan for improving the standard of antenatal care at scale to reduce stillbirth. Progressive implementation over 2.5 years, commenced from mid-2019. This qualitative study aims to understand the process enablers and barriers influencing the effectiveness of SBB implementation. Method(s): Semi-structured interviews with SBB site leads/champions (17 maternity services) and state program leads across Queensland (QLD), Victoria (VIC) and New South Wales (NSW) were conducted post-implementation of SBB to explore local, regional, and state implementation strategies, processes and experiences. Normalization Process Theory was used as an analytic framework for thematic analysis to understand different approaches and contexts for SBB implementation. Result(s): 17 site leads (6 VIC, 5 NSW, 6 QLD) and 6 state program leads were interviewed from Sept to mid-Nov 2022. Findings indicate strong local leadership, shared regional and state-based learning opportunities, consistency of information and endorsement by clinical networks were key implementation supports. Barriers included limitations and inconsistencies across data and information systems, and constraints with protected time for training and improvement activities. High turnover and re-deployment of leads due to external factors (e.g., COVID, extreme weather events) disrupted timelines, impacting commitment and engagement with the initiative. Conclusion(s): Despite differences in resources, approaches, and timelines between sites and states for SBB implementation, similar key barriers and enablers were identified. An in-depth understanding of the factors underpinning successful implementation of the SBB will guide future activities to support sustainable change.

The Selah study protocol of three interventions to manage stress among clergy: a preference-based randomized waitlist control trial.

INTRODUCTION: Like many helping professionals in emotional labor occupations, clergy experience high rates of mental and physical comorbidities. Regular stress management practices may reduce stress-related symptoms and morbidity, but more research is needed into what practices can be reliably included in busy lifestyles and practiced at a high enough level to meaningfully reduce stress symptoms. METHODS AND ANALYSIS: The overall design is a preference-based randomized waitlist control trial. United Methodist clergy in North Carolina will be eligible to participate. The intervention and waitlist control groups will be recruited by email. The interventions offered are specifically targeted to clergy preference and include mindfulness-based stress reduction, Daily Examen, and stress inoculation training. Surveys will be conducted at 0, 12, and 24 weeks with heart rate data collected at 0 and 12 weeks. The primary outcomes for this study are self-reported symptoms of stress and heart rate at week 12 for each intervention compared to waitlist control; the secondary outcome is symptoms of anxiety comparing each intervention vs waitlist control. ETHICS AND DISSEMINATION: Ethical approval was obtained from the Duke University Campus IRB (2019-0238). The results will be made available to researchers, funders, and members of the clergy community. STRENGTHS AND LIMITATIONS OF THIS STUDY: While evidence-based stress reduction practices such as mindfulness-based stress reduction (MBSR) exist, a wider variety of practices should be tested to appeal to different individuals. Clergy in particular may prefer, and consequently enact, spiritual practices like the Daily Examen, and individuals such as clergy who spend most of their time thinking and feeling may prefer experiential-based practices like stress inoculation training. If efficacious, the Daily Examen and stress inoculation training practices have high feasibility in that they require few minutes per day. This study is limited by the inclusion of Christian clergy of only one denomination. TRIAL REGISTRATION: ClinicalTrials.gov NCT04625777 . November 12, 2020.

Novel Trial Design: CHIEF-HF.

BACKGROUND: The expense of clinical trials mandates new strategies to efficiently generate evidence and test novel therapies. In this context, we designed a decentralized, patient-centered randomized clinical trial leveraging mobile technologies, rather than in-person site visits, to test the efficacy of 12 weeks of canagliflozin for the treatment of heart failure, regardless of ejection fraction or diabetes status, on the reduction of heart failure symptoms. METHODS: One thousand nine hundred patients will be enrolled with a medical record-confirmed diagnosis of heart failure, stratified by reduced (≤40%) or preserved (>40%) ejection fraction and randomized 1:1 to 100 mg daily of canagliflozin or matching placebo. The primary outcome will be the 12-week change in the total symptom score of the Kansas City Cardiomyopathy Questionnaire. Secondary outcomes will be daily step count and other scales of the Kansas City Cardiomyopathy Questionnaire. RESULTS: The trial is currently enrolling, even in the era of the coronavirus disease 2019 (COVID-19) pandemic. CONCLUSIONS: CHIEF-HF (Canagliflozin: Impact on Health Status, Quality of Life and Functional Status in Heart Failure) is deploying a novel model of conducting a decentralized, patient-centered, randomized clinical trial for a new indication for canagliflozin to improve the symptoms of patients with heart failure. It can model a new method for more cost-effectively testing the efficacy of treatments using mobile technologies with patient-reported outcomes as the primary clinical end point of the trial. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT04252287.

The Reports of my Death are Greatly Exaggerated: An Evaluation of Futility Cut-Points in Massive Transfusion.

BACKGROUND: Following COVID and the subsequent blood shortage, several investigators evaluated futility cut-points in massive transfusion. We hypothesized that early, aggressive use of damage control resuscitation, including whole blood (WB), would demonstrate that these cut-points of futility were significantly underestimating potential survival among patients receiving >50 units of blood in the first four hours. METHODS: Adult trauma patients admitted from 11/2017-10/2021 who received emergency-release blood products in prehospital or ED setting were included. Deaths within 30 min of arrival were excluded. Total blood products were defined as total RBC, plasma, WB in the field and in the first 4 hours after arrival. Patients were first divided into those receiving ≤50 or > 50 units of blood in the first 4 hours. We then evaluated patients by whether they received any WB or received only component therapy (COMP). 30-day survival was evaluated for all included patients. RESULTS: 2,299 patients met inclusion (2,043 in ≤50 U, 256 in >50 U groups). While there were no differences in age or gender, the >50 U group was more likley to sustain penetrating injury (47 vs 30%, p < 0.05). Patients receiving >50 U of blood had lower field and arrival blood pressure and larger prehospital and ED resuscitation volumes (p < 0.05). Patients in the >50 U group had lower survival than those in the ≤50 cohort (31 vs 79%; p < 0.05). Patients who received WB (n = 1,291) had 43% increased odds of survival compared to those who received COMP (n = 1,008)(1.09-1.87, p = 0.009) as well as higher 30-day survival at transfusion volumes >50 U. CONCLUSION: Patient survival rates in patients receiving >50 units of blood in the first 4 hours of care are as high as 50-60%, with survival still at 15-25% after 100 units. While responsible blood stewardship is critical, futility should not be declared based on high transfusion volumes alone. LEVEL OF EVIDENCE: Level III, Retrospective comparative study without negative criteria.