[Do not resuscitate orders and limitation of therapeutic effort: Ethical challenges in healthcare teams in Chile]. / Orden de no reanimar y limitación del esfuerzo terapéutico: desafíos éticos en equipos sanitarios en Chile.

The purpose of this paper is to delve into the ethical aspects experienced by the healthcare team when they receive the directive to limit therapeutic effort or a do-not-resuscitate order. From an interpretative, qualitative paradigm with a content analysis approach, a process based on three phases was conducted: pre-analysis in which categories were identified, the projection of the analysis, and inductive analysis. During 2023, interviews were conducted in the clinical setting of a high-complexity hospital in Chile with 56 members of the healthcare teams from critical and emergency units, from which four categories emerged: a) the risk of violating patients' rights by using do-not-resuscitate orders and limiting therapeutic effort; b) the gap in the interpretation of the legal framework addressing the care and attention of patients at the end of life or with terminal illnesses by the healthcare team; c) ethical conflicts in end-of-life care; and d) efficient care versus holistic care in patients with terminal illness. There are significant gaps in bioethics training and aspects of a good death in healthcare teams facing the directive to limit therapeutic effort and not resuscitate. It is suggested to train personnel and work on a consensus guide to address the ethical aspects of a good death.

El propósito de este trabajo es profundizar en los aspectos éticos que experimenta el equipo de salud cuando reciben la indicación de limitar el esfuerzo terapéutico o la orden de no reanimar. Desde un paradigma interpretativo, cualitativo y con un enfoque de análisis de contenido, se realizó un proceso basado en tres fases: preanálisis en el que se identificaron las categorías, la proyección del análisis y el análisis inductivo. Durante 2023, se realizaron entrevistas en el entorno clínico de un hospital de alta complejidad en Chile a 56 miembros de equipos de salud de unidades críticas y urgencias, de las que emergieron cuatro categorías: a) riesgo de vulnerar los derechos de los pacientes al utilizar la orden de no reanimar, y limitación del esfuerzo terapéutico; b) brecha en la interpretación del marco legal que aborda la atención y cuidado de pacientes al final de la vida, o con enfermedades terminales por parte del equipo de salud; c) conflictos éticos de la atención al final de la vida; y d) el cuidado eficiente o el cuidado holístico en pacientes con enfermedad terminal. Existen brechas importantes en la formación en bioética y aspectos del buen morir en los equipos de salud que se enfrentan a la orden de limitar el esfuerzo terapéutico y no reanimar. Se sugiere capacitar al personal, y trabajar una guía de consenso para abordar los aspectos éticos del buen morir.

One-year longitudinal changes of peripheral CD4+ T-lymphocyte counts, gut microbiome, and plaque vulnerability after an acute coronary syndrome.

Background: Longitudinal changes in gut microbiome and inflammation may be involved in the evolution of atherosclerosis after an acute coronary syndrome (ACS). We aimed to characterize repeated profiles of gut microbiota and peripheral CD4+ T lymphocytes during the first year after an ACS, and to address their relationship with atherosclerotic plaque changes. Methods: Over one year we measured the microbiome, peripheral counts of CD4+ T populations and cytokines in 67 patients shortly after a first ACS. We compared baseline measurements to those of a matched population of 40 chronic patients. A subgroup of 20 ACS patients underwent repeated assessment of fibrous cap thickness (FCT) of a non-culprit lesion. Results: At admission, ACS patients showed gut dysbiosis compared with the chronic group, which was rapidly reduced and remained low at 1-year. Also, their Th1 and Th2 CD4+ T counts were increased but decreased over time. The CD4+ T counts were related to ongoing changes in gut microbiome. Unsupervised clustering of repeated CD4+ Th0, Th1, Th2, Th17 and Treg counts in ACS patients identified two different cell trajectory patterns, related to cytokines. The group of patients following a high-CD4+ T cell trajectory showed a one-year reduction in their FCT [net effect = -24.2 µm; p = 0.016]. Conclusions: Patients suffering an ACS show altered profiles of microbiome and systemic inflammation that tend to mimic values of chronic patients after 1-year. However, in one-third of patients, this inflammatory state remains particularly dysregulated. This persistent inflammation is likely related to plaque vulnerability as evident by fibrous cap thinning (Clinical Trial NCT03434483).

A clinical practice guideline for the management of the foot and ankle in rheumatoid arthritis.

Rheumatoid arthritis causes progressive joint destruction in the long term, causing a deterioration of the foot and ankle. A clinical practice guideline has been created with the main objective of providing recommendations in the field of podiatry for the conservative management of rheumatoid arthritis. Thus, healthcare professionals involved in foot care of adults with rheumatoid arthritis will be able to follow practical recommendations. A clinical practice guideline was created including a group of experts (podiatrists, rheumatologists, nurses, an orthopaedic surgeon, a physiotherapist, an occupational therapist and patient with rheumatoid arthritis). Methodological experts using GRADE were tasked with systematically reviewing the available scientific evidence and developing the information which serves as a basis for the expert group to make recommendations. Key findings include the efficacy of chiropody in alleviating hyperkeratotic lesions and improving short-term pain and functionality. Notably, custom and standardized foot orthoses demonstrated significant benefits in reducing foot pain, enhancing physical function, and improving life quality. Therapeutic footwear was identified as crucial for pain reduction and mobility improvement, emphasizing the necessity for custom-made options tailored to individual patient needs. Surgical interventions were recommended for cases which were non-responsive to conservative treatments, aimed at preserving foot functionality and reducing pain. Moreover, self-care strategies and education were underscored as essential components for promoting patient independence and health maintenance. A series of recommendations have been created which will help professionals and patients to manage podiatric pathologies derived from rheumatoid arthritis.

Cardiac Stasis Imaging, Stroke and Silent Brain Infarcts in Patients with Non-Ischemic Dilated Cardiomyopathy.

Cardioembolic stroke is one of the most devastating complications of non-ischemic dilated cardiomyopathy (NIDCM). However, in clinical trials of primary prevention, the benefits of anticoagulation are hampered by the risk of bleeding. Indices of cardiac blood stasis may account for the risk of stroke and be useful to individualize primary prevention treatments. We performed a cross-sectional study in patients with NIDCM and no history of atrial fibrillation (AF) from two sources: 1) a prospective enrollment of unselected patients with left ventricular (LV) ejection fraction <45% and 2) a retrospective identification of patients with a history of previous cardioembolic neurological event. The primary endpoint integrated a history of ischemic stroke or the presence intraventricular thrombus, or a silent brain infarction (SBI) by imaging. From echocardiography, we calculated blood flow inside the LV, its residence time (RT) maps and its derived stasis indices. Of the 89 recruited patients, 18 showed a positive endpoint: 9 had a history stroke or TIA and 9 were diagnosed with SBIs in the brain imaging. Averaged RT, performed good to identify the primary endpoint (AUC (95% CI)= 0.75 (0.61-0.89), p= 0.001). When accounting only for identifying a history of stroke or TIA, AUC for was 0.92 (0.85-1.00) with and odds ratio= 7.2 (2.3 - 22.3) per cycle, p< 0.001. These results suggest that, in patients with NIDCM in sinus rhythm, stasis imaging derived from echocardiography may account for the burden of stroke.

Are local public expenditure reductions associated with increases in inequality in emergency hospitalisation? Time-series analysis of English local authorities from 2010 to 2017.

BACKGROUND: Reductions in local government funding implemented in 2010 due to austerity policies have been associated with worsening socioeconomic inequalities in mortality. Less is known about the relationship of these reductions with healthcare inequalities; therefore, we investigated whether areas with greater reductions in local government funding had greater increases in socioeconomic inequalities in emergency admissions. METHODS: We examined inequalities between English local authority districts (LADs) using a fixed-effects linear regression to estimate the association between LAD expenditure reductions, their level of deprivation using the Index of Multiple Deprivation (IMD) and average rates of (all and avoidable) emergency admissions for the years 2010-2017. We also examined changes in inequalities in emergency admissions using the Absolute Gradient Index (AGI), which is the modelled gap between the most and least deprived neighbourhoods in an area. RESULTS: LADs within the most deprived IMD quintile had larger pounds per capita expenditure reductions, higher rates of all and avoidable emergency admissions, and greater between-neighbourhood inequalities in admissions. However, expenditure reductions were only associated with increasing average rates of all and avoidable emergency admissions and inequalities between neighbourhoods in local authorities in England's three least deprived IMD quintiles. For a LAD in the least deprived IMD quintile, a yearly reduction of £100 per capita in total expenditure was associated with a yearly increase of 47 (95% CI 22 to 73) avoidable admissions, 142 (95% CI 70 to 213) all-cause emergency admissions and a yearly increase in inequalities between neighbourhoods of 48 (95% CI 14 to 81) avoidable and 140 (95% CI 60 to 220) all-cause emergency admissions. In 2017, a LAD average population was ~170 000. CONCLUSION: Austerity policies implemented in 2010 impacted less deprived local authorities, where emergency admissions and inequalities between neighbourhoods increased, while in the most deprived areas, emergency admissions were unchanged, remaining high and persistent.

Measurement properties of the Foot Function Index (FFI) questionnaire: A systematic review.

OBJECTIVE: Large numbers of people are subject to alterations and pathologies in the foot. To quantify how these problems of foot function affect the quality of life, clinicians and researchers have developed measures such as the Foot Function Index (FFI). Our aim is to determine the methodological quality of the FFI including adaptations to other languages. DATA SOURCES: The studies considered in this review were extracted from the PubMed, Embase and CINAHL databases. The inclusion criteria were followed: (1) studies of patients with no previous foot or ankle pathology and aged over 18 years; (2) based on English-language patient-reported outcome measures that assess foot function; (3) the patient-reported outcome measures should present measurement properties based on COnsensus-based Standards for the selection of health Measurement Instruments (COSMIN) criteria. REVIEW METHODS: The systematic review was conducted following the COSMIN criteria to establish the methodological quality of the original FFI, together with its variants and adaptations. The last search was carried out in May 2024. RESULTS: Of the 1994 studies obtained in the preliminary search, 20 were eligible for inclusion in the final analysis. These results are the validations and cross-cultural adaptations to the following languages: the original FFI has cross-cultural adaptation in 13 languages and the FFI-Revised Short Form has been adapted and validated for use in 2 languages. CONCLUSION: In terms of methodological quality, the FFI-Revised Short Form questionnaire is a valuable instrument for evaluating ankle and foot function and could usefully be expanded to be available in more languages.

Correction: Socioeconomic, demographic and geographic determinants of food consumption in Mexico.

[This corrects the article DOI: 10.1371/journal.pone.0288235.].

Impaired islet function and normal exocrine enzyme secretion occur with low inter-regional variation in type 1 diabetes.

Histopathological heterogeneity in the human pancreas is well documented; however, functional evidence at the tissue level is scarce. Herein, we investigate in situ glucose-stimulated islet and carbachol-stimulated acinar cell secretion across the pancreas head (PH), body (PB), and tail (PT) regions in donors without diabetes (ND; n = 15), positive for one islet autoantibody (1AAb+; n = 7), and with type 1 diabetes (T1D; <14 months duration, n = 5). Insulin, glucagon, pancreatic amylase, lipase, and trypsinogen secretion along with 3D tissue morphometrical features are comparable across regions in ND. In T1D, insulin secretion and beta-cell volume are significantly reduced within all regions, while glucagon and enzymes are unaltered. Beta-cell volume is lower despite normal insulin secretion in 1AAb+, resulting in increased volume-adjusted insulin secretion versus ND. Islet and acinar cell secretion in 1AAb+ are consistent across the PH, PB, and PT. This study supports low inter-regional variation in pancreas slice function and, potentially, increased metabolic demand in 1AAb+.

Daily compliance of the ABCDEF liberation bundle for patients in the intensive care unit: A retrospective descriptive study / Cumplimiento diario del bundle ABCDEF de liberación de los pacientes de cuidados intensivos: estudio descriptivo retrospectivo.

Introduction: Implementing the ABCDEF bundle has demonstrated improved outcomes in patients with critical illness. This study aims to describe the daily compliance of the ABCDEF bundle in a Chilean intensive care unit. Methods: Retrospective observational study of electronic clinical records of nursing, physiotherapy, and medical professionals who cared for patients over 18 years of age, admitted to an intensive care unit for at least 24 hours, with or without mechanical ventilation. Daily bundle compliance was determined by considering the daily records for each element: Assess pain (element A), both spontaneous awakening trials (element B1) and spontaneous breathing trials (element B2), choice of sedation (element C), delirium assessment (element D), early mobilization (element E), and family engagement (element F). Results: 4165 registered bundle elements were obtained from nursing (47%), physiotherapy (44%), and physicians (7%), including 1134 patient/days (from 133 patients). Elements E and C showed 67 and 40% compliance, while D, A, and B2 showed 24, 14 and 11%, respectively. For B1 and F, 0% compliance was achieved. Compliance was higher in patients without mechanical ventilation for A and E, while it was similar for D. Conclusions: Early mobilization had the highest compliance, while spontaneous awakening trials and family engagement had absolute non-compliance. Future studies should explore the reasons for the different degrees of compliance per bundle element in clinical practice.

Introducción: La implementación del ABCDEF ha demostrado mejores resultados en los pacientes críticos. El objetivo de este trabajo es identificar el cumplimiento del registro diario del ABCDEF en una unidad de cuidados intensivos chilena. Métodos: Estudio observacional retrospectivo de los registros clínicos electrónicos de profesionales de enfermería, kinesiología y medicina que trataron a pacientes mayores de 18 años, hospitalizados en una unidad de cuidados intensivos durante al menos 24 horas, con o sin requerimiento de ventilación mecánica. Se determinó el cumplimiento diario del considerando la presencia del registro en la ficha clínica de cada elemento: evaluación del dolor (elemento A), prueba de interrupción de la sedación (elemento B1) y ventilación espontánea (elemento B2), elección de la sedación (elemento C), evaluación del (elemento D), movilización temprana (elemento E) y empoderamiento de la familia (elemento F). Resultados: Se obtuvieron 4165 elementos del registrados provenientes de enfermería (47%), kinesiología (44%) y medicina (7%), incluyendo 1134 días/paciente (133 pacientes). Los elementos E y C mostraron un cumplimiento del 67 y 40%, mientras que D, A, y B2 mostraron 24, 14 y 11%, respectivamente. Para B1 y F se obtuvo 0% de cumplimiento. El cumplimiento fue mayor en los pacientes sin ventilación mecánica para A y E, mientras que para D fue similar. Conclusiones: La movilización temprana fue el elemento con mayor cumplimiento, mientras que las pruebas de interrupción de sedación y el empoderamiento de la familia tuvieron incumplimiento absoluto. Futuros estudios deberían explorar las razones que expliquen los diferentes grados de cumplimiento por elemento del en la práctica clínica.

Aetiology, epidemiology and treatment of musculoskeletal injuries in badminton players: a systematic review and meta-analysis.

The present study has two main goals: to conduct a systematic review of musculoskeletal injuries experienced by badminton players, and to examine the management of such injuries. Searches were conducted of the PROSPERO, PubMed, Scopus, and SPORTDiscus databases, from their inception until March 2023. The papers analysed were all based on a study population consisting of individuals aged 18 years or more, diagnosed with badminton-related injuries. The methodological quality assessments was using the Newcastle-Ottawa Scale and REVMAN. A total of 28 studies were included in the systematic review. In total, the analysis included 2435 participants. Of these athletes, 35.6% (1012) were female and 64.4% (1503) were male. By type of injury, sprains were the most commonly studied and the most prevalent, accounting for 36.06% of the sample. These were followed by muscle injuries, representing 23.86% of the total. Injuries to the joints were the least prevalent, accounting for 4.97% of the sample. Lower limb injuries accounted for 52.15% of the total. Of these, ankle injuries were the most common. Despite the generally low quality of the studies considered, the evidence suggests that musculoskeletal injuries, especially to the lower limb, most commonly affect badminton players of all levels.

Liver respiratory-induced motion estimation using abdominal surface displacement as a surrogate: robotic phantom and clinical validation with varied correspondence models.

PURPOSE: This work presents the implementation of an RGB-D camera as a surrogate signal for liver respiratory-induced motion estimation. This study aims to validate the feasibility of RGB-D cameras as a surrogate in a human subject experiment and to compare the performance of different correspondence models. METHODS: The proposed approach uses an RGB-D camera to compute an abdominal surface reconstruction and estimate the liver respiratory-induced motion. Two sets of validation experiments were conducted, first, using a robotic liver phantom and, secondly, performing a clinical study with human subjects. In the clinical study, three correspondence models were created changing the conditions of the learning-based model. RESULTS: The motion model for the robotic liver phantom displayed an error below 3 mm with a coefficient of determination above 90% for the different directions of motion. The clinical study presented errors of 4.5, 2.5, and 2.9 mm for the three different motion models with a coefficient of determination above 80% for all three cases. CONCLUSION: RGB-D cameras are a promising method to accurately estimate the liver respiratory-induced motion. The internal motion can be estimated in a non-contact, noninvasive and flexible approach. Additionally, three training conditions for the correspondence model are studied to potentially mitigate intra- and inter-fraction motion.

Primary central nervous system lymphoma: A mirror type presentation in an immunocompetent patient.

Background: Primary central nervous system (CNS) lymphoma is a very rare extranodal non-Hodgkin lymphoma. The bilateral pattern, as we call it "mirror type", has been identified in other CNS lesions such as gliomas, metastases, and demyelinating lesions, so the differential diagnosis includes imaging studies such as magnetic resonance imaging contrasted with spectroscopy, ruling out immunodeficiency or metastatic disease. Case Description: A 65-year-old female presented progressing headache, loss of memory and language alterations, as well as sensory alterations. Neuroimaging showed the presence of two equidistant periventricular lesions at the level of both ventricular atria, a spectroscopy study suggestive of malignancy. Serological studies showed no evidence of immunodeficiency or the presence of positive tumor markers; however, a biopsy was performed, which revealed a histopathological result of primary lymphoma of the CNS. Conclusion: In neuro-oncology, primary CNS tumors with multiple lesions are rare, even more, the "mirror type" lesions. Lymphomas are lesions that can present in different ways on imaging and clinical presentation. These tumors that present a vector effect due to their size, perilesional edema, or that lead to loss of neurological function are highly discussed in diagnostic and surgical treatment. Due to their prognosis, action on diagnosis and treatment must be taken as quickly as hospital resources allow.

Biosensor capability of the endometrium is mediated in part, by altered miRNA cargo from conceptus-derived extracellular vesicles.

We tested the hypothesis that the biosensor capability of the endometrium is mediated in part, by the effect of different cargo contained in the extracellular vesicles secreted by the conceptus during the peri-implantation period of pregnancy. We transferred Bos taurus taurus embryos of different origin, in vivo (high developmental potential (IV)), in vitro (intermediate developmental potential (IVF)), or cloned (low developmental potential (NT)), into Bos taurus indicus recipients. Extracellular vesicles (EVs) recovered from Day 16 conceptus-conditioned medium were characterized and their microRNA (miRNA) cargo sequenced alongside RNA sequencing of their respective endometria. There were substantial differences in the endometrial response to in vivo versus in vitro and in vivo versus cloned conceptuses (1153 and 334DEGs respectively) with limited differences between in vitro Vs cloned conceptuses (36 DEGs). The miRNA cargo contained in conceptus-derived EVs was similar between all three groups (426 miRNA in common). Only 8 miRNAs were different between in vivo and cloned conceptuses, while only 6 miRNAs were different between in vivo and in vitro-derived conceptuses. Treatment of endometrial epithelial cells with mimic or inhibitors for miR-128 and miR-1298 changed the proteomic content of target cells (96 and 85, respectively) of which mRNAs are altered in the endometrium in vivo (PLXDC2, COPG1, HSPA12A, MCM5, TBL1XR1, and TTF). In conclusion, we have determined that the biosensor capability of the endometrium is mediated in part, by its response to different EVs miRNA cargo produced by the conceptus during the peri-implantation period of pregnancy.

The consequences of the new European reclassification of non-invasive brain stimulation devices and the medical device regulations pose an existential threat to research and treatment: An invited opinion paper.

A significant amount of European basic and clinical neuroscience research includes the use of transcranial magnetic stimulation (TMS) and low intensity transcranial electrical stimulation (tES), mainly transcranial direct current stimulation (tDCS). Two recent changes in the EU regulations, the introduction of the Medical Device Regulation (MDR) (2017/745) and the Annex XVI have caused significant problems and confusions in the brain stimulation field. The negative consequences of the MDR for non-invasive brain stimulation (NIBS) have been largely overlooked and until today, have not been consequently addressed by National Competent Authorities, local ethical committees, politicians and by the scientific communities. In addition, a rushed bureaucratic decision led to seemingly wrong classification of NIBS products without an intended medical purpose into the same risk group III as invasive stimulators. Overregulation is detrimental for any research and for future developments, therefore researchers, clinicians, industry, patient representatives and an ethicist were invited to contribute to this document with the aim of starting a constructive dialogue and enacting positive changes in the regulatory environment.

Mixed Methods EvAluation of the high-volume low-complexity Surgical hUb pRogrammE (MEASURE): a mixed methods study protocol.

INTRODUCTION: The waiting list for elective surgery in England recently reached over 7.8 million people and waiting time targets have been missed since 2010. The high-volume low complexity (HVLC) surgical hubs programme aims to tackle the backlog of patients awaiting elective surgery treatment in England. This study will evaluate the impact of HVLC surgical hubs on productivity, patient care and the workforce. METHODS AND ANALYSIS: This 4-year project consists of six interlinked work packages (WPs) and is informed by the Consolidated Framework for Implementation Research. WP1: Mapping current and future HVLC provision in England through document analysis, quantitative data sets (eg, Hospital Episodes Statistics) and interviews with national service leaders. WP2: Exploring the effects of HVLC hubs on key performance outcomes, primarily the volume of low-complexity patients treated, using quasi-experimental methods. WP3: Exploring the impact and implementation of HVLC hubs on patients, health professionals and the local NHS through approximately nine longitudinal, multimethod qualitative case studies. WP4: Assessing the productivity of HVLC surgical hubs using the Centre for Health Economics NHS productivity measure and Lord Carter's operational productivity measure. WP5: Conducting a mixed-methods appraisal will assess the influence of HVLC surgical hubs on the workforce using: qualitative data (WP3) and quantitative data (eg, National Health Service (NHS) England's workforce statistics and intelligence from WP2). WP6: Analysing the costs and consequences of HVLC surgical hubs will assess their achievements in relation to their resource use to establish value for money. A patient and public involvement group will contribute to the study design and materials. ETHICS AND DISSEMINATION: The study has been approved by the East Midlands-Nottingham Research Ethics Committee 23/EM/0231. Participants will provide informed consent for qualitative study components. Dissemination plans include multiple academic and non-academic outputs (eg, Peer-reviewed journals, conferences, social media) and a continuous, feedback-loop of findings to key stakeholders (eg, NHS England) to influence policy development. TRIAL REGISTRATION: Research registry: Researchregistry9364 (https://www.researchregistry.com/browse-the-registry%23home/registrationdetails/64cb6c795cbef8002a46f115/).

Sudden cardiac death triggered by minimal alcohol consumption in the context of novel PPA2 mutations in 2 unrelated families.

Biallelic variants in PPA2 gene cause a rare but lethal mitochondrial disorder. We describe the first four cases reported in Spain of PPA2 disease in two unrelated families. We have conducted a revision of the clinical history, necropsies, and postmortem genetic testing from probands, and clinical evaluation, genetic testing and blood transcript analysis in family members. All the cases harbored biallelic PPA2 variants in compound heterozygous status. Two brothers from family 1 suffered sudden death after a small first intake of alcohol in 2013 and 2022. The sister remains alive but affected with cardiomyopathy, extensive scar on cardiac imaging, and high sensitivity to alcohol intake. The three siblings carried PPA2 c.290A > G (p.Glu97Gly) novel missense variant and PPA2 c.513C > T (p.Cys171 = ) altering splicing site variant, both probably leading to mRNA degradation based on in-silico and transcript analyses. A teenager from family 2 suffered sudden death after a small intake of alcohol in 2018 and carried PPA2 c.683C > T (p.Pro228Leu) missense and PPA2 c.980_983del (p.Gln327fs) novel frameshift variant, both probably leading to abnormal protein structure. All cases were asymptomatic until adolescence. Furthermore, the sister in family 1 has survived as an asymptomatic adult. PPA2 disease can manifest as cardiac arrest in the young, especially after alcohol exposure. Our results show that PPA2 deficiency can be related to different pathogenicity mechanisms such as abnormal protein structure but also mRNA decay caused by synonymous or missense variants. Strict avoidance of alcohol consumption and early defibrillator implantation might prevent lethal arrhythmias in patients at risk.

Synergism between remdesivir and ribavirin leads to SARS-CoV-2 extinction in cell culture.

BACKGROUND AND PURPOSE: There is a need for effective anti-COVID-19 treatments, mainly for individuals at risk of severe disease such as the elderly and the immunosuppressed. Drug repositioning has proved effective in identifying drugs that can find a new application for the control of coronavirus disease, in particular COVID-19. The purpose of the present study was to find synergistic antiviral combinations for COVID-19 based on lethal mutagenesis. EXPERIMENTAL APPROACH: The effect of combinations of remdesivir and ribavirin on the infectivity of SARS-CoV-2 in cell culture has been tested. Viral populations were monitored by ultra-deep sequencing, and the decrease of infectivity as a result of the treatment was measured. KEY RESULTS: Remdesivir and ribavirin exerted a synergistic inhibitory activity against SARS-CoV-2, quantified both by CompuSyn (Chou-Talalay method) and Synergy Finder (ZIP-score model). In serial passage experiments, virus extinction was readily achieved with remdesivir-ribavirin combinations at concentrations well below their cytotoxic 50 value, but not with the drugs used individually. Deep sequencing of treated viral populations showed that remdesivir, ribavirin, and their combinations evoked significant increases of the number of viral mutations and haplotypes, as well as modification of diversity indices that characterize viral quasi-species. CONCLUSION AND IMPLICATIONS: SARS-CoV-2 extinction can be achieved by synergistic combination treatments based on lethal mutagenesis. In addition, the results offer prospects of triple drug treatments for effective SARS-CoV-2 suppression.

SARS-CoV-2 mutant spectra as variant of concern nurseries: endless variation?

Introduction: SARS-CoV-2 isolates of a given clade may contain low frequency genomes that encode amino acids or deletions which are typical of a different clade. Methods: Here we use high resolution ultra-deep sequencing to analyze SARS-CoV-2 mutant spectra. Results: In 6 out of 11 SARS-CoV-2 isolates from COVID-19 patients, the mutant spectrum of the spike (S)-coding region included two or more amino acids or deletions, that correspond to discordant viral clades. A similar observation is reported for laboratory populations of SARS-CoV-2 USA-WA1/2020, following a cell culture infection in the presence of remdesivir, ribavirin or their combinations. Moreover, some of the clade-discordant genome residues are found in the same haplotype within an amplicon. Discussion: We evaluate possible interpretations of these findings, and reviewed precedents for rapid selection of genomes with multiple mutations in RNA viruses. These considerations suggest that intra-host evolution may be sufficient to generate minority sequences which are closely related to sequences typical of other clades. The results provide a model for the origin of variants of concern during epidemic spread─in particular Omicron lineages─that does not require prolonged infection, involvement of immunocompromised individuals, or participation of intermediate, non-human hosts.