Background: Understanding past successes in reaching unvaccinated or “zero-dose” children can help inform strategies for improving childhood immunization in other settings. Drawing from positive outlier methods, we developed a novel approach for identifying potential exemplars in reducing zero-dose children. Methods: Focusing on 2000–2019, we assessed changes in the percentage of under-one children with no doses of the diphtheria–tetanus–pertussis vaccine (no-DTP) across two geographic dimensions in 56 low- or lower-middle-income countries: (1) national levels; (2) subnational gaps, as defined as the difference between the 5th and 95th percentiles of no-DTP prevalence across second administrative units. Countries with the largest reductions for both metrics were considered positive outliers or potential ‘exemplars’, demonstrating exception progress in reducing national no-DTP prevalence and subnational inequalities. Last, so-called “neighborhood analyses” were conducted for the Gavi Learning Hub countries , comparing them with countries that had similar no-DTP measures in 2000 but different trajectories through 2019. Results: From 2000 to 2019, the Democratic Republic of the Congo, Ethiopia, and India had the largest absolute decreases for the two no-DTP dimensions—national prevalence and subnational gaps—while Bangladesh and Burundi registered the largest relative reductions for each no-DTP metric. Neighborhood analyses highlighted possible opportunities for cross-country learning among Gavi Learning Hub countries and potential exemplars in reducing zero-dose children. Conclusions: Identifying where exceptional progress has occurred is the first step toward better understanding how such gains could be achieved elsewhere. Further examination of how countries have successfully reduced levels of zero-dose children—especially across variable contexts and different drivers of inequality—could support faster, sustainable advances toward greater vaccination equity worldwide. The expansion of routine immunization is heralded as a global success story , enablinOver the last few years, a growing body of research has sought to assess characteristics of zero-dose children and their families or households, as well as potential drivers of high zero-dose prevalence at different geographic levels ,15,16,17Good Health at Low Cost case studies first in 1985 [Positive outlier, or so-called ‘positive deviance’, methodologies have been used at the unit or organizational level in healthcare settings ,22,23, a in 1985  and then in 1985 , sought  in 1985 . In 2016 in 1985 ,30,31; s in 1985 ,33,34,35 in 1985  and Gavi in 1985 ), adoptiWith this study, we develop a novel approach for identifying positive outliers in reducing zero-dose children over time. This analysis currently takes a geographic focus, one of many important dimensions of inequality, by comparing patterns in both national and subnational declines in the percentage of under-one children with no doses of DTP (no-DTP) from 2000 to 2019 among 56 low- and lower-middle-income countries (LMICs). Based on this approach, identified ‘exemplar’ countries or subnational locations that substantially reduced zero-dose children could be targeted for further examination into the policy or program factors behind such gains.We used estimates of DTP1 among children under 1 year of age at the national and second administrative levels from the Institute for Health Metrics and Evaluation (IHME). The methods used to estimate DTP1 at different geospatial resolutions are detailed elsewhere ; in brieFor this analysis, we focused on 56 LMICs . These cWe conducted three analyses to characterize potential exemplars in reducing zero-dose children over time, as summarized below. R version 4.2.1 was used for data processing, analyses, and visualizations .Quantifying changes in zero-dose children across geographies. We assessed changes in the percentage of under-one children without any doses of DTP (no-DTP) between 2000 and 2019 across two geographic dimensions: (1) national levels; (2) subnational gaps among second-level administrative units. For the latter—subnational gaps—we used the 5th and 95th percentile values of the prevalence of no-DTP children estimated across second-level administrative units and computed the difference for a given country–year. We opted to use the 5th and 95th percentiles rather than absolute minimum and maximum values of no-DTP prevalence to offset the potential for undue influence of outliers for a given subnational unit–year. Furthermore, how countries define second-level administrative units widely varies  in Nigeria); using percentiles to define subnational gaps may also help mitigate the degree to which having more (or fewer) administrative units could affect measures of subnational inequality.Identifying potential exemplars in reducing zero-dose children. Second, countries with the largest declines for both no-DTP metrics between 2000 and 2019 were considered as potential exemplars in reducing zero-dose children. Prior research conducted under the EGH program has typically used one progress measure per geographic unit [hic unit ,34,35, aWe ranked each country ordinally, 1 to 56, based on their national and subnational reductions in no-DTP prevalence from 2000 to 2019, with 1 being the largest reduction and 56 being the smallest reduction or, if applicable, the largest increase since 2000. We took the mean of those rankings to identify which countries had achieved the most progress across both geographic dimensions. We applied these rankings and calculations for absolute and relative progress separately: computing percentage point changes for absolute progress from 2000 to 2019 and then percentage change from 2000 to 2019 for relative progress. We opted to consider both progress metrics—absolute and relative progress—as they could better represent a range of successful approaches used to reduce no-DTP prevalence from different starting points , and thus likely mirror different stages of immunization delivery needs and strategies.Comparing divergent no-DTP trajectories since 2000 for select locations. Third, we conducted so-called “neighborhood analyses” for select countries, comparing them to other countries that had similar levels for both no-DTP measures in 2000 but different trajectories through 2019. Such analyses are thought to be supportive of potential cross-location learning and knowledge translation around what could work to address zero-dose challenges when starting from similar baseline levels of no-DTP prevalence. At the country-level, we focused on Nigeria, Mali, Uganda, and Bangladesh—the four countries selected for the Gavi Learning Hubs [ing Hubs  and souging Hubs ; in brieAmong the 56 LMICs included in this analysis, 44 (78.6%) had some kind of reduction in both national levels of no-DTP and subnational gaps in no-DTP prevalence between 2000 and 2019 ; Table 2Absolute progress. The DRC, Ethiopia, and India registered the largest absolute reductions in national no-DTP prevalence and subnational gaps from 2000 to 2019 . By 2019, national no-DTP prevalence levels fell to 6.9%, a 45.0 percentage-point decline. The DRC’s subnational gaps narrowed by 30.8 percentage-points by 2019, decreasing to a total of 16.0 percentage points across the 5th and 95th percentiles of territories . As highlighted by For Ethiopia nationally, 63.4% of under-one children lacked any doses of DTP in 2000, but no-DTP prevalence fell 51.6 percentage points to 11.7% by 2019. Across its zones, Ethiopia had a 47.9 percentage-point gap between the 5th and 95th percentile levels of no-DTP prevalence in 2000, spanning from 39.6% to 87.5%. This subnational gap decreased by 17.2 percentage points by 2019, to a 30.8 percentage-point difference between the 5th and 95th percentile no-DTP levels across zones . However, amid such marked gains over the last 19 years, Ethiopia’s reductions in subnational gaps have stagnated from 2016–2019 .In India, national no-DTP prevalence was 30.9% in 2000 with a 52.5 percentage-point gap between the 5th and 95th percentile for no-DTP levels across districts . By 2019, 7.2% of under-one children had no doses of DTP in India nationally, a 23.6 percentage-point decline. Subnational gaps in India decreased 32.7 percentage points between 2000 and 2019, falling to 19.7 percentage-point difference in 2019 . Although overall subnational gaps have narrowed , severalRelative progress. As measured by the percentage change in no-DTP metrics between 2000 and 2019, Bangladesh and Burundi achieved the largest relative reductions in no-DTP prevalence . By 2019, this subnational gap fell to 2.4 percentage points, representing an 85.9% decline across communes at the 5th and 95th percentile . Progress accelerated after 2005 , when thFocusing on the four Gavi Learning Hub countries—Nigeria, Mali, Uganda, and Bangladesh—we mapped their no-DTP trajectories from 2000 to 2019 against potential exemplars in reducing zero-dose children ; the excFor Nigeria, Ethiopia was its closest ‘neighbor’ in terms of national no-DTP prevalence in 2000—55.5% in Nigeria and 63.4% in Ethiopia—with diverging no-DTP trajectories through 2019 . From 2000 to 2019, Nigeria consistently had among the highest subnational no-DTP disparities in the world; even in 2000, when Ethiopia had the fourth highest subnational gap in no-DTP among included countries  to other settings. As highlighted by the so-called “neighborhood” analysis, comparing divergent no-DTP trajectories among peer locations could support deeper study around what catalyzed faster progress for some places—and how those lessons could be applied elsewhere. The combination of this positive outlier methodology and cross-country platforms supported by the Gavi Learning Hubs offers unique opportunities to better understand ‘what works’ for accelerating progress in reaching unvaccinated children worldwide.Considering positive outliers—or potential exemplars—in reducing no-DTP prevalence for both absolute and relative progress can better reflect the range of successful strategies implemented from a range of different no-DTP prevalence starting points. After all, the types of programmatic and policy decisions that may occur when more than 30–50% of under-one children have had no doses of DTP could differ from those occurring when high zero-dose communities are more clustered and national levels of no-DTP are well below 10%. In health service delivery, these differences may unfold around more widespread intervention introduction and scale-up activities  versus more tailored service provision to individuals or communities who still lack access to or demand for an intervention . For inDespite their marked progress since 2000, further improvements in vaccination reach and uptake are needed in the DRC, Ethiopia, India, and other countries still experiencing large populations of unvaccinated children. Accordingly, it is possible that lessons learned from countries with exceptional relative reductions from 2000 to 2019 could be applicable to countries such as the DRC, Ethiopia, and India today; after all, 2019 no-DTP estimates for the latter countries are quite similar to the 2000 estimates for countries such as Bangladesh and Burundi. For Bangladesh, reductions in national no-DTP and subnational gaps nearly paralleled each other time, charting a path toward nearly 0% no-DTP nationally and negligible subnational differences by 2019. These trends may reflect the country’s concerted efforts to better reach rural communities with lower levels of vaccination , among oThere is ample opportunity—and need—to characterize what drives successful vaccine delivery and uptake across the spectrum of past and current challenges, particularly around vaccination inequalities. One key consideration that emerged from this analysis involves the pathways by which no-DTP changed both nationally and sub-nationally from 2000 to 2019. Particularly among countries that started with higher levels of no-DTP , subnational gaps often remained unchanged or increased while national no-DTP prevalence began improving. Such pathways suggest that explicit equity program targets and implementation practices may not occur until later. Other countries, including India and Bangladesh, had more consistent declines for both metrics from 2000 to 2019—a potential signal into the ways in which countries are concurrently addressing both national vaccination priorities and at least geographic inequalities. Nonetheless, it is also possible that countries such as India and Bangladesh experienced similar pathways of minimal changes in or rising subnational inequality amid decreasing national no-DTP prior to 2000. Developing a more formalized characterization or framework around ‘pathways of progress’ toward greater vaccination equity should be considered in future studies, both by geography and across other crucial factors . While assessing progress metrics is a necessary first step to better identify potential exemplars in reducing zero-dose burdens, they alone cannot shed light on what countries have executed and how such actions were associated with further improvements. Formally applying methods such as that of the EGH program, with qualitative examination of policy and programs alongside quantitative analyses around drivers of progress , should Past work has sought to synthesize and/or assess particular characteristics of successful immunization programs; nonetheless, few studies have expressly focused on both zero-dose children and incorporating mixed-methodologies with a positive outlier lens. For example, qualitative research in Senegal, Zambia, and Nepal points to factors including strong community engagement, integrated delivery, adaptive service provision, and robust data systems as central to improving and/or maintaining high levels of DTP1 and/or DTP3 ,50,51,52It is worth noting that declines in no-DTP prevalence—and thus increased coverage of DTP1, a marker of program reach—do not inherently equate to gains in broader program retention or complete immunization. For instance, in much of Ethiopia, DTP3 coverage has not improved in parallel amid sizeable increases in DTP1 ,54,55. TThis analysis is subject to a number of limitations. First, this study focuses on changes in geographic inequalities at the second administrative level or higher, which results in representing only one of many critical factors that contribute to inequities in immunization delivery . While gSecond, focusing on the second-administrative level likely masks important differences experienced at more granular levels , ,54 and tr = 0.47 in 2000 and r = 0.38 in 2019) or change metrics from 2000 to 2019 . Since first- or second-level administrative units are often meaningful for health program implementation , we viewed using country administrative units as having more benefits and relevance than the potential drawbacks around variable subnational geographies. However, exploring alternative units of analysis  could be beneficial for future work.Third, country-to-country comparisons of subnational gaps and changes in these gaps over time may be affected by a country’s total number of second-level administrative units rather than meaningful differences in vaccination equity at comparable areal units. For instance, subnational gaps in no-DTP may seem higher among in a country divided into more second-level administrative units than those with fewer units . At leasFourth, we opted to use estimates from IHME for this analysis rather than administrative data sources  or alternative sources . Because the primary goal of this study was to be able to directly compare national and subnational levels and trends in no-DTP across countries, IHME estimates provided the greatest number of countries with subnational estimates for the full time period (2000–2019). Fifth, DTP estimates draw from household surveys and other data sources in which groups or communities with higher rates of unvaccinated children may be systematically under-represented . Accordingly, current no-DTP estimates may not fully capture the ‘true’ magnitude or trends in zero-dose children among populations with disproportionately high vulnerabilities and risks for not being vaccinated. Sixth, the time period of analysis focused on 2000 to 2019, and thus the identification of potential exemplars may be sensitive to estimated levels of childhood vaccination at either end of the 19-year range. Importantly, this analysis does not account for the ongoing effects of the COVID-19 pandemic, of which has had differential impacts across countries and communities since March 2020 ,60,61. RLastly, these analyses currently lack deeper contextual information from and by the communities most affected by higher rates of un- and under-vaccination. Our aim is to receive critical feedback on the potential applications of these positive-outlier methods for cross-country learning and synthesis around what works to reduce high rates of zero-dose prevalence, and to work with country and regional leadership to improve these approaches going forward.Recognizing where exceptional progress in reducing zero-dose children has occurred is the first step toward better understanding what countries did to attain such improvements. Such insights then can inform strategy adaptions to other settings, and further reinforce successful strategies in places that achieved large reductions historically but still have large populations of unvaccinated children today. Characterizing pathways to greater vaccination equity, as well ensuring mechanisms by which effective knowledge translation and cross-country learning can be supported, will strengthen efforts toward ensuring all children can fully benefit from vaccines.


Calcitonin gene-related peptide (CGRP) monoclonal antibodies (mAbs) are approved in Europe as preventive treatment of migraine in patients with at least four monthly migraine days. Migraine gives rise to direct healthcare expenditures, but most of the economic burden of migraine is socioeconomic. Evidence on the socioeconomic implications of CGRP-mAbs is, however, limited. There is an increasing interest in supplementing evidence from randomised controlled trials (RCTs) with real-world evidence (RWE) to aid clinical decision making and inform decision making for migraine management. The objective of this study was to generate RWE on the health economic and socioeconomic implications of administering CGRP-mAbs to patients with chronic migraine (CM) and episodic migraine (high-frequency episodic migraine (HFEM), and low-frequency episodic migraine (LFEM)).Real-world data (RWD) on Danish patients with CM, HFEM, and LFEM were collected via two Danish patient organisations and two informal patient networks and used in a tailored economic model. Treatment effects of CGRP-mAbs on health economic and socioeconomic outcomes were estimated using a sub-sample of patients with CM who receive CGRP-mAb treatment.A total of 362 patients  were included in the health economic model , and 303 patients were included in the socioeconomic model (15.2% received treatment with CGRP-mAbs). Health economic savings from initiating CGRP-mAb treatment totalled €1,179 per patient with CM per year on average . Socioeconomic gains from initiating CGRP-mAb treatment totalled an average gross domestic product (GDP) gain of €13,329 per patient with CM per year .Our results indicate that CGRP-mAbs have the potential to reduce both health economic expenditures and the socioeconomic burden of migraine. Health economic savings are used as a basis for health technology assessments (HTAs) of the cost-effectiveness of new treatments, which implies that important socioeconomic gains may not be given enough importance in decision making for migraine management.The online version contains supplementary material available at 10.1186/s12883-023-03302-7. Migraine is a serious condition with debilitating symptoms , 2. MigrCalcitonin gene-related peptide (CGRP) monoclonal antibodies (mAbs) have been shown to significantly reduce the number of monthly migraine days (MMD) in patients with chronic and episodic migraine . In addiIn several European countries, reimbursement of CGRP-mAbs is restricted to specific sub-populations within the approved indication, e.g., chronic migraine patients with at least two  prior trThere is a growing interest in the use of real-world evidence (RWE) as a supplement to evidence from randomised controlled trials (RCTs) and to aid clinical decision making and inform decision makers for migraine management . Real-woA tailored economic model was developed to estimate the savings and gains from initiating treatment with CGRP-mAbs on a range of outcomes that are affected by migraine following an approach from the literature . A distiWe collected RWD on Danish adult patients with CM, HFEM, and LFEM to generate retrospective RWE on the societal implications of CGRP-mAbs. Before initiating the survey for gathering RWD, the participants provided informed consent to participate in the survey. RWD were collected through the social media networks of the Danish patient organisations, Migraine and Headache Association, and Migraine Denmark from 11 January to 7 March 2022. Followers of the two patient organisations’ social media networks do not have to be members of the patient organisations to follow them on social media. In addition, RWD were collected in the same period through the social media networks of two informal patient groups, and local physicians helped spread the word about the research study. Inclusion criteria included at least four self-reported MMD on average over the last three months or at least four self-reported MMD on average per month before initiating their current preventive treatment, at least 18 years of age, and completion of either the health economic part of the survey or both the health and socioeconomic part. CM is defined according to ICHD-3 ; HFEM isPatients who received CGRP-mAb-treatment at the time of the data collection were asked about the implications of their migraines on their lives “today” at the time of data collection and before initiating CGRP-mAb-treatment. By comparing these two time periods, treatment effects on a broad range of clinical and non-clinical outcomes were estimated for patients with CM receiving CGRP-mAb-treatment. These were estimated both as numerical treatment effects  and as percentage treatment effects . Baseline estimates of the same outcomes were estimated for patients with chronic migraine who did not receive treatment with CGRP-mAbs at the time of the data collection, and for patients with HFEM and LFEM who are not recommended for treatment with CGRP-mAbs in Denmark but are within the indication approved by the European Medicines Agency (EMA) –18. The Patients with CM may not be receiving treatment with CGRP-mAbs in Denmark because they do not fulfill the requirement to have experienced treatment failure on at least two preventive treatments (one antihypertensive and one anti-epileptic medication), have medication overuse headache, are on the waiting list to receive treatment with CGRP-mAbs, or have chosen not to initiate the treatment.The RWD used in the health economic model include the use of acute medication, preventive medication, and healthcare resources. Healthcare resource use consists of general practitioner (GP) visits, specialist visits, outpatient visits, hospitalisations, and emergency room (ER) visits. These have all been shown to be significantly higher in patients with migraine , 8, 19. The RWD used in the socioeconomic model include absenteeism (sick days), presenteeism (decreased productivity) , workingThe savings and gains are reported as the averages per patient initiating treatment according to the Danish guidelines . Health All monetary values are reported in Euros, using a conversion rate from Danish Kroner (DKK)  to Euros of €0.1344 per DKK. The statistical analysis was carried out in StataIC 15.1. Statistical significance was estimated using two-sided paired Student’s t-tests on the null hypothesis of equal means for sub-samples with at least 30 respondents. For sub-samples with fewer than 30 patients, Wilcoxon signed-rank test on the null hypothesis of no difference in population mean ranks was used.A total of 665 individuals initiated the survey. Of these, 303 patients were excluded due to the following: only looking at the front page (83), self-identifying as having fewer than four monthly migraine days 142), failing to complete the survey (62), and being omitted in data cleaning (16). The dataset for the health economic analysis totaled 362 patients, whereof 59 (16.3%) received treatment with CGRP-mAbs at the time of the data collection. Additionally, 59 patients failed to complete the questions for the socioeconomic model, totaling 303 patients in the socioeconomic analysis. Of these, 46 (15.2%) received treatment with CGRP-mAbs at the time of the data collection. Descriptive characteristics for the full sample size (N = 362) included in this study are shown in Table 42, failiPatients who received treatment with CGRP-mAbs at the time of the data collection reported a 34.92% reduction in MMD  = 4.19–7.23) compared to before they initiated treatment.The estimated treatment effects in the health economic model show significant reductions in a wide range of outcomes, as shown in Table The estimated treatment effects in the socioeconomic model show large reductions in adverse socioeconomic outcomes, as shown in Table The estimated differences in Tables Our estimates of the total health economic savings from initiating treatment with CGRP-mAbs in patients with CM, HFEM, and LFEM totalled €1,179, €264, and €175 per patient per year on average, respectively, as shown in Table Our estimates of the total socioeconomic gains from initiating treatment with CGRP-mAbs in patients with CM, HFEM, and LFEM totalled an average GDP gain of €13,329, €10,449, and €9,947 per patient per year on average, respectively, as shown in Table We found health economic savings from initiation of treatment with CGRP-mAbs of €1,179, €264, and €175 for patients with CM, HFEM, and LFEM, respectively. Socioeconomic gains from initiating treatment with CGRP-mAbs amounted to €13,329 €10,449, and €9,947 for patients with CM, HFEM, and LFEM, respectively. For comparison, an estimate of the confidential annual net price of CGRP-mAbs in Denmark is approximately €3,562 per patient per year, and €4,131 per patient per year taking the extra outpatient visits associated with the treatment into account [Our results indicate that socioeconomic gains are important drivers for the societal gains associated with CGRP-mAb-treatment. This is in line with Sussman et al. 2018) and Autio et al. (2021), both of whose studies find substantial socioeconomic gains associated with treatment with CGRP-mAbs , 8. Suss and AutiSocioeconomic implications of new treatments are most often not explicitly considered in health technology assessments (HTAs) in Europe, and productivity effects are excluded . Our resThe present study design has some limitations. The sample was a non-random sample of patients who self-selected into participation in the research project, which may give rise to self-selection bias . Even thA total of 97.5% of patients in the RWD are female, which is higher than prevalence estimates would suggest . This maThe percentage treatment effects estimated in patients with CM who receive treatment with CGRP-mAbs are used to estimate the numerical treatment effects in patients with CM, HFEM, or LFEM who do not receive treatment with CGRP-mAbs by assuming the same percentage reduction that is observed in patients with CM who receive CGRP-mAb-treatment. There is no formal test of this assumption, but to show the comparability of patients with CM who receive treatment with CGRP-mAbs to patients with CM who do not, descriptive statistics split between patients receiving treatment with CGRP-mAbs and patients with CM not receiving treatment with CGRP-mAbs are summarised in Supplementary Table The reimbursement practice in Denmark did not enable us to analyse the potential of CGRP-mAbs to prevent chronification of migraine from episodic migraine (LFEM or HFEM) to CM, since only patients with CM are eligible for treatment. An addition, the observed implications of CM, HFEM, and LFEM on, e.g., educational attainment and being active in the labour market (cf. Table Our results indicate that CGRP-mAbs have the potential to reduce both the direct health economic expenditures and the socioeconomic burden of migraine, thus having significant labour market implications. Further research is needed to confirm the health and socioeconomic implications of CGRP-mAbs in patients with CM, HFEM, and LFEM using, e.g., longitudinal research designs. Health technology assessments currently use health economic savings as a basis for evaluating the cost-effectiveness of new treatments, which implies that important societal gains in the form of socioeconomic savings may not be given enough importance in decision making.Below is the link to the electronic supplementary material.Supplementary Material 1


Background: COVID-19 vaccine mandates are considered a controversial public health policy both in public debate and among healthcare workers (HCWs). Thus, the objective of this systematic review is to give a deep insight into HCWs’ views and attitudes towards COVID-19 vaccination mandates amid the ongoing COVID-19 pandemic. Methods: A systematic literature search of five databases  was conducted between July 2022 and November 2022. Original quantitative studies that addressed the attitudes of HCWs regarding COVID-19 vaccine mandates were considered eligible for this systematic review. All the included studies (n = 57) were critically appraised and assessed for risk of systematic bias. Meta-analyses were performed, providing a pooled estimate of HCWs’ acceptance towards COVID-19 vaccine mandates for: 1. HCWs and 2. the general population. Results: In total, 64%  of HCWs favored COVID-19 vaccine mandates for HCWs, while 50%  supported mandating COVID-19 vaccines for the general population. Conclusions: Our findings indicate that mandatory vaccination against COVID-19 is a highly controversial issue among HCWs. The present study provides stakeholders and policy makers with useful evidence related to the compulsory or non-compulsory nature of COVID-19 vaccinations for HCWs and the general population. Other: The protocol used in this review is registered on PROSPERO with the ID number: CRD42022350275. According to the World Health Organization (W.H.O.), policies that encourage voluntary vaccination, such as public information campaigns, should take priority over mandatory vaccination policies . HoweverVaccination hesitancy among HCWs was a public health concern even before the COVID-19 pandemic . Common COVID-19 vaccines have been shown to be effective in reducing both disease spread and the risk of severe clinical outcomes . With thThe aim of our study is to understand HCWs’ attitudes towards COVID-19 vaccine mandates, to assist policy makers in evidence-based decision making. We summarize the complex interactions between various determinants that play an important role in the chain of events concerning HCWs and their stance towards the COVID-19 vaccine mandates .This systematic review and meta-analysis was reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) .A systematic literature search was performed between July 2022 and November 2022 including articles from 5 databases: PubMed, Embase, Scopus, CINAHL, and Web of Science. In our search, only articles written in English were included. The following search strategy was used for all data bases:  OR  OR  OR  OR (nurse students) OR  OR (medicine students) OR  OR (dentistry students)) AND ((mandatory) OR (compulsory) OR (obligatory) OR (required)) AND ((COVID-19) OR (SARS-CoV-2)) AND ((vaccination) OR (immunization)). EndNote Web was used to import references from all databases and remove duplicates. After the removal of duplicates, the manual screening of abstracts/titles was performed by two independent researchers (M.P and G.R.), who applied the inclusion/exclusion criteria for each citation, and disagreements were resolved by consensus. During the abstract/title screening, 1597 articles were excluded as they were irrelevant to the research questions. A double screening strategy followed by a consensus (M.P and G.R.) was also adopted for the full text screening and the application of the inclusion/exclusion criteria to the screened studies. Last, all included studies were evaluated for risk of systematic bias by two independent researchers  using Loney’s criteria, and disagreements were solved by consensus .Population: For this review, HCWs were defined as active professionals from all health-related professions , with any vaccination status against COVID-19.Study design: Original quantitative studies were included.Outcomes: Articles that investigated the views and attitudes of HCWs towards mandatory COVID-19 vaccines of any type were included in this systematic review.Population: Non HCW populations were not eligible for this systematic review. In addition, studies on retired HCWs did not meet the eligibility criteria.Study design: Studies of qualitative design were excluded.Outcomes: Studies that did not analyze data on HCWs’ views and attitudes towards mandatory COVID-19 vaccines of any type were not included.Single-author data extraction (M.P.) was adopted, while a second author was responsible for quality checking of the extracted data (G.R.). The main themes used to determine which data were extracted were: 1. HCWs’ acceptance towards COVID-19 vaccination mandates for the general population, 2. HCWs’ acceptance towards COVID-19 vaccination mandates for HCWs. Moreover, in the third and last section of the study, we summarized broad evidence regarding the views and attitudes of HCWs towards mandatory COVID-19 vaccinations. Details of the studies, such as names of first authors, year of publication, sample size, and country, were extracted, along with various socio-demographic factors . Tabulated data of the available information were used for comparison between the included studies. Sample size in meta-analyses may differ from those tabulated, as not all the responders answered the questions regarding COVID-19 mandates in each individual study. The total number of participants in the papers assessed in our study was 77,466 HCWs.2 > 95%), which is common in meta-analyses of proportions [Narrative synthesis was used for combining evidence from all the included studies when the data were not compatible with meta-analysis, but also for summarizing the main findings of the meta-analyses, according to the Cochrane Consumers and Communication Review Group guidelines . STATA 1portions . The outportions .Sensitivity analysis was performed at two levels: 1. alternative dichotomization and 2. exclusion of the studies at high risk of systematic bias. Regarding alternative dichotomization, a random effects meta-analysis model was performed using the data as follows: for a 3-point scale, agree and neutral = 0, while disagree = 1. For a 5-point scale, strongly agree, agree, and neutral = 0, while disagree and strongly disagree = 1 ,24.A second sensitivity analysis was conducted based on the results of critical appraisal of the individual studies . Loney’sDue to the high observed heterogeneity between the individual studies, a subgroup analysis was deemed appropriate. Three subgroup analyses were conducted by stratifying data by year of publication, W.H.O. region, and health profession .Many authors have discussed the need for a different approach to the funnel plot when it comes to publication bias risk assessment in meta-analyses of proportions ,26. WithAfter conducting systematic searches in PubMed, CINAHL, Web of Science, Scopus, and Embase, we retrieved 1903 articles. After duplicate removal (221 results), 1682 articles were retrieved for title and abstract screening. Our screening resulted in a collection of 85 articles. After applying the inclusion/exclusion criteria, we excluded 28 articles for specific reasons . FinallyRegarding the acceptance of HCWs towards mandatory COVID-19 vaccination for the general population, 22 studies were considered for quantitative evidence synthesis. The total number of participants was 24,882 HCWs, coming from 13 different countries . ConsideRegarding the acceptance of HCWs towards COVID-19 vaccine mandates for the general population, 22 studies with 24,882 participants from 13 different countries were considered for quantitative evidence synthesis.2 = 99.6%, Cochrane Q test p-value = 0.000), we calculated a pool estimate of 50%  HCWs’ acceptance towards COVID-19 vaccine mandates for the general population .The publication bias risk assessment was conducted using both a DOI plot and a funnel plot. According to the LFK index, with a cut-off point of |2| (minor vs. major asymmetry), no asymmetry was observed, indicating that no risk of publication bias was detected . ConsideA total of 30 studies with 38,839 participants were considered for meta-analysis regarding the outcome “acceptance of HCWs towards mandatory COVID-19 vaccination for HCWs” . DespiteA total of 30 studies with 38,839 participants from 18 different countries were considered for the quantitative synthesis of evidence regarding the outcome “HCWs’ acceptance of COVID-19 vaccination mandates for HCWs”.2 = 99.6%, Cochrane Q test p-value = 0.000). The estimated pooled proportion was 64%   .The publication bias risk was assessed using a DOI plot and the corresponding LFK index, along with a funnel plot and Egger’s test. As can be seen below, major positive asymmetry is observed (LFK index = 2.42), indicating a high risk of publication bias . Even ifA comparison between the primary analysis results and the results of the sensitivity analyses was conducted to control for the effect of data dichotomization, as well as for the risk of systematic bias in the individual studies . The genp-value = 0.028), whereas for the outcome “HCWs’ acceptance of COVID-19 vaccination mandates for the general population”, no asymmetry was observed .Alternative dichotomization sensitivity analysis resulted in a pooled estimate of 55% for HCW acceptance of mandatory COVID-19 vaccines for the general population, while for HCWs, the corresponding acceptance rate was 67%. Regarding publication bias risk assessment during the alternative dichotomization sensitivity analysis, for the outcome “HCWs’ acceptance of COVID-19 vaccination mandates for HCWs”, we identified major asymmetry using both the DOI and funnel plots  regarding HCWs’ acceptance towards COVID-19 vaccine mandates for HCWs; meanwhile, no asymmetry was observed regarding HCWs’ acceptance of mandatory COVID-19 vaccination for the general population .A second sensitivity analysis was carried out that excluded studies at high the risk of systematic bias. According to the risk of bias sensitivity analysis, HCWs’ COVID-19 vaccine mandates acceptance rates were 45% for the general population and 55% for HCWs. As for the publication bias risk assessment in the risk of bias sensitivity analysis, minor asymmetry was observed using both the DOI and funnel plots , while the within-subgroup heterogeneity remained considerably high .A sub-group analysis was performed by stratifying the data according to W.H.O. region . No statp-value = 0.148, 0.822), while within-subgroup heterogeneity remained high in all sub-groups  , publication year did not have a significant subgroup effect (= 0.000) .In our third approach to subgroup analysis, we sought to explore the effect of occupational status on acceptance of COVID-19 vaccine mandates. Only four studies reported stratified data in relation to the outcome “HCWs’ acceptance of COVID-19 vaccine mandates for HCWs”. Thus, only one subgroup analysis was performed, yielding an odds ratio of 1.29  . AlthougIn the last section, we include studies that were not considered for meta-analysis due to a lack of compatible data. Therefore, only a narrative synthesis of evidence was conducted. The demographic features and main findings of these studies are tabulated in Four studies addressed the attitudes of HCWs towards mandatory COVID-19 vaccination as a working requirement. In a large US study, 90.5% of HCWs decided to be vaccinated after a COVID-19 vaccine working requirement was announced, while 26.7% remained unvaccinated when the COVID-19 vaccine was not a working requirement . FurtherRegarding HCWs’ COVID-19 vaccine acceptance, mandatory COVID-19 vaccinations did not seem to be an adequate factor in opinion change, as reported by a study from Jordan . In aligIn the single included study from Poland, most of the participants agreed that the COVID-19 vaccine should be mandatory for HCWs , in alignment with the findings of Ciliberti et al., where Italian healthcare students supported COVID-19 vaccine mandates for the whole community and for students ,80. In aTo our knowledge, this is the first published systematic review and meta-analysis that investigates the attitudes of HCWs towards COVID-19 vaccine mandates for the general population and HCWs. Our results indicate that COVID-19 vaccine mandates are a highly controversial topic among healthcare professionals. HCWs were divided regarding the imposition of COVID-19 vaccine mandates on the general population, while only a weak majority of them supported the imposition of COVID-19 vaccination on HCWs. Regarding the causes of the controversy, Rodger et al. highlighted that concerns about the safety and effectiveness of the COVID-19 vaccine, as well as distrust in the pharmaceutical industry and governmental/public health officials, were crucial factors in the opposition to COVID-19 vaccine mandates . Other aAs already mentioned, safety concerns were prominent among HCWs, and although the benefits of COVID-19 vaccination clearly outweigh the risks, the latter are worth mentioning. Many countries decided to suspend the Oxford/AstraZeneca and the Johnson & Johnson vaccines due to a small risk of blood clots, while the mRNA vaccines were related with mild cases of myocarditis . The argVaccine mandates are at the center of interest when it comes to limitations to individual liberties, but this becomes more complex when we consider HCWs’ ethical and professional obligation to minimize the risk of harming their patients . Indeed,Wide global differences have also been seen in healthcare workers’ willingness to be vaccinated with the COVID-19 vaccine, as revealed in a review of 26 studies . AccordiOther authors have attempted to assess HCWs’ acceptance of mandatory vaccinations for diseases other than COVID-19. In a meta-analysis, the pooled proportion of HCWs favoring mandatory influenza vaccinations was 61%  . AlthougOn the other hand, findings from an Australian study conducted in 2009 showed that the vast majority (78%) of health officials supported a new mandatory vaccination policy against certain infectious diseases  for HCW, while only 3.6% were against it . This alAccording to Buse et al., central decision makers, such as governmental officials and policy makers, have prominent roles in top-down implementation policy strategies, leaving not much room for other actors, such as regional authorities, the private sector, and frontline workers, to be engaged in decisions that have an important impact on them . The curBy many authors, it has been proposed that vaccine mandates during the COVID-19 pandemic may threaten social cohesion by cultivating social polarization ,110. ManA very important finding is that our review is possibly biased by small studies that reported extremely high rates (>80%) of HCWs’ acceptance of COVID-19 vaccine mandates for HCWs. All of these studies were classified as being at high risk of systematic bias, and therefore, the magnitude of publication bias was thoroughly assessed during the risk of bias sensitivity analysis ,112. PooEvidence from a study conducted in Italy revealed that university students’ attitudes about COVID-19 vaccine mandates changed during the pandemic following pandemic trends . AlthougThe relationship between professional status and the acceptance of mandatory vaccinations has been the subject of other scientific articles . RegardiOur study has several strengths and limitations. First, our study greatly contributes to the current literature by providing novel evidence of healthcare workers’ attitudes towards mandatory COVID-19 vaccinations for both HCWs and the general population. We identified and reported a high risk of publication bias in our meta-analysis. However, through our thorough sensitivity analysis, we assessed both the risk of systematic bias in the individual studies and the publication bias, providing three levels of evidence. Last, we included 57 studies with more than 75,000 participants, which significantly contributes to the reliability of our results.However, our study has several limitations. First, as with observational studies, great heterogeneity was observed among the included studies ,119. ThiOverall, 50% of HCWs opposed mandatory vaccination of the general population, and 36% of them opposed vaccine mandates for HCWs. These figures indicate that the mandatory COVID-19 vaccination of HCWs and the general population is a highly controversial topic among HCWs. In line with the W.H.O.s’ recommendations, our findings suggest that policy makers should prioritize other alternatives  over mandatory vaccination policies.We recognize the well-intentioned efforts of researchers to contribute to the scientific literature during emerging situations. In this context, policymakers should think critically before translating new evidence into policies, as our findings show that low-quality evidence with intriguing results tends to be published.Further research is needed on the roles of different healthcare professionals regarding acceptance towards COVID-19 vaccine mandates.Understanding the relationship between vaccination status and/or intention to be vaccinated and the acceptance of vaccine mandates is expected to provide useful evidence for future vaccination policies.Our findings indicate that HCWs’ attitudes towards COVID-19 mandates may have changed during the pandemic. Determining the reasons for this change will inform policy makers as to the appropriate time of decision making.Important societal benefits are expected to arise as a result of clarifying the complex interactions between the different types of policy implementation and the role of the public engagement in the decision-making process during an epidemic/pandemic.The present study provides stakeholders and policy makers with useful evidence related to the compulsory or non-compulsory nature of COVID-19 vaccination policy among HCWs and the general population. Moreover, the present findings may also inform vaccination policies in future pandemics.


Artificial cells are based on dynamic compartmentalized systems. Thus, remodeling of membrane-bound systems, such as giant unilamellar vesicles, is finding applications beyond biological studies, to engineer cell-mimicking structures. Giant unilamellar vesicle fusion is rapidly becoming an essential experimental step as artificial cells gain prominence in synthetic biology. Several techniques have been developed to accomplish this step, with varying efficiency and selectivity. To date, characterization of vesicle fusion has relied on small samples of giant vesicles, examined either manually or by fluorometric assays on suspensions of small and large unilamellar vesicles. Automation of the detection and characterization of fusion products is now necessary for the screening and optimization of these fusion protocols. To this end, we implemented a fusion assay based on fluorophore colocalization on the membranes and in the lumen of vesicles. Fluorescence colocalization was evaluated within single compartments by image segmentation with minimal user input, allowing the application of the technique to high-throughput screenings. After detection, statistical information on vesicle fluorescence and morphological properties can be summarized and visualized, assessing lipid and content transfer for each object by the correlation coefficient of different fluorescence channels. Using this tool, we report and characterize the unexpected fusogenic activity of sodium chloride on phosphatidylcholine giant vesicles. Lipid transfer in most of the vesicles could be detected after 20 h of incubation, while content exchange only occurred with additional stimuli in around 8% of vesicles. The development of artificial cells based on phospholipid giant unilamellar vesicles (GUVs) has widened the application of these compartments beyond their role as membrane models . GUV-basAs artificial cells gain prominence, rigorous quantification and evaluation of experimental reproducibility is becoming a pressing issue. Previous quantification approaches of vesicle fusion products relied on the manual inspection of few events, such as by observation of fluorophore exchanges between compartments in microscopy, coupled with fluorometric techniques to measure the average properties of large and small vesicles ,9,25. ThThe rapid development of artificial cell research has prompted interest in the statistical analysis of GUV populations, with recent works tackling the problem from various angles. Flow cytometry was one of the first techniques applied to this end, but it is limited due to its lack of spatial resolution ,28,29. IIn line with these advances, tools to statistically study GUV-GUV fusion are needed. Previous techniques approached the problem using fluorogenic reactions or protein expression, which are triggered following content mixing. Exchange of lipidic components has been monitored by changes in fluorescent lipid emission due to Förster resonance energy transfer ,31. TakiWe present a flexible analytical pipeline for content and lipid exchange quantification in GUV populations based on high-throughput imaging. Sedimented vesicles in a sealed chamber are imaged with a motorized stage to record a large sample surface, containing thousands of GUVs. GUV images are elaborated with ImageJ to identify and label the compartments based on their membrane fluorescence. The only assumption made in this step is the presence of at least one channel related to the fluorescence of a membrane probe. Adaptation of GUV detection to brightfield or phase contrast images is straightforward, but we focused on fluorescence, since this can resolve hemifused and aggregated GUVs. Information on fluorescence channels for lipid or content markers, together with morphological parameters for single GUVs, are saved for statistical analysis. This is performed by scripts developed in the R programming language for statistical computing. Raw images were used by an R script to compute the correlation coefficient between pairs of channels as measurements of the colocalization of various probes. This method was then compared with IFC to validate the quantitative reliability of this approach in identifying content and lipid exchange frequencies, vesicle concentration estimates, and size distributions, highlighting not only the advantages, but also the possible blind spots and pitfalls of the method. Finally, this new approach was applied to the analysis of aggregated GUVs, reporting the fusogenicity of sodium chloride, which was previously thought to be non-fusogenic.GUV imaging data was acquired as multi-channel multi-point .nd2 files. For each image, labelled masks of content markers and lipid markers were produced by the ImageJ macro by sequentially filtering, thresholding, and combining the image channels  of GUV Imaging of free-standing GUVs can be challenging, motivating the use of various immobilization techniques ,41. To iSample preparation was critical to ensure the reproducible evaluation of GUVs concentration. Nevertheless, the quantification of GUVs in a sealed imaging chamber with a defined thickness was deemed unreliable due to the incomplete sedimentation of the particles even under long timescales, when the vesicles on the bottom of the chamber reached a dynamic equilibrium with those that were out of focus.® AF488)  (DOPE–Af488), or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 5) (DOPE–Cy5) as lipid markers, and Dextran–Cascade Blue™  as content marker. The datasets contained samples of the isolated P1, P2, and PC populations, and mixed samples containing various ratios of all three populations. Specifically, NC contained 1:1 P1 and P2, while M1 to M4 contained P1:P2:PC at 1:1:1, 1:1:0.5, 1:1:0.1, and 1:1:0.05, respectively. Analysis parameters for microscopy were optimized by screening a variety of values. For each one, false-positives were evaluated by counting negative GUVs in a sample of 121 positive vesicles. Similarly, false negatives were counted in a sample of 121 positive GUVs. Window width, use of masks, correlation coefficient, and thresholding parameters were sequentially optimized by choosing the parameter that yielded the lowest false-negatives and false-positives. The data shown for MCC refers to the correlation of the Dex–Af647 signal relative to the Dex–Af488 signal. The results of each optimization are shown in The accuracy of GUV classification in positive and negative populations for content/lipid exchange was evaluated as described in To validate the quantification of GUVs content and lipid exchange by the analysis method developed here, dilutions of positive control (PC) GUVs mixed in various ratios with P1 and P2 GUVs were acquired on the same day by microscopy and IFC.Additionally, sample sizes may affect the accuracy of thresholding, especially in the dataset here, where the GUVs number in some controls tended to be lower  potentials, which are present even when composed of zwitterionic lipids due to the specific orientation of the headgroups dipoles . ElectroCharacterization of lipid exchange in phosphatidylcholine GUVs is shown in No content exchange could be detected from the qualitative observation of POPC GUVs micrographs. MPPC-DOPE 9-1 was used to assess content exchange since less aggregates persisted after HS washing with this composition. Additionally, the transition of MPPC to the liquid-ordered state, and de-mixing of DOPE can provide membrane tension, which is known to facilitate content exchange in hemifused systems ,48,49,50Overall, GUVs-GUVs fusion conditions lead to the production of heterogeneous samples via aggregation, hemifusion, mixing, and dilution of various probes. The optimal strategy identified in the previous sections was applied to these highly heterogeneous conditions, highlighting the differences between the control samples used previously, and a real-case scenario. Nevertheless, quantification of these fusion products was shown to be reliable both in terms of lipid and content exchange, with some caveats concerning the sensitivity of lipid exchange detection to aggregation.We present a novel strategy to quantify lipid and content exchange in GUVs using high-throughput image analysis. Large sample sizes and robust statistical analysis permitted the application of optimization strategies to GUVs experiments. Analysis of GUVs usually involves a few handpicked vesicles from the same population to extrapolate results, yielding large uncertainties, and leaving doubts on their reproducibility. With the method we developed here, the correlation between the fluorescent signals in the membrane and the lumen of GUVs provided a robust criterion for the detection of fusion products. We applied colocalization analysis to measure content exchange and lipid exchange in GUVs fusion experiments, first by validating the method in control experiments using IFC, and then by measuring lipid and content exchange in aggregated GUVs. By analysing large populations of GUVs, it was possible to optimize the imaging conditions for quantification, and to characterize GUVs sedimentation in a sealed imaging chamber. After optimization of the analysis parameters, our microscopy-based approach yielded results that were in quantitative agreement with IFC, which is the current state-of-the-art for the in-depth characterization of fluorescence in large populations of GUVs. GUVs concentration estimates were consistently lower in microscopy. Loss in cases of recall lower than one, out of focus GUVs, and vesicles excluded from analysis due to them lying on image boundaries, all contributed to this discrepancy. Better characterization of each contribution may lead to a compensation factor between the two techniques, thereby accounting for these losses, and yielding accurate GUVs concentration estimates. The approach described here was designed to work under a variety of conditions and with samples of heterogeneous membranes, by applying local contrast enhancement and thresholding. We demonstrate that GUVs detection was robust with respect to the fluorophore type and concentration, and to the method used to make GUVs. No GUVs shape assumption was made, aside from a convexity threshold, which was set to exclude noise generated from empty images. If no empty images are included in the acquisition, this shape filtering step can be removed as well, leading to unbiased detection of GUVs with a variety of shapes. The analysis of multi-channel images of GUVs was designed to be flexible, with the ability to analyze any number of fluorescent channels. This can be applied to multiple content or lipid marker transfer events, or to design a joint lipid-content exchange assay by carefully selecting fluorescent probes. With the appropriate controls, this method could quantify GUVs fusion, hemifusion, leakage, and lysis in one experiment. Establishing relationships between multiple fluorescence signals and their colocalization may also be of use in studies of GUVs hosting complex reactions or expressing multiple gene products, thereby providing valuable tools to characterize individual GUVs as reaction vessels.Lipid and content exchange between the fusing GUVs were quantified for a variety of lipid systems upon sodium chloride-induced aggregation. No fusogenic activity of monovalent cations on phospholipid GUVs has been reported thus far, probably due to the long timescales involved in the process. Compared to other fusogens, such as calcium ions, the activity of sodium chloride was weak. Long incubation times and large concentration gradients were required to observe appreciable effects. In non-fusogenic compositions, such as pure POPC, only lipid exchange was observed, while content exchange required other inputs such as temperature–sensitive lipid compositions. With the tools presented here, hemifusion and fusion of GUVs using other alkali halides, lipid compositions, and osmotically induced membrane tension can be easily screened and utilized to optimize a protocol for efficient GUVs-GUVs content exchange and lipid exchange. We found indications that content exchange upon the incubation of MPPC–DOPE 9-1 GUVs with sodium chloride was due to the partial exchange in the hemifusion intermediate, rather than full fusion. The lack of full fusion may not be an issue in artificial cell applications, as it can be used to deliver contents between compartments while also maintaining size homeostasis without the need for division. Lipid and content transfer have been used, up to now, as a proxy for vesicle fusion, but the results presented here indicate that transfer of these materials may not be univocally linked to the full fusion of GUVs. Further characterization of GUVs-GUVs interactions is required to describe these fusion events.Whether or not morphological data based on a single GUVs image can be used to extract meaningful information is still an open question. We performed size compensation on GUVs radii to account for the fact that the compartments were not imaged on their equatorial plane  but did ® AF488)  (DOPE–Af488), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-Rhodamine  (DPPE-Rh), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 5) (DOPE–Cy5), which were all purchased from Avanti Polar Lipids (USA) as well . All water-based solutions were prepared with MilliQ water purified with a purelab flex elga system. Glucose, sucrose, sodium hydroxide, hydrochloric acid, and sodium chloride were purchased from Sigma. N-(2-Hydroxy ethyl)-Piperazine ethane Sulfonic acid (HEPES), Dextran–Cascade Blue™ , Dextran-Alexa Fluor™ 647 , and Dextran-Alexa Fluor™ 488  were all obtained from Thermo Fisher. Novec 7500 engineered fluid used in sample preparation was bought from 3M. Mineral Oil  and squalene (TCI) were used as lipid carriers for the formation of the GUVs. PlusOne Repel Silane ES (Cytiva) was used following the manufacturer’s instructions to treat the microscopy slides, making them hydrophobic.1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-myristoyl-2-palmitoyl-glycero-3-phosphocholine (MPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-ditridecanoyl-sn-glycero-3-phosphocholine (13PC), and 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) were purchased from Avanti Polar Lipids , either in powder or chloroform solution. They were dissolved in chloroform  and used without further purification. Membranes were fluorescently marked with the fluorescent-tagged lipids 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-; Dex–Af647 ; and Dex–CB . All experiments were conducted with POPC GUVs containing a small fraction of fluorescent lipid: 0.4% mol/mol DPPE–Rh for content exchange experiments, and 0.6% mol/mol of DOPE–Af488 minor DOPE–Cy5 for lipid exchange experiments. To test the segmentation performance with phase-separated membranes, GUVs were prepared with a mix of 90% DPPC and 10% DOPE. The protocol was modified to work above the liquid-ordered to liquid-disordered transition temperature of DPPC by carrying out the incubations at 55 °C and performing all centrifugation steps at 40 °C with a force of 16,100× g for 1 min. Vesicles were cooled down to room temperature at a rate of 0.5 °C per minute. The same protocol was adopted to prepare MPPC-DOPE 9-1 GUV for the content exchange experiments. Gentle rehydration was performed following [GUVs were prepared by the emulsion transfer method (ET) and gentle rehydration (GR) to compare the performance of vesicle segmentation under various conditions. The emulsion transfer method was modified from our previous work . A chlorollowing  by dryinVesicle samples were imaged by placing a small volume of suspension on a glass slide (treated with Repel Silane) with an adhesive imaging spacer ; 120 µm thick SecureSeal imaging spacers, Grace BioLabs). The imaging chamber was sealed with a silanized 1.5H coverslip (22 * 22 mm Marienfield), and vesicles were allowed to sediment by inverting the chamber and incubating for at least an hour unless otherwise specified. To limit evaporation and ensure the homogeneous coverage of the slide’s surface, the chamber was filled with fluorinated oil Novec 7500, ensuring no air was trapped with the samples. Imaging was carried out with a Nikon Ti2 microscope equipped with a CREST Optics X-Light V2 Spinning Disk module. Samples were imaged with a Plan Apochromatic 60 × Oil (NA 1.4), and the images were recorded by an Andor iXon Ultra 888 EMCCD camera. Monochromatic light was provided by a Lumencore SpectraX light engine equipped with several LEDs paired with dichroic beam splitters. Emission from various fluorophores was collected through bandpass filters paired with the appropriate excitation light . For each of the 12 datasets, a panel of 121 GUVs was randomly sampled from the positive and negative portions of the dataset. The GUVs that did not exhibit fluorescence in both channels in the positive sample were considered false-positives, while GUVs fluorescence observed in both channels but drawn from the negative population were considered as false-negatives. The parameters were sequentially optimized by selecting the condition that yielded the lowest average false-positive and false-negative percentages. Masks were used to isolate information for the GUV membrane, content, or both regions by excluding the pixels in the window that had a different label value from the one of the tested GUVs in the label maps. Similarly, constructed datasets were used to compare the GUVs quantification carried out by microscopy with IFC.IFC measurements were taken on an Amnis ImagestreamX Mk II imaging flow cytometer. The optical configuration used to record fluorescence data is summarized in GUVs aggregation was induced by incubating 10 µL of 1:1 P1-P2 mixes in 90 µL of sodium chloride solution . The osmolarity of all solutions used was adjusted by measuring it using a Typ3M osmometer (Löser). After 20 h of incubation at room temperature (or 37 °C for MPPC–DOPE 9-1 content exchange), the top 90 µL of solution was removed by aspiration and 90 µL of HS were used to wash the GUVs sediment and induce GUVs separation. After resuspension in an appropriate volume (around 20 µL), the GUVs were imaged as described in We demonstrate a high-throughput microscopy method that permits the quantification of vesicle subpopulations with different fluorescent probes and minimal user input. This method defines vesicle subpopulations based on their membrane and lumen fluorescence properties to identify fusion products and quantify their prevalence in heterogeneous GUVs samples. We highlight the criticalities in sample preparation and compare various analytical approaches to minimize false-negatives and false-positives in the fused population. Our methodology performs on par with state-of-the-art techniques and is readily applicable, since it is based on widely used software including ImageJ and R. Furthermore, our findings can be used and integrated in existing analysis systems to enhance their flexibility in future applications. We applied this fusion quantification method to aggregated GUVs populations in sodium chloride, demonstrating its previously unreported fusogenic activity on a variety of membrane compositions. Fusion events induced during sodium chloride aggregation may be unwanted artefacts in GUVs assembly experiments, or desirable in artificial cell application, where content exchange can be induced in hemifused GUVs clusters.


A relationship between walking ability and self-efficacy has been demonstrated in various rehabilitation patient populations. In experienced prosthetic ambulators, walking ability is related to self-efficacy of balance, however, this relationship has not been quantified for those with newly acquired lower limb amputations (LLA).To investigate the association between walking performance (objective) and self-reported walking abilities (subjective) on balance self-efficacy in those with LLA.Cross-sectional study of 27 people  at discharge from inpatient prosthetic rehabilitation for first major unilateral LLA. Individuals completed 6m straight path walking and the L-Test under single- and dual-task conditions. The Prosthesis Evaluation Questionnaire (PEQ) was administered, and the Ambulation subscale provided subjective measures of walking ability. A single PEQ question on satisfaction with walking (16B) was also used as a proxy for subjective walking ability. The Activities-specific Balance Confidence Scale measured balance self-efficacy. Multivariable linear regression was used to evaluate the strength of association between walking ability (objective and subjective) and balance self-efficacy (dependent variable).Walking velocity on the 6m straight path under single-task (p=0.011) and dual-task conditions (p=0.039), the single-task L-Test (p=0.035) and self-reported satisfaction with walking (p=0.019) were associated with self-efficacy of balance.Objective measures of walking ability that were independently associated with balance self-efficacy included straight path walking velocity under single and dual-task conditions and the single-task L-Test. Satisfaction with walking was also associated with balance self-efficacy. This highlights the interplay between physical and psychological factors during rehabilitation. More research in the area of self-efficacy and walking ability is needed to establish self-efficacy as a target during prosthetic rehabilitation for those with LLA. There is an established trend in walking ability following prosthetic rehabilitation for LLA; the largest improvements in self-reported functional mobility occur between 6 weeks and 4 months post-surgery, after which time function plateaus.5Individuals encounter significant physical, psychological and social consequences following the loss of a limb.7 Falls are a particular concern in this population, as over half of those with LLA fall at least once annually.9 After a fall, older individuals without LLA may limit their walking due to impact on self-efficacy and a fear of falling.9 However, walking is crucial for social participation and maintaining QOL in those with LLA.10 As such, the impact of psychological factors on walking ability in this population have been investigated. Research indicates that factors such as self-efficacy may impact upon the rehabilitation goals of social activity and community integration for those with LLA.12While mobility and function improve during rehabilitation, the LLA population may experience difficulties with gait that persist long-term.13 Developing self-efficacy may be done through observing successful peers or through verbal persuasion from credible individuals, such as physicians.14 However, the strongest sources of self-efficacy are mastery experiences; other sources are generally weak and are likely to deteriorate.15 Mastery experiences are the successful completion of a specific task.15 These experiences can build upon each other as an individual accomplishes progressively more difficult activities.15 As such, the mastery experience of ambulating with a prosthetic device would be expected to strongly impact a person's sense of competence and self-efficacy.Self-efficacy is an individual's belief in their ability to perform a certain task.16 in those with knee osteoarthritis,18 for those with diabetes and peripheral arterial disease,19 and in individuals with multiple sclerosis.20 However, the experience following LLA is different to these populations as the latter have previous mastery experiences from which they derive their self-efficacy. At the onset of rehabilitation, all tasks relating to the prosthetic device are novel. Prosthetic training provides a series of mastery experiences for new skills related to the use of a prosthesis, allowing those with LLA to develop self-efficacy. Individuals also receive encouragement from clinicians during their time in prosthetic rehabilitation and are able to observe fellow patients. Levels of self-efficacy during this rehabilitation process have not been reported, however, for more experienced prosthetic ambulators, walking ability is related to balance self-efficacy.12Research has demonstrated a positive relationship between walking performance and self-efficacy following a stroke,Physical and psychological factors are both important considerations for rehabilitation following LLA. For the psychological factor of self-efficacy to be addressed as a target during the rehabilitation process, a better understanding of the relationship between walking ability and self-efficacy is needed. To quantify the relationship between self-efficacy of balance and walking ability in those with LLA, self-reported measures of ability and objective walking performance must be carried out. The objective of this study was to investigate the association between walking performance (objective) and self-reported walking ability (subjective) on balance self-efficacy in those with LLA at discharge from inpatient prosthetic rehabilitation. It is hypothesized that at this stage in the rehabilitation process straight path walking and complex path walking under single-task conditions, as well as patient satisfaction with walking, will be related to balance self-efficacy.This was a cross-sectional study of individuals completing inpatient prosthetic rehabilitation for first major LLA. Participants were recruited from the Regional Amputee Rehabilitation Program at Parkwood Institute in London, Ontario between March 2016 and April 2017. The study was approved by the University of Western Ontario Ethics Board, and by the Lawson Health Research Institute Clinical Resources Impact Committee. All individuals provided written informed consent prior to participating in the study.21 A total of 27 participants were recruited during the study period.Individuals participating in an inpatient rehabilitation program for their first major, unilateral LLA were recruited prior to discharge. LLA at the level of transtibial or above were considered major amputations; these were selected as the represent the most common levels seen in prosthetic rehabilitation programs. All individuals were new prosthetic ambulators as they were fitted with a prosthesis upon commencement of inpatient rehabilitation. Admission criteria to inpatient rehabilitation include achievable rehabilitation and prosthetic goals, medically stable and sufficiently conditioned to undergo a training program, ability to learn and retain new skills, and emotionally and socially  prepared to participate. Training involved prosthetic limb fitting, limb care and management, and safe skill learning to progress from transfers to gait training with the use of various gait aids. Eligibility criteria for the current study were: ≥50 years of age, functional use of English and ability to walk 10m without assistance from another person. Exclusion criteria included any physical problem that significantly limited movement or presence of severe depression. Those were severe depression were excluded as depression can negatively impact performance on cognitive tasks requiring attention and visuospatial abilities, especially in older adults.The following demographic and medical history information was obtained: age, sex, height, weight, level of amputation, etiology of amputation, time since amputation, use of walking aid at discharge, level of education, medications, comorbidities, history of falls in the past 12 months, global cognitive status as measured by the Montreal Cognitive Assessment (MoCA).22 Testing was carried out under single- and dual-task conditions with standardized instructions.23 Using standard instructions, participants began sitting on an armless chair and upon the word “go” rose to standing and walked 3 meters, turned 90 degrees, walked 7 meters, turned 180 degrees, and walked the same path to return to a seated position. Patients walked at their usual pace and were timed to the nearest 100th of a second.The L-Test is a complex walking task that evaluates functional mobility for those with LLA.® System electronic walkway . One-meter acceleration and deceleration zones were provided to ensure only steady state walking was captured. Assessment of gait velocity using the GAITRite® System was performed separately to the walking assessment using the L-Test.A 6m straight path assessed gait velocity (cm/s) under single- and dual-task conditions. Participants walked at their usual pace along the GAITRite24 It consists of 9 stand-alone subscales including Ambulation, Appearance, Frustration, Perceived Response, Residual Limb Health, Social Burden, Sounds, Utility and Well Being. The Ambulation subscale was used to assess self-perceived walking ability .24 The following standalone question relating to participant satisfaction (question 16B) was also used: “Over the past four weeks, rate how satisfied you have been with how you are walking”. Questions utilize a 100mm Visual Analog Scale (VAS) from “cannot” (0) to “no problem” (100). Higher scores indicate a more positive response.24 The complete PEQ was administered to participants.The Prosthesis Evaluation Questionnaire (PEQ) is a self-administered tool that evaluates prosthetic related QOL.25 Participants rate how confident they are in performing activities without losing their balance or becoming unsteady, from 0% to 100%. A mean of the 16 items gives the overall score, with higher scores indicating better balance self-efficacy.25 The scale has good test-retest reliability and internal consistency in the LLA population.26The ABC Scale is a 16-item tool that evaluates balance confidence, or self-efficacy of balance.27 The task chosen to represent this increased cognitive load was serial subtraction by 3's, starting from a randomly selected number between 100 and 150. Participants were instructed to count aloud as they walked, and responses were recorded. No instructions on task prioritization were given. All assessments occurred within 48 hours of discharge. Patients used their usual gait aids to perform walking tests. A standardized protocol, based on Hunter et al.,23 was used during testing.Single-task  and dual-task  walking assessments were performed in this study. Demographic and clinical information was collected first. Following this, walking tasks were demonstrated by a research assistant. Participants then completed single-task assessments and received a 5-minute break before completing dual-task testing. Only one trial per walking test was completed. Dual-task testing using a cognitively demanding task was used to more closely approximate real-world walking.29 Due to the small sample size of the current study, no subgroup analysis based on level of amputation was performed. Significance level was set to p<0.05. Statistical analysis was performed using the IBM SPSS Statistics version 24.0 .Variables were calculated as means and SDs or frequencies and percentages, as appropriate. Multivariable linear regression was used to evaluate the strength of association between walking ability (objective and subjective) and balance self-efficacy (dependent variable). Regression diagnostics were performed to ensure assumptions for linear regressions were met. To generate the most parsimonious model, analysis was adjusted for age, level of amputation, number of comorbidities and number of medications. These variables were selected based on the literature and their clinical significance.Table 1. Twenty-seven participants were included in the study. Many participants  had transtibial amputations; the remainder of the participants had transfemoral amputations. Almost 2/3rds of the study participants were male . The mean balance self-efficacy rating using the ABC scale was 69.48 ± 14.50, with scores ranging from 35.63 to 89.69.Demographics are summarized in Table 2. After adjusting for confounders, single-task and dual-task walking velocity along the 6m straight path were both found to be independently associated with self-efficacy of balance (Table 3). For every 1cm/s increase in walking velocity along a 6m path, balance self-efficacy increases by 0.32 for single-task (p=0.011) and 0.28 for dual-task conditions (p=0.039).Results from objective and subjective measures of walking ability are presented in Table 3). For every one second decrease in performance on the L-Test balance self-efficacy increases by 0.17 (p=0.035), and for every 1-point increase in satisfaction with walking self-efficacy increases by 0.60 (p=0.019). No associations between the other objective or subjective measures and balance self-efficacy were found.Single-task performance on the L-Test and satisfaction with walking were also independently associated with self-efficacy of balance  concept or design of the work, or acquisition, analysis or interpretation of data; (2) drafted or critically revised the article; (3) approved the version to be published; (4) participated sufficiently to take public responsibility for appropriate portions of the content.This work was supported by the St. Joseph's Healthcare Foundation Cognitive Vitality and Brain Health Seed Funding Opportunity in London, Ontario, Canada. The funding body had no involvement in the conduct of the study.The study was approved by the University of Western Ontario Ethics Board, and by the Lawson Health Research Institute Clinical Resources Impact Committee. All individuals provided written informed consent prior to participating in the study.


Background and aimsSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can exacerbate hyperglycemia and can cause life-threatening diabetic ketoacidosis (DKA) in patients with diabetes mellitus (DM). The objective of this study is to compare the characteristics of diabetic COVID-19 patients with and without DKA and to determine the predictors of mortality in the setting of COVID-19 and DKA.MethodsThis is a retrospective single-center cohort study including patients admitted to our hospital with COVID-19 and DM from March 2020 to June 2020. Patients with DKA were filtered as per the diagnostic criteria set by the American Diabetes Association (ADA). Patients with hyperosmolar hyperglycemic state (HHS) were excluded. A retrospective analysis was performed, which included those who developed DKA and those with neither DKA nor HHS. The primary outcome measurement was mortality rate and predictors of mortality for DKA.ResultsOut of 301 patients with COVID-19 and DM, 30 (10%) had DKA and five (1.7%) had HHS. Mortality was significantly higher in the DKA group compared to the non-DKA/HHS group . After adjusting for parameters used for multivariate logistic model for mortality, DKA was no longer associated with mortality . The independent predictors for mortality were age, platelet count, serum creatinine, C-reactive protein, hypoxic respiratory failure, need for intubation, and need for vasopressors.ConclusionOur study demonstrates higher mortality rate in diabetic COVID-19 patients with DKA. Though direct and independent statistical association of mortality with DKA could not be proven in our multivariate logistic model, physicians must be vigilant in risk-stratifying and managing these patients in a timely manner. The coronavirus disease 2019 (COVID-19) caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evoked a whole new perspective on the wide range of metabolic derangements caused by a viral infection. Within a span of three months since its genesis, the World Health Organization (WHO) declared this viral outbreak as a global pandemic . A meta-The hyperinflammatory states of both COVID-19 and DKA synergistically lead to the clinical downfall of such patients. A large retrospective study of 658 hospitalized patients with COVID-19 demonstrated that this infection can generate ketosis and ketoacidosis even in those without DM . StudiesWe hypothesized worse clinical outcomes with an increased mortality in patients with DKA precipitated by SARS-CoV-2 infection. The objective of this study is to understand the characteristics of diabetic patients with COVID-19 and DKA and to determine the predictors of their mortality.This is a retrospective single-center cohort study involving patients admitted during the first peak of COVID-19 from March 2020 to June 2020. Following approval from our IRB (study #20:56) and ethics committee, we identified a total of 301 subjects, aged 18 and above, in our electronic medical record system using the International Classification of Diseases 10th Revision (ICD-10) codes for the diagnosis of COVID-19 and DM. All of them were confirmed to have a positive nasopharyngeal RT-PCR for SARS-CoV-2. Data on basic demographic factors, medical history, laboratory values, hospital course including the modalities of treatment and final clinical outcome were manually extracted. All the laboratory values were from the day of admission, except for d-dimer, C-reactive protein (CRP), and lactate dehydrogenase (LDH), which were the highest recorded values during the hospitalization. We also obtained a list of diabetic patients who had positive blood cultures and/or respiratory viral panel during June 2018 to June 2020 using the ICD-10 codes.We defined DKA cases as those who had serum glucose > 250 mg/dL, serum bicarbonate ≤ 18 mEq/L, anion gap > 10 mEq/L, and positive serum/urine ketones. We used the definition criteria set by the American Diabetes Association (ADA) . Those wContinuous variables are described as median and interquartile range except for age, which is described as mean and SD, as it was the only normally distributed variable. Categorical variables were described as counts and percentages. Wilcoxon rank-sum test was used for analyzing continuous variables except for age, for which the logistic regression was used with DKA as the outcome. Univariate and multivariate logistic regression models were used to analyze for mortality. A p-value of <0.05 was considered significant. All data were analyzed using the software Stata/SE Version 17 .Out of 301 patients with COVID-19 and DM, we found that 30 (10%) had DKA, of whom eight (26.7%) were diagnosed after admission. There were five (1.7%) with HHS who were excluded from analysis, leaving 266 patients in the non-DKA/HHS group.In the comparative analysis performed between the DKA and non-DKA/HHS groups Table , there wPatients with DKA were predominantly insulin-dependent , medication non-compliant , and poorly controlled DM . We did not see any statistically significant difference in the race and other comorbidities including congestive heart failure, chronic obstructive pulmonary disease, asthma, chronic kidney disease, and malignancy between both groups. There was also no statistically significant difference in other home medications between both groups, including SGLT2 (sodium-glucose cotransporter-2) inhibitors. Among the laboratory values, we found that the DKA subgroup had higher lactic acid , CRP , and LDH .Patients with DKA were significantly more likely to be septic with two or more systemic inflammatory response syndrome (SIRS) criteria satisfied upon presentation  and to require ICU stay , intubation , and vasopressors . A significantly higher proportion of patients with DKA received steroids . Mortality was observed in 11 (36.6%) patients with DKA, which was significantly higher than those with neither DKA nor HHS .Using a multivariate logistic regression analysis Table , we deteOut of a total of 286 patients with DM and positive blood cultures and/or respiratory viral panel during June 2018 to June 2020, 15 (5.2%) patients had developed DKA and two (0.7%) patients had developed HHS.Our study corroborates the previously established data that diabetic patients with COVID-19 who develop DKA have a significantly higher mortality compared to those without DKA/HHS. However, it has not yet been established as an independent predictor of mortality. Even in our study, though DKA had significantly higher mortality, we could not establish it as an independent predictor of mortality. A recently published review article on this subject warrants the need for more studies with specific variables and larger sample size to establish a more definitive correlation . While bA state of acute insulin deficiency and profound insulin resistance in association with a surge of inflammatory response triggered by SARS-CoV-2 seems to be the primary mechanism by which these patients are predisposed to glycemic crisis . This noYang et al. also suggested an acute damage of islets leading to insulin-dependent DM in patients with SARS . There wSARS-CoV-2 can dysregulate the immune system and produce an inflammatory response characterized by high serum levels of pro-inflammatory cytokines  and chemokines (IL-8) . In sevePatients with DM and obesity are characterized by a chronic state of low-grade inflammation with defects in their innate and adaptive immunity . Obese iThe gastrointestinal manifestations of COVID-19, such as anorexia, nausea, vomiting, and diarrhea, can contribute to volume depletion and increased lipolysis resulting in ketogenesis. These patients are admitted in a catabolic state, which is exacerbated by an inability to compensate for metabolic acidosis through renal and pulmonary mechanisms due to concurrent acute kidney injury (AKI) and impaired pulmonary gas exchange. Unopposed angiotensin II increases pulmonary vascular permeability and worsens the injury to lung parenchyma . Fluid cWhile our study shows that patients with T1DM were significantly more likely to develop DKA, majority of patients in both cohorts had T2DM due to the worldwide higher relative prevalence. Development of AKI is a frequent complication in COVID-19 patients and is associated with an increased risk of adverse outcomes including mortality . In our In patients with SARS-CoV infection, it was noted that more than 50% of them developed DM during hospitalization while not receiving any corticosteroids. Follow-up after three years showed that only 5% of these patients remained diabetic, indicating the transient nature of damage of islets by SARS-CoV infection . SimilarThe limitation of our study was related to the retrospective nature of the study due to which causality could not be assigned. Some laboratory data were not available for all patients, which limited our ability to extend the statistical analysis to explore other associations and contributing factors for mortality. There is currently insufficient data to determine if SARS-CoV-2 poses an increased risk over other severe infectious diseases in predisposition to DKA. A collaborative prospective, multi-center approach would help in studying the long-term impact of COVID-19 on DM.In conclusion, physicians must be vigilant at all times to diagnose DKA with high degree of clinical suspicion in diabetic patients with COVID-19, especially in those with AKI, elevated CRP, hypoxic respiratory failure, need for intubation, and need for vasopressors. These factors should be considered while risk-stratifying diabetic patients to decrease worse clinical outcomes by a timely management. Insulin dosage should be carefully adjusted during follow-up due to the risk of hypoglycemia from tapering steroid doses and simultaneous recovery of pancreatic β-cell function.