Asthma is a growing health problem in children in marginalised urban settings in low-income and middle-income countries. Asthma attacks are an important cause of emergency care attendance and long-term morbidity. We designed a prospective study, the Asthma Attacks study, to identify factors associated with recurrence of asthma attacks (or exacerbations) among children and adolescents attending emergency care in three Ecuadorian cities.Prospective cohort study designed to identify risk factors associated with recurrence of asthma attacks in 450 children and adolescents aged 5–17 years attending emergency care in public hospitals in three Ecuadorian cities . The primary outcome will be rate of asthma attack recurrence during up to 12 months of follow-up. Data are being collected at baseline and during follow-up by questionnaire: sociodemographic data, asthma history and management (baseline only); recurrence of asthma symptoms and attacks (monthly); economic costs of asthma to family; Asthma Control Test; Pediatric Asthma Quality of life Questionnaire; and Newcastle Asthma Knowledge Questionnaire (baseline only). In addition, the following are being measured at baseline and during follow-up: lung function and reversibility by spirometry before and after salbutamol; fractional exhaled nitric oxide (FeNO); and presence of IgG antibodies to SARS-CoV-2 in blood. Recruitment started in 2019 but because of severe disruption to emergency services caused by the COVID-19 pandemic, eligibility criteria were modified to include asthmatic children with uncontrolled symptoms and registered with collaborating hospitals. Data will be analysed using logistic regression and survival analyses.Ethical approval was obtained from the Hospital General Docente de Calderon (CEISH-HGDC 2019-001) and Ecuadorian Ministry of Public Health (MSP-CGDES-2021-0041-O N° 096-2021). The study results will be disseminated through presentations at conferences and to key stakeholder groups including policy-makers, postgraduate theses, peer-review publications and a study website. Participants gave informed consent to participate in the study before taking part. Asthma attacks are a growing cause of morbidity in children and burden on health systems in low-income and middle-income countries, and there is limited information on factors associated with asthma attacks in poor urban settings in Latin America.This protocol describes a study designed to investigate risk factors associated with recurrence of asthma attacks in children and adolescents living in marginalised settings in three Ecuadorian cities.This study is recruiting children and adolescents with uncontrolled asthma symptoms and asthma attacks and following them prospectively for 6–12 months to identify factors associated with recurrence of attacks in the context of the COVID-19 pandemic.The COVID-19 pandemic has had a major impact on health-seeking behaviours, while mitigation strategies to control transmission of the novel coronavirus, SARS-CoV-2, have resulted in marked declines in the circulation of respiratory viruses that are considered to underlie a high proportion of asthma attacks. Such factors may interfere with recruitment and follow-up of study participants.Asthma is the most common chronic disease of childhood and is estimated to affect more than 350 million people worldwide.2Asthma attacks or exacerbations are frequent in children and are a common cause of hospitalisation,8 12To understand better factors contributing to the risk of asthma attacks, it is important to identify those factors contributing to suboptimal management, which can be improved through better healthcare.13 16 18The Asthma Attacks Study (ATTACK) aims to identify factors related to recurrence of asthma attacks in Ecuadorian children attending emergency care in three Ecuadorian cities. Recruitment into the study started in March 2019, but was interrupted by the COVID-19 pandemic that had a major impact on use of and access to emergency care.Severe asthma in children is associated with loss of lung function,13Asthma attacks represent an acute or subacute increase in respiratory symptoms2 agonist for moderate-to-severe asthma.26 32Asthma attacks may be prevented either by avoiding previously identified triggers or by appropriate preventive treatment. Patients using ICS were three times more likely to have well-controlled asthma,Soon after the emergence of the novel coronavirus, SARS-CoV-2, in humans and its spread into a global pandemic, the question was raised as to whether this novel viral infection might alter the risk of asthma symptoms because of deficient antiviral immune responses and the tendency to increase exacerbations during infections with other respiratory viruses.A prospective multicentre study is being done in children aged 5–17 years in low-income settings in three Ecuadorian cities  to study factors associated with acute asthma attacks. Because of the impact of the COVID-19 pandemic on risk of asthma attacks and healthcare access, the study was divided into two phases of recruitment and follow-up: (1) phase I (or pre-COVID-19 from May 2019 to March 2020) evaluated risk factors associated with recurrence of acute asthma attacks among children and adolescents attending ERs of public hospitals in low-income settings in the three cities and (2) phase II (or peri-COVID-19 from March 2021 to June 2022) is evaluating risk factors associated with attacks among children and adolescents with asthma and current symptoms who have previously attended specialist care at public hospitals in Cuenca and Portoviejo. Phase II is also evaluating the effects of exposures to SARS-CoV-2 on asthma attacks and symptoms. In addition, both study phases are evaluating the impact of asthma symptoms and adequate control of asthma on the quality of life and economic costs for the family of children and adolescents with asthma. The objectives of phases I and II are detailed in 41This is a multicentre prospective study conducted in three Ecuadorian cities . Quito, the capital, and Cuenca, the third largest city, are located in the Andean highlands  where the annual average temperature is ~16°C, while Portoviejo is in the coastal region (at sea level) with an annual average temperature of ~25°C.44The study was initially based in tertiary care hospitals in each of the three cities .Children and adolescents aged 5–17 years were recruited either while attending ERs with an asthma attack or through daily registries and contact telephone numbers of patients attending the ERs with an asthma attack. From May 2019, eligible patients  were proThe COVID-19 pandemic had a dramatic impact on our ability to recruit children with asthma attacks in the three study hospitals that resulted in the suspension of recruitment in March 2020. This was owing to the reassignment of all three hospitals to COVID-19-only activities for prolonged periods and the imposition of a legally enforced lockdown nationally. A study manager supervised day-to-day activities of dedicated teams (physician and nurse) of trained study personnel based in each of the public hospitals. Each participant was followed up for 12 months in phase I and at least 6 months in phase II. Standardised procedures were used throughout . SubjectThe following questionnaires were used as indicated in Lung function testing is being done according to American Thoracic Society criteria using a portable ultrasonic spirometer . Global Lung Function Initiative 2012 reference values will be used to calculate z scores depending on age, sex, height and ethnicity.Blood sample are being taken at baseline, 6 and 12 months in phase II. Blood is drawn from an antecubital vein into a 4 mL plastic tube containing EDTA (ethylenediaminetetraacetic acid) as anticoagulant . After centrifugation, plasma is stored at −20°C until analysis. Levels of IgG specific for SARS-CoV-2 are being measured using a validated enzyme linked immunosorbent assay.55Personnel gathering data during face-to-face visits have been trained to use appropriate personal protective equipment according to international guidelines.A sample size of 450 patients was proposed for phase I, based on a binary outcome variable of any recurrence of an asthma attack within 12 months or so. We used an estimated recurrence risk of about 50% during 6-month follow-up from a similar population in Ecuador.2-agonist bronchodilator treatment. The original definition for an asthma attack in phase I that included ER attendance had to be modified in the light of changes in health-seeking behaviour caused by the COVID-19 pandemic and fear of contagion in hospitals and other acute health facilities.The definition of an asthma attack in both phases of the study is an episode of acute wheezing requiring an unscheduled visit to a health professional (including visits to ERs) and which is controlled by βThe statistical models to be used by study objective are summarised in Hosmer-Lemeshow measure of goodness of fit would be employed for logistic regression models with a p value of less than 0.05 indicating a poor fit to the data. Model validation will be done by splitting the data set into training–testing subsets (logistic regression setting). Cross validation techniques will be used to understand the prediction power of the corresponding logistic regression model using area under the receiver operator curve, prior to propose the model entering clinical practice with the view of elaborating a prognostic model. In a survival setting, it is also possible that the precise time of the attacks may be missed but recorded as between two time points. This would fall into interval censored data as an alternative to survival setting in which the precise time of the event is required.Multiple recurrences of asthma attacks within the follow-up period will generate a longitudinal binary outcome, with multiple records for each participant over follow-up. The hierarchical structure of the data requires special inferential techniques, which account for the two sources of variability: that of between participants and within participants. Three different modelling approaches will be considered: generalised estimation equation, mixed models and random effects survival models. The first approach provides population average estimates, the second provides subject-specific estimates, while the third is also known as shared frailty survival model. A frailty is a latent random effect, which enters multiplicatively on the hazard function and accounts for the multiple failures setting and/or for the clustered aspect of the data.Complete data analyses will be done under missing completely at random assumptions. However, patterns in missing data will be explored and methods to evaluate potential impact of missing data on estimates will be used as appropriate such as multiple imputations using chained equations accompanied by a sensitivity analysis to the missing data assumptions.The prevalence of childhood asthma has increased in many LMICs over the past 50 or so years where asthma attacks are a growing burden to health systems.This study provides a range of climatic settings in Ecuador from high altitude (Cuenca and Quito) to sea level (Portoviejo). Study activities were interrupted in March 2020 by the COVID-19 pandemic resulting in a suspension of recruitment in hospital ERs owing to dramatic changes in access to and use of health facilities. As a consequence, the study protocol was modified to allow reinitiation of recruitment and also the evaluation of the effect of exposures to SARS-CoV-2 on risk of asthma attacks and symptoms in children. The study will help fill an unmet knowledge gap on the effects of SARS-CoV-2 on paediatric asthma, particularly from low-resource LMIC settings.19A potential limitation is recruitment of sufficient subjects with an acute asthma attack attending ERs at the three public hospitals and ensuring monthly follow-up in phase I of the study. The COVID-19 pandemic had a major impact on health-seeking behaviours while mitigation strategies to control transmission of the novel coronavirus, SARS-CoV-2, resulted in marked declines in the circulation of respiratory viruses that are considered to cause a high proportion of asthma attacks.Ethical approval for phase I was obtained from the Ethics Committee of the Hospital General Docente de Calderon (CEISH-HGDC 2019-001) and for phase II from the Ecuadorian Ministry of Public Health COVID-19 Ethics Committee (MSP-CGDES-2021-0041-O N° 096-2021). The study results will be disseminated through presentations at conferences and to key stakeholder groups including policy-makers, postgraduate student theses, peer-review publications and on a study website. Participants gave informed consent to participate in the study before taking part.


The COVID-19 crisis has disrupted health systems all over the world. In a survey by the WHO, 93% of the countries reported disruption in their mental health services. This research assessed the extent to which mental health was included in the national response to the COVID-19 pandemic in African countries. It also explored barriers and enablers to mental health integration into the COVID-19 response. Lessons learned from COVID-19 can help improve the response to future public health emergencies.A web-based survey was sent to mental health focal points in 55 African countries. The survey assessed the perceived degree of implementation of the Inter-Agency Standing Committee (IASC) “14 Globally Recommended Activities” for mental health response to COVID-19. This was followed by in-depth interviews to explore barriers and enablers to mental health integration into the COVID-19 response.Responses were received from 28 countries. Lack of political will, poor funding, limited human resources, and weak pre-existing mental health systems were the key challenges in addressing mental health needs during COVID-19. Participants highlighted the need to capitalize on the increased attention to mental health during COVID-19 to support its integration into the emergency preparedness and response plans and strengthen health systems in the longer term. They have also stressed the importance of sustaining and strengthening the new partnerships and service delivery models that emerged during the COVID-19 pandemic.The number of recommended mental health activities implemented during the COVID-19 pandemic varied considerably across African countries. Several factors limit mental health integration into emergency response. However, there are signs of optimism, as mental health gained some attention during COVID-19, which can be built on to integrate mental health into emergency response and strengthen health systems in the long term.• Capitalize on the increased attention to mental health during COVID-19 to support its integration into the emergency preparedness and response plans and strengthen health systems in the long term.• Sustain and strengthen the new partnerships and service delivery models that emerged during the COVID-19 pandemic.


Developing psychotherapeutic competencies is an essential part of psychiatric training. All core trainees in LYPFT until 2021 saw a patient for Psychodynamic Psychotherapy. The pandemic led to unprecedented changes to clinical practice and medical education. In LYPFT all face-to-face appointments in the Medical Psychotherapy Service were paused in March 2020. Patients were offered the choice to continue therapy remotely or postpone therapy. Supervision was also moved to a remote format. Face-to-face psychotherapy sessions resumed from August 2020, with new departmental procedures around infection control and the use of PPE. This project aimed to establish the junior doctors’ experience of delivering psychodynamic psychotherapy in LYPFT during the COVID-19 pandemic.The project was carried out via a two-step methodology: Firstly with an online survey which included a quantitative analysis of the impact of the pandemic; and secondly via semi-structured interviews with a resultant thematic analysis.22 junior doctors who were invited to participate, 15 completing the survey (68%). Four patients had deferred therapy; the mean length of deferral was 2 months. Ten respondents had sessions cancelled due to infection or self-isolation. Face-to-face delivery was experienced by 13 respondents, 5 respondents had delivered therapy via phone and 6 had delivered therapy with PPE. Thirteen were concerned about attaining their psychotherapy competencies. Seven preferred face-to-face supervision, and 4 preferred remote working.Thematic Analysis of the semi-structured interviews identified three themes regarding the impact of the COVID-19 pandemic on Junior Doctors experience of Psychodynamic Psychotherapy, with sub-themes detailed below. Throughout the themes, the challenges and difficulties with delivering therapy in the COVID-19 pandemic, as well as areas of good practice and opportunities were identified.The Structure of Therapy The Therapist's Training The Work of Therapy To create a short guide for junior doctors delivering Psychodynamic Psychotherapy during a pandemic.To consider the types of supervision delivery within the Medical Psychotherapy ServiceTo ensure there is space for junior doctors within the Medical Psychotherapy department or a private space within their base placement, should remote therapy be required.To ensure future plans related to possible pandemic restrictions address the need for good quality and strong internet connections/WIFIRecommendations:


Sickle cell disease (SCD) is a heterogeneous group of inherited disorders characterized by the production of sickle hemoglobin which is less soluble than an adult or fetal hemoglobin. Voxelotor is a hemoglobin S polymerization inhibitor that has been approved for sickle cell disease treatment in the adult and adolescent populations. It acts as a hemoglobin modulator by increasing its affinity to oxygen which prevents red blood cells from sickling. Chronic kidney disease is a common but under-reported complication of SCD and it is a leading cause of morbidity and mortality. The data about the safety and efficacy of voxelotor use in chronic kidney disease is limited. Herein we report a 49-year-old man, with sickle cell disease and stage IV chronic kidney disease, who was managed successfully with voxelotor and resulted in decreasing transfusion requirement and vaso-occlusive painful crisis without affecting kidney function. Sickle cell disease (SCD) is a heterogeneous group of inherited disorders characterized by the production of sickle hemoglobin, which polymerizes when deoxygenated resulting in rigid and very fragile red blood cells. Hemoglobin SS is the most common genotype, however, other genotypes like C, D, and E present with different phenotypes of chronic hemolysis and recurrent vaso-occlusive crisis , 2. SCD Voxelotor is a hemoglobin S polymerization inhibitor that has been approved for sickle cell disease treatment by the United States Food and Drug Administration (FDA) November 2019 for the adult and adolescent population (≥12 years) . It reveChronic kidney disease is a common but under-reported complication of SCD and is a leading cause of morbidity and mortality. The annual incidence of acute renal failure and chronic kidney disease was found to be 2–3 times higher compared to the non-sickle group in a retrospective study . Renal iThe data about the safety and efficacy of voxelotor in chronic kidney disease is limited. Herein we report a case of a 49-year-old man, a known case of sickle cell disease and stage IV chronic kidney disease who was requiring frequent transfusion and suffering from severe body pain, managed successfully with voxelotor without affecting kidney function.2), splenectomy at age 20, osteonecrosis of the left femoral head status post left hip replacement at age of 25. The patient required recurrent hospital visits for vaso-occlusive pain crises and blood transfusions despite being compliant with hydroxyurea 1000 mg twice daily.A patient is a 49-year-old man with SCD (hemoglobin S/D double heterozygous), diabetes mellitus, and hypertension. His clinical course was complicated by chronic kidney stage IV, due to sickle glomerulopathy  and phase II (glucuronidation). Around two-thirds of voxelotor and its metabolites are excreted in feces (62.6%) while around one-third are excreted in the urine (35.4%) after oral administration  for the publication of any potentially identifiable images or data included in this article.AA contributed to acquisition of data and drafted the manuscript. Both authors equally contributed to writing and editing.


Acute myocarditis is commonly associated with viral infections, including severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Myocarditis following mRNA COVID-19 vaccination has also been reported, however this is rare and usually resolves within days or weeks. We present a case of acute myocarditis reported after vaccination with mRNA-1273 COVID-19 vaccine (Moderna) diagnosed using cardiac magnetic resonance imaging (CMR). This report describes the utility of CMR in the diagnosis and follow-up of such patients using parameters which could suggest the clinical course of myocarditis.A 23-year-old male presented in the emergency department with complaints of chest pain radiating to the left arm following vaccination with the second dose of COVID-19 mRNA-1273 vaccine (Moderna). Patient's history revealed an incidence of myocarditis in the past. CMR showed a mid-range left ventricular ejection fraction (38%) and subepicardial late gadolinium enhancement (LGE) in the inferolateral and apical myocardial segments with diffuse elevation of native T1 mapping relaxation times in all myocardial segments. The patient was admitted briefly in the intensive care unit and after a favorable clinical course was discharged from the hospital in stable condition. A follow-up CMR after 3 months revealed normalization of LVEF (57%) and native T1- times in most segments. Scarred myocardium reflecting chronic myocarditis continued to show elevated T1 times.Our patient presenting with acute myocarditis after recent COVID-19 mRNA vaccination reported a favorable clinical course. CMR revealed increased T1 mapping relaxation times diffusely spread across the myocardium and an impairment of the left ventricular function (LVEF) during the acute phase. However, the LVEF as well as the T1 times normalized at follow-up in all segments except for myocardium affected by chronic myocarditis. The use of BNT163b2 mRNA (Pfizer-Biontech) and mRNA-1273 (Moderna) vaccines to curb the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has gained traction worldwide . We presA 23-year-old male initially presented in our emergency department with symptoms suggestive of angina pectoris. The patient reported the sudden onset of chest pain radiating to the left arm as well as headache 1 day after vaccination with the second dose of the mRNA-1273 (Moderna) COVID-19 vaccine. Dyspnoea, fever, or excessive sweating were denied. Further examination revealed a relevant past-history of perimyocarditis in 2018 and 2019 (possibly post-infectious). The patient was not on medication at the time of presentation.The clinical examination of the patient was unremarkable. The body temperature recording was 37.5°C. The heart rate on admission was 96 beats/min and the blood pressure was 110/60 mmHg. The oxygen saturation was 99% on room air. An electrocardiogram (ECG) showed sinus rhythm with mild concave ST-elevations in II, III, and aVF. Laboratory data see  revealedThe troponin I level peaked on the second day  and NT-proBNP levels showed moderate elevation . A thoracic CT revealed no obvious pulmonary infiltrates and no evidence of coronary plaques or significant stenoses in the coronaries. An echocardiogram performed in the ICU revealed a moderately reduced left ventricular ejection fraction (LVEF) with hypokinetic inferolateral and apical segments.The echocardiographic findings were confirmed using a cardiac MRI (CMR) . Considering the medical history of the patient, images from the CMR scan during the earlier bout of myocarditis  were comThe patient was started on a therapy with Ibuprofen 400 mg (twice daily), beta-blockers (Bisoprolol 2.5 mg once daily) as well as an ACE-Inhibitor (Ramipril 2.5 mg once daily). There was rapid improvement of clinical symptoms and a repeat echocardiogram performed on day 6 showed only a mildly reduced LVEF (52%) thus facilitating a timely discharge. The patient was stable throughout the course of hospital stay and no complications were documented. A follow-up CMR performed after 3 months revealed a markedly improved LVEF (57%). Videos documenting this improvement have been added as The occurrence of post-vaccination myocarditis has most notably been associated with the smallpox vaccine; however, isolated cases have also been reported with the use of influenza, tetanus, human papillomavirus, and hepatitis B vaccines . AlthougA recent study suggested that the onset of clinical myocarditis symptoms  was probably shorter for COVID-19 vaccine associated cases as compared to those reported after a viral illness. Presenting symptoms also appeared to resolve earlier in the post-vaccination myocarditis group  in addition to a beta-blocker and an ACE-Inhibitor. This led to the successful resolution of clinical symptoms and complete recovery on follow-up. In one case series, patients were administered colchicine or prednisolone, which also showed similar favorable outcomes .CMR has proven to be an essential tool in the clinical management of myocarditis. In a recent multicenter study, it could be shown that myocardial injury during acute COVID-19 infection requiring hospital admission was associated with a CMR abnormality in approximately half of the patients. Additionally, almost 27% had pathological CMR findings consistent with a non-infarct myocarditis pattern of injury . The useA study by Ferreira et al., has suggested that the diffuse increase of T1 mapping indices is influenced by myocardial edema seen in acute myocarditis. Additionally, the acute inflammation as revealed by T1 mapping was shown to be superior to edema imaging with T2-weighted sequences . An incrThe Vaccine Adverse Event Reporting System (VAERS) has already received 1226 preliminary reports of myocarditis and pericarditis after about 300 million doses of the Pfizer and Moderna vaccines (until mid-2021) . A recenRecently, the increased incidence of myocarditis after vaccination with mRNA-1273 COVID-19 vaccine (Moderna) had led many countries in western Europe to temporarily suspend its use in adults under 30 years of age. This case study highlights the clinical course of one such patient diagnosed with post-vaccination myocarditis (Moderna). The transient increase in native T1 mapping, as well as the transient LVEF deterioration during the acute phase reflects a similar pattern of a self-limiting and reversible cardiac injury. This underscores the favorable benefit-risk assessment for COVID-19 mRNA vaccination and the importance of CMR in the clinical management of these patients.This case report reaffirms the important role of CMR in the diagnosis of post-vaccination myocarditis. There was a transient increase in native T1 mapping relaxation times, as well as a transient LVEF deterioration during the acute phase of myocarditis in our patient, which was induced by inoculation with the COVID-19 mRNA-1273 vaccine (Moderna). Nevertheless, these values normalized at follow-up in all segments except for residual myocardium affected by chronic myocarditis.This case report is not reflective of an entire study population. Large multi-centric trials would be required to establish the true nature of adverse effects of COVID-19 mRNA vaccines. Furthermore, the follow-up period would need to be considerably longer to draw any definitive conclusions.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Medizinische Ethikkommmision II, Medizinische Fakultät Mannheim, Universität Heidelberg. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.The authors take responsibility for all aspects of the reliability and freedom from bias of the data presented and their discussed interpretation.UA and TP wrote the article. UA, TP, SR, SB, and DD analyzed the data, discussed the results, and reviewed and approved the article. All authors contributed to the report and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Amid growing recognition of the importance of transitioning adolescents and young adults (AYA) from pediatric- to adult-oriented health care systems, residency programs are being tasked with educating residents on best transition practices. However, consensus on how to approach training residents in transition of care (TOC) is limited. Our academic residency program therefore created and implemented a TOC of AYA curriculum for pediatric residents in an effort to increase provider knowledge and comfort with this topic. Three classes of post-graduate year one (PGY1) pediatric residents participated in this curriculum from 2017-2019 (n=35) and subsequently completed a problem-based learning (PBL) exercise in a primary care clinic with adolescent patients based on core goals in transitioning AYA. Residents completed pre-PBL and post-PBL surveys quantifying provider comfort in several aspects of the transition process. The majority of residents (94%) identified the PBL exercise as being useful, with no significant difference between classes. Eighty-nine percent (n=31) identified 1) earlier introduction of TOC and/or 2) incorporation of TOC discussions during AYA well visits as intended areas of future practice change. Overall provider comfort in transitioning AYA increased significantly from matched pre-PBL to post-PBL surveys (p=0.004). Paired mean differences also showed a significant increase in provider comfort based on several identifiable skillsets in transitioning AYA. This study suggests that a formal curriculum for pediatric residents significantly increases resident comfort in transitioning AYA and encourages change in future clinical practice. Future directions include evaluating the implementation of a formal longitudinal curriculum across several PGY levels and expansion of the curriculum to include internal medicine residents. Standardized curricula on this topic may improve resident comfort on a national level. Transition of care (TOC) is defined as the “purposeful, planned movement of adolescents and young adults (AYA) with chronic physical and medical conditions from child-centered to adult-oriented health care systems.” . With imAlthough residency programs are encouraged to incorporate specific training on TOC of AYA, there is sparse research and thus limited consensus as to how this should best be accomplished . SurveysIn response, we have created a longitudinal curriculum aimed at training pediatric residents about aspects of TOC of AYA in the primary care setting. This paper describes the first phase of the longitudinal curriculum which was delivered to residents in the first post-graduate year of their program (PGY1). Residents engaged in a three-pronged curriculum, with 1) a self-paced didactic on TOC, 2) clinical encounters with AYA where topics pertaining to TOC were discussed, and 3) a self-reflective exercise. The goal of this curriculum was to improve resident knowledge, familiarity, and comfort in the process of transitioning AYA.This study was determined to be exempt from human subject research by the University of Maryland Baltimore Institutional Review Board . Subjects included PGY1 categorical Pediatric, combined Internal Medicine and Pediatrics (Med-Peds), and combined Pediatrics and Emergency Medicine (Peds-EM) residents from the 2017-18, 2018-19, and 2019-20 academic years. Subjects participated in the AYA TOC curriculum at the University of Maryland Medical Center in Baltimore, Maryland, and completed: 1) pre-curriculum surveys, 2) a self-paced PowerPoint, 3) Got TransitionTM Readiness Assessments with one to three patients, 4) problem-based learning (PBL) exercises, and 5) post-curriculum surveys. Each subject was assigned a unique coded identifier, allowing the research team to link survey responses while maintaining anonymity. TM Initiative and joint consensus physician organization statements [The pre-curriculum survey consisted of three baseline questions focused on prior training, exposure, and experience in transitioning AYA, as well as 10 questions evaluating resident comfort in several aspects of transitioning patients  completed both pre- and post-curriculum surveys and 35 (56%) completed the PBL exercise. Subjects who completed only one of the pre- or post-surveys were excluded. At baseline, of those who participated, two residents (9%) were exposed to formal curricula and/or various AYA TOC models prior to our intervention.Comfort in resident self-ability significantly improved in several measures from pre- to post-curriculum, including discussing the differences between pediatric- and adult-centered models of care p=0.02), accessing resources pertaining to transition of AYA (p=0.006), and more specifically accessing transition resources for chronic conditions (p=0.019)  identified that the PBL exercise changed their perception on the importance of introducing TOC topics during AYA clinic visits. Of the 33 residents who responded to the question on utility of the PBL, 94% (n=31) identified the PBL as being overall useful. No differences were noted between annual PGY1 cohorts Table . After c0 identifThrough the PBL exercise, residents identified one or more barriers in transitioning care of AYA patients, including healthcare literacy n=16, 46%), socioeconomic factors , provider or AYA/family perception on differences in pediatric versus adult care , and time constraints   of the residents requested resources for patients such as printed handouts related to TOC, lists of adult providers, and insurance information. Thirty-one percent (n=11) also requested further information for themselves such as transitioning patients with specific health conditions, how to engage in a warm handoff, and improving knowledge on insurance.Though transitioning care of AYA from pediatric-centered to adult-oriented health care systems is of the utmost importance, consensus on the ideal curriculum to teach resident trainees about this topic is lacking. Prior studies have evaluated curricula focused on transitioning AYA with specific disorders or chronic disease. Our study is the first to evaluate a formal longitudinal transitional care curriculum with a primary care focus. After implementation of the first phase of our curriculum, results from our pilot study showed a statistically significant increase in resident comfort level in skills required to transition AYA such as the ability to discuss the transition process with patients and to identify barriers that AYA may face during the transition process.Review of the literature shows few formal curricula addressing TOC of AYA among residency programs despite reported discomfort and desire to learn further about this topic among both pediatric and internal medicine providers -7. SpeciIn addition to statistically significant increased individual comfort level in the process of transitioning AYA after implementation of the curriculum, residents self-identified areas of intended change in future clinical practice. Though we did not study patient outcomes, we hope that increased provider comfort and identifiable ways of improving future clinical practices may impact patient outcomes around the transition and transfer periods in favorable ways.The research team anticipated time constraint to be a large barrier in initiating the discussion of transitioning AYA, but only one resident identified this as a barrier. These findings suggest that introducing and discussing the topic of TOC is plausible during a routinely scheduled AYA visit, even with often packed clinic schedules.The only measure that did not significantly increase was resident comfort in ability to care for a transition-ready AYA patient, though overall comfort in transitioning patients did significantly increase. We suspect that residents were not aware of the difference in nomenclature of transitioning patients versus transferring patients prior to participating in the curriculum and that may have played a role in how residents answered pre-curriculum vs post-curriculum surveys.Our study has several limiting factors. First, there is a small sample size due to incomplete resident participation, with only ~50% for the 2017-18 and 2018-19 cohorts completing all aspects of the curriculum. The 2019-20 cohort had even less participation due to impact of COVID-19 on clinical rotations. Our results therefore may not reflect the views of our entire resident cohort. The study was also conducted at a single urban academic center and may not be generalizable to all residency programs. Third, post-survey responses were delayed by six to 12 months from the time of introduction of the curriculum and may reflect increased comfort due to the clinical nature of residency rather than a formal curriculum. Responses from former graduates of the program who did not participate in a formal curriculum on AYA TOC will serve as a baseline to assess for difference in timing in ongoing analyses of this curriculum. Lastly, our residents evaluated the educational aspect of the curriculum rather than evaluating the process of transitioning AYA itself and therefore cannot be used to gauge if the curriculum has positive effects on patient outcomes.We will continue to expand on our curriculum in order to address the reported discomfort in the literature among both pediatric and internal medicine providers on TOC of AYA -6. The sWe developed a longitudinal curriculum focused on the TOC of AYA for pediatric residents. The first phase of the curriculum aimed at PGY1 included learning through didactic, clinical experiences, and self-reflections. Residents subsequently demonstrated significantly increased comfort across various measures of the process of transitioning patients after implementation of the first phase of our curriculum. Significant increases were seen post-curriculum overall in self-determined resident ability to transition AYA. Though our study was performed at a single academic urban institution, we hope that our curriculum can serve as a guide for curricula at other pediatric residency programs in the future.


Spirituality and religiousness are not so far extensively investigated in patients after stroke.The aim of this preliminary study is to explore whether self-reports in two questionnaires measuring the personal experience of spirituality and religiousness can influence cognition and more specifically performance on neuropsychological tests examining attention.Fifteen male stroke patients participated voluntarily one year after their hospitalization. The mean age of the patients was 75.58 years , level of education 15.47 years (SD = 3.82). In addition to that, fifteen controls with similar demographics, free of physical and mental diseases, were also examined. Depressive symptoms of the participants were assessed with the 15-item Geriatric Depression Scale. The Daily Spiritual Experience Scale, the Systems of Belief Inventory (SBI-15R) and a number of standardized tests examining attention were administered: Trail Making Test-Part A (TMT-A) time to completion, the Digit Span (WAIS-III) greatest forward span, the Ruff 2 & 7 Selective Attention Test automatic detection speed (ADS) and controlled search speed (CSS).indicated that there was a statistically significant difference between the control group and the stroke group in attention. No statistically significant difference was found between the two groups regarding the levels of spirituality and religiousness.Although spirituality and religiousness may be related with quality of life, cognitive functioning such as attention does not seem to be influenced by these variables one year post-stroke. Future research should further investigate the possible influence of the abovementioned factors in post-stroke recovery and rehabilitation.


Prone positioning significantly improved the oxygenation both during invasive and non-invasive ventilation. For invasively ventilated patients reduced intrapulmonary shunt fraction, ventilation dead space and respiratory resistance were observed in the prone position. Oxygenation is improved during non-invasive and invasive ventilation with prone positioning in patients with C-ARDS. Different mechanisms may underly this benefit during these two ventilation modalities, driven by improved distribution of lung regional aeration, intrapulmonary shunt fraction and ventilation-perfusion matching. However, the differences in the severity of C-ARDS may have biased the sensitivity of electrical impedance tomography when comparing positional changes between the protocol groups.Severe COVID-19-related acute respiratory distress syndrome (C-ARDS) requires mechanical ventilation. While this intervention is often performed in the prone position to improve oxygenation, the underlying mechanisms responsible for the improvement in respiratory function during invasive ventilation and awake prone positioning in C-ARDS have not yet been elucidated. In this prospective observational trial, we evaluated the respiratory function of C-ARDS patients while in the supine and prone positions during invasive (n = 13) or non-invasive ventilation (n = 15). The primary endpoint was the positional change in lung regional aeration, assessed with electrical impedance tomography. Secondary endpoints included parameters of ventilation and oxygenation, volumetric capnography, respiratory system mechanics and intrapulmonary shunt fraction. In comparison to the supine position, the prone position significantly increased ventilation distribution in dorsal lung zones for patients under invasive ventilation  and Registered 24 April 2020,  Similar to non-COVID-19-related acute respiratory distress syndrome (ARDS), atelectasis develops mainly in the dependent lung regions in C-ARDS. Furthermore, pulmonary micro-thromboembolism also contributes to the severely deteriorated lung function in C-ARDS due to pulmonary endothelial damage5. Thus, the vascular pathology augments the development of large dependent regions7. This gravity-related ventilation-perfusion mismatch can be potentially alleviated by placing the patient in prone position9, since this position is associated with improved oxygenation for patients with ARDS10 and C-ARDS11. Accordingly, a potential lung homogenising effect of prone positioning can be anticipated in critically ill patients with COVID-19 and thus change in body position has been suggested in the guidelines both for intubated and non-intubated patients12. Nevertheless, this recommendation is based mainly on meta-analysis from randomised controlled trials investigating patients with non-COVID-19-related ARDS10.Patients with SARS-CoV-2-related acute respiratory distress syndrome (C-ARDS) present an unusual pattern of lung injury characterised by relatively well-preserved lung gas volume, but severely impaired ventilation-perfusion matchingDespite expert consensus on promoting the prone position for patients with C-ARDS, there is a lack of firm evidence to support the beneficial effects of this therapeutic strategy in SARS-Cov-2 pneumonia, and its value has not been verified by quantitative assessment of regional ventilation distribution. In addition, there is a lack of knowledge about the mechanisms that potentially improve the lung function of mechanically ventilated C-ARDS patients in prone position. Therefore, our aim was to elucidate the differences in regional ventilation distribution between the prone and supine positions in two groups of patients with C-ARDS ventilated invasively or receiving non-invasive ventilation (NIV). We hypothesize that the prone position favours the regional redistribution of lung ventilation in comparison to the supine position, through mechanisms that might differ between invasive and non-invasive ventilation.After obtaining approval from the Ethics Committee of the Canton of Geneva, Switzerland (2020-00896), written informed consent to participate in the study was obtained from the patients or from their legal representatives. The study was performed in accordance with the ethical standards specified in the 1964 Declaration of Helsinki and its later amendments and reporting followed STROBE guidelines. The research protocol was registered at Clinicaltrials.gov under the number NCT04359407.In this single-centre prospective observational study we enrolled patients with C-ARDS who required respiratory support in the prone position while at the intensive or intermediate care units of the University Hospitals of Geneva between April 27th 2020 and May 10th 2021. Patients under NIV were recruited from the intermediate care unit; whereas, invasively-ventilated patients were recruited from the intensive care unit (ICU). Each patient was assessed in supine and prone positions in consecutive order depending on their position at the time of enrolment.13, (2) they had SARS-CoV-2 infection confirmed by PCR test, (3) they were scheduled to undergo prone positioning, and (4) they were between 18 and 80 years old. Patients with pacemakers, defibrillators or other electrically active implants, with damaged skin or wound dressings that impaired skin contact of the electrodes, with chest tubes or with a history of thoracic surgery or lung resection were not included in this study.The study flow chart is shown in Fig. see below).For both groups of patients receiving NIV and invasive ventilation, the primary endpoint was the relative change in regional ventilation between supine and prone positions measured with electrical impedance tomography  and the peripheral oxygen saturation (SpO2) resulting from postural changes. Since mechanically ventilated patients were equipped with arterial and central venous catheters and mainstream volumetric capnography was performed as part of the standard procedure, the secondary endpoint in these patients were the intrapulmonary shunt fraction (Qs/Qt), ratio of arterial partial pressure of oxygen (PaO2) and FiO2, phase 3 slope of the volumetric capnogram (SIII) and ventilation dead space fractions resulting from postural change. Total respiratory system mechanics including resistance (Rrs) and compliance (Crs), blood pressure, heart rate, rhythm and vasopressor dose were also recorded.The secondary endpoints for non-invasively ventilated patients were the inspired fraction of oxygen , in accordance to the international consensus statement on EIT measurements14. A medium, large or extra-large belt containing 16 surface electrodes was placed around the patient’s chest in the 5th intercostal space, according to the size of the thoracic cage. EIT measurements were performed in both supine and prone position for a period of five minutes, while the belt was kept in place. EIT data were generated by the injection of small electrical currents (5 mA at 50 Hz). Images of 32 × 32 pixels were reconstructed from the EIT data using the manufacturer’s algorithm. Tidal impedance variation (∆Z) from each minute were averaged in four regions of interest (ROI), defined as either quadrants or layers, and expressed as percentage of the global ∆Z . This variable has been demonstrated to be correlated with VT15 and local compliance variation16.EIT was performed using the Dräger PulmoVista2 and CPAP pressure were registered. Intubated patients were ventilated in volume-controlled mode using a Hamilton C6 ventilator . Respiratory parameters measured by the ventilator or set by the ICU staff to provide an adequate low-tidal ventilation were recorded, . Rrs was calculated as the ratio between the peak to plateau pressure drop and the resulting flow rate, whereas Crs is determined as the ratio between the tidal volume and the corresponding change in the difference between plateau pressure and PEEP. All patients received neuromuscular blocking agents.Non-intubated patients received ventilation support with either continuous positive airway pressure (CPAP) with a facemask  or with nasal high-flow therapy . Respiratory rate, FiO2, arterial saturation of oxygen (SaO2) and partial pressure of carbon-dioxide (PaCO2), arterial and central venous haemoglobin concentrations, and central venous oxygen partial pressure and saturation (ScvO2) were measured. The PaO2/FiO2 ratio was calculated as the lung oxygenation index. The Qs/Qt ratio was assessed based on the modified Berggren equation172 and CvO2 are the oxygen content of the arterial and central venous blood, respectively, and CcO2 is the capillary oxygen content.Blood gas data were collected concomitantly with EIT measurements for the intubated patients both before and after changing position. Blood gas data were not available for patients under NIV since an arterial catheter was not part of their standard of care. PaO®)18. The ventilation dead space according to Fowler19 was calculated as the volume expired until the inflection point of the phase II slopes of the volumetric capnogram. The ventilation dead space fraction according to Bohr20 was calculated as2 is the mean alveolar partial pressure of CO2 estimated from the midpoint of phase III of the capnogram. PĒCO2 is the mixed expired partial pressure of CO2, determined as integrating the area under the volumetric capnogram curves and dividing the resulting values by VT.Capnographic measurements in the intubated patients were carried out by the FluxMed mainstream volumetric capnograph . The mainstream capnography sensor (Capnostat) together with the flow sensor were inserted proximally between the Y piece and the endotracheal tube. The phase III slope of the volumetric capnogram (SIII) reflecting the emptying of alveolar gas was calculated using the FluxMed software  was used for statistical analysis. A level of p < 0.05 was considered statistically significant, all reported p values are two-tailed.Numerical data are reported as mean and standard deviation. Graphical figures (boxplots) are reported using median and interquartile ranges. The normal distribution of the data was checked with the Shapiro–Wilk test. Paired t-tests were applied to assess the effect of patient position on the primary and secondary outcome variables. To test the effect of patient position in different ROI’s, two-way repeated measures of analysis of variance (ANOVA) were used with Holm–Šídák The present study was approved by the Ethics Committee of the Canton of Geneva, Switzerland, under the number CCER 2020-00896. Written informed consent to participate in the study was obtained from the patients or from their legal representatives. Forty-seven patients with C-ARDS requiring respiratory support in the prone position were enrolled in the study. Of the 16 patients under NIV, one patient was excluded due to respiratory distress during the observation period and two because they did not tolerate the prone position, thus 13 non-intubated patients were included in the final analysis. Data from 15 intubated patients were included in the analysis after exclusion of 16 patients due to protocol disruption for medical treatment or incomplete data acquisition for technical reasons  and invasive mechanical ventilation (lower panels). There was no evidence for a statistical difference in the regional lung aeration between the prone and supine positions in any ROI  for the COVID-19 patients under NIV. Conversely, for patients under invasive ventilation, prone positioning led to a significant redistribution of regional lung aeration in favour of dorsal zones  and quadrants ). Further examining the regional changes in lung aeration revealed that gas redistribution occurred primarily in the left region of the lung (quadrant ROI 1 and 3).Figure 2 (p < 0.01) following the assumption of the prone position . There was no evidence for a difference in the respiratory rate between prone and supine position under NIV , which resulted in a significantly higher PaO2/FiO2 in comparison to the supine position (p < 0.001). In addition, the prone position led to a significantly lower Qs/Qt in these patients (p < 0.05) with a negative correlation between postural changes in Qs/Qt and PaO2/FiO2 .Figure p < 0.05).Respiratory and haemodynamic data are provided in Table In this prospective observational trial, we observed differences in the regional ventilation redistribution between invasive and NIV following changes from supine to prone position in patients with COVID-19-related ARDS. While no ventilation redistribution was evidenced in non-invasively ventilated patients, prone positioning of invasively ventilated patients led to redistribution of regional ventilation from the ventral to the dorsal lung areas. Prone positioning improved oxygenation both for patients under NIV and invasive ventilation. The improvements in ventilation and oxygenation in prone position were accompanied by a significantly lower physiological dead space, intrapulmonary shunt and respiratory resistance for intubated COVID-19 patients.22. The regional ventilation distribution was assessed in the present study with EIT, a well-validated dynamic, real-time imaging technique24, which has been used to characterise or to guide the ventilation strategy in COVID-19 patients27. The dorsal redistribution of regional ventilation observed only for invasively ventilated patients with C-ARDS may be attributed in part to recruitment of atelectatic areas, which are more pronounced in invasively ventilated patients than those under awake prone positioning. Interestingly, lateral differences between right and left lungs were observed in the redistribution of ventilation  during awake prone positioning36, we observed no change in RR in our cohort of patients under NIV despite an improvement in oxygenation.A hallmark finding of this study is that oxygenation was improved both under invasive and non-invasive ventilation and was associated even with a simultaneous reduction in FiO37.Since regional aeration was not significantly modified by prone positioning in NIV patients, we can assume that the benefit in oxygenation is predominantly explained by the redistribution of pulmonary blood flow and optimisation of ventilation-perfusion matching, rather than alveolar recruitment or aeration change as speculated by previous literature38. While a markedly decreased phase 3 slope was reported earlier for patients with ARDS18, our findings suggest that C-ARDS differs from non-COVID ARDS in this aspect. This finding suggests that, in C-ARDS, regional ventilation and perfusion of the open lung regions might be more homogeneous than in non-COVID ARDS. Conversely, in terms of Bohr and Enghoff dead space, the changes are similar to those observed in non-COVID ARDS39 and were markedly higher than those obtained for ventilated patients with healthy lungs40. A marked elevation in the difference between the Bohr and Fowler dead spaces (VDB–VDF) is observed regardless of the body position, which suggests the presence of large lung regions that are ventilated but not perfused. Similarly, the difference between the Enghoff and Bohr dead spaces (VDE–VDB) is much higher than in healthy ventilated patients40, indicating the development of alveolar regions with intrapulmonary shunt. Moreover, prone positioning significantly decreased the Enghoff’s dead space VDE for intubated patients, suggesting a beneficial redistribution of lung aeration. The Enghoff’s dead space VDE incorporates the intrapulmonary shunt in addition to the VDB41. Altogether, these findings demonstrate that simultaneous respiratory and circulatory changes are responsible for the ventilation–perfusion mismatch observed for patients with C-ARDS.The data on volumetric capnography provides additional insight into the respiratory effects of C-ARDS and body position. The phase 3 slope is not significantly modified by body position and presents values that are normal or even lower than normalF without affecting changes in the other outcomes. Third, the heterogeneous nature of both the lung injury and the disease severity between patients led to considerable variances in the estimated endpoints. Thus, the precise benefits of prone position for each individual may vary over time, depending on the rapidly changing pathological aspects of the disease43. Nevertheless, despite the aforementioned variability, we demonstrated a consistent improvement in oxygenation that is in accordance with previous reports44. Fourth, despite an estimated sample size of 13 patients per group, we had to enrol 31 intubated patients to have complete datasets for both prone and supine positions, due to the fact that these ICU patients often required medical care interventions that disrupted the research protocol procedures. Finally, this study reports a single-centre trial; however, the magnitude of the improvement in oxygenation and lung function along with the clinical characteristics of the study subjects likely permit a confident generalisability of our findings to the population of patients with moderate-to-severe C-ARDS requiring ventilatory support in the prone position.Some limitations of this study warrant consideration. First, the measurements with EIT were solely presented as the percentage of aeration instead of as absolute impedance or volume units; this data presentation reduces the bias related to technical issues related to intra- and inter-subject impedance variability. Second, the respiratory circuit used in this study  comprised the endotracheal tube, the closed-circuit suction, the flowmeter, the mainstream capnograph and a bacterial filter with continuous humidification; this circuit dead space was similar in all patients and corresponds to the standard circuit in routine clinical practice. This instrumental dead space may have contributed to the VDIn summary, our results provide additional evidence for the benefit of prone positioning for patients with moderate-to-severe C-ARDS. The benefits under NIV and invasive ventilation are governed by different underlying mechanisms. For invasively ventilated patients the improvement in oxygenation is driven by the redistribution of lung aeration and optimised ventilation-perfusion matching, leading to a decrease in physiological dead space and in intrapulmonary shunt fraction. During awake prone positioning, the lack of change in the distribution of aeration suggests that other mechanisms are responsible for the oxygenation benefits. Further studies are warranted to evaluate the factors contributing to the improved gas exchange during awake prone positioning.


This cohort study examines a clinical decision model for penicillin allergies among pediatric patients; the model considers when reactions occurred; whether patients experienced angioedema, anaphylaxis, or a severe cutaneous adverse reaction; and whether treatment was required. The aim of this cohort study was to examine the previously validated PEN-FAST adult score6 in children.The pediatric prevalence of self-reported drug allergies is 10%,3 we examined the PEN-FAST score in 2028 children with 2031 penicillin allergy labels  age for the 2028 children in the cohort was 4.3 (2.1-8.0) years, with mostly male participants (1091 [53.7%]). Most reported reactions occurred in the past 5 years or at an unknown time (1661 [81.8%]), with amoxicillin suspected in 2022 reactions (99.6%) . AnaphylChanging the coding for timing (<1 year) or removing the angioedema reported symptom did not improve the performance of the PEN-FAST tool in this pediatric population . A subgrIn this Canadian pediatric prospective multicenter cohort, the PEN-FAST tool did not help identify low-risk penicillin allergies. This previously validated tool in an adult population was not useful for risk stratification in children younger than 12 years. In teenagers (≥13 years), the predictive ability of the tool increased , indicating that the tool could have some value in this population following further study. The extrapolation of the results is limited by the small number of teenagers included.The PEN-FAST performed similarly for immediate and delayed reactions, and the timing of the reported reaction was not associated with the outcome of the allergy investigation. In this context, it is possible that the low validity of the PEN-FAST tool in children is explained by the increased prevalence of viral-induced reactions compared with true drug hypersensitivity. This analysis adds to the evidence that true drug allergies are rare among children and that they are often incorrectly labeled during a viral infection. Furthermore, the criteria included in the PEN-FAST score might not provide adequate information considering different index reactions in the pediatric population compared with the adult population. This study highlights that children are not little adults and clinical decision rules need to be derived and validated in the target population.These findings suggest that the PEN-FAST drug allergy clinical decision rule should not be adapted to a pediatric population younger than 12 years at this time. New validated point-of-care clinical tools are required to identify low-risk penicillin allergies in a pediatric population, and validation of PEN-FAST in adolescents requires further examination in extended international cohorts.


One of the immediate impacts of the pandemic was the increase in unemployment and the reduction of working hours - especially among women, those with lower job security and in countries with a weaker social welfare system. The aging society is at further risk, which is already challenged by a growing shortage of workers and the rising costs of pensions. To understand the long-term consequences of COVID-19 for the aging workforce, this paper advances existing research by exploring from a life-course perspective how employment after the COVID-19 outbreak is anchored in larger employment histories (2011–2021).Sequence-analyses are applied using retrospective life history data combined with both COVID-19 Surveys (2020–2021) from SHARE including respondents aged 50+. Using cluster-analyses, the resulting sequences are classified into six groups. By applying descriptive-analyses, I examine how these groups differ by gender, work quality, and country.Older workers – especially men – with continuous full-time employment histories are less frequently affected by unemployment and fewer working hours during COVID-19. Whereas those – especially women – with disruptive employment and part-time work histories are more frequently affected by unemployment after the outbreak of COVID-19. Respondents in countries characterized by social democratic welfare compared to liberal welfare regimes are less likely affected by unemployment during the pandemic. Discussion: The pandemic particularly puts older workers with disruptive employment histories at additional risk of labor market exit. Therefore policymakers need to address inequalities in earlier life to prevent long-term consequences of social inequality caused by the pandemic.


Beta-thalassemia is one of the most common monogenic disorders. Standard treatment of the most severe forms, i.e., transfusion-dependent thalassemia (TDT) with long-term transfusion and iron chelation, represents a considerable medical, psychological, and economic burden. Allogeneic hematopoietic stem cell transplantation from an HLA-identical donor is a curative treatment with excellent results in children. Recently, several gene therapy approaches were evaluated in academia or industry-sponsored clinical trials as alternative curative options for children and young adults without an HLA-identical donor. Gene therapy by addition of a functional beta-globin gene using self-inactivating lentiviral vectors in autologous stem cells resulted in transfusion independence for a majority of TDT patients across different age groups and genotypes, with a current follow-up of multiple years. More recently, promising results were reported in TDT patients treated with autologous hematopoietic stem cells edited with the clustered regularly interspaced short palindromic repeats-Cas9 technology targeting erythroid BCL11A expression, a key regulator of the normal switch from fetal to adult globin production. Patients achieved high levels of fetal hemoglobin allowing for discontinuation of transfusions. Despite remarkable clinical efficacy, 2 major hurdles to gene therapy access for TDT patients materialized in 2021: (1) a risk of secondary hematological malignancies that is complex and multifactorial in origin and not limited to the risk of insertional mutagenesis, (2) the cost—even in high-income countries—is leading to the arrest of commercialization in Europe of the first gene therapy medicinal product indicated for TDT despite conditional approval by the European Medicines Agency. The resulting imbalance in alpha/beta chains produces anemia, mainly driven by ineffective erythropoiesis. Beta-thalassemia represents a group of disorders which constitute a considerable medical, psychological, and economic burden in a wide range of countries, including many with limited resources and an underdeveloped healthcare infrastructure.1-3 Additionally, an increased understanding of the molecular mechanisms underlying both dyserythropoiesis and iron overload resulted in the development of new drugs that alleviate disease phenotype.4 The current life expectancy of TDT patients is greater than 50 years with optimal monitoring and treatment of iron overload.6 Nonetheless, quality of life (QOL) is still greatly impaired by iron overload-related morbidity—particularly in older patients—residual anemia despite regular transfusions and required adherence to lifelong treatment.8 Furthermore, access to complex, multidisciplinary, and expensive care is only available in high-income countries, while a large number of patient candidates live in low- to middle-income countries.In transfusion-dependent thalassemia (TDT), the most severe form of beta-thalassemia, patients require lifelong transfusion and iron chelation therapy due to anemia present since infancy. With the proper administration of supportive care, the life expectancy of TDT patients has dramatically improved since the 1970s. Over the past 20 years, the introduction of orally active iron chelators and increasing use of cardiac magnetic resonance imaging in detecting presymptomatic cardiac iron overload have improved the prevention of both mortality due to heart failure (the leading cause of death in TDT patients before these advances) and morbidity.9-12 It remains the only viable curative treatment and is increasingly used outside of western countries; however, it is limited by a lack of related/unrelated donors for a large proportion of patient candidates. The European Society for Blood and Marrow Transplantation (EBMT) and other groups have reported excellent results in transplant patients younger than 14 years with an HLA-matched sibling donor (MSD).12 In most countries, MSD HSCT is a standard of care in children with TDT; it should be performed before iron overload occurs in order to decrease the risk of transplant-related mortality. Several studies have demonstrated that the quality of treatment that patients received throughout their life before allo-HSCT impacts the results of HSCT, by protecting tissues from chronic iron-related oxidative stress.13 Thalassemia-free survival (TFS) now routinely reaches >90%, especially when cord blood transplantation from an HLA-identical sibling donor is used resulting in a low incidence of acute and chronic graft versus host disease (GVHD).14 Excellent results were also reported in pediatric patients receiving transplants from 10/10 HLA-matched unrelated donors with 2-year overall survival and TFS similar to those obtained with MSD, despite a higher incidence of acute and chronic GVHD.15 In addition to HSCT with HLA-identical donors, the use of alternative donors was recently reported when using transplants from haploidentical transplantation yielded encouraging results,16 while an excess of graft failure was reported by international registries for unrelated cord blood transplantation.17 Allogeneic HSCT alleviates the need for lifetime supportive care and improved QOL,19 provided that the risks of transplant complications are lowered with transplantation at a young age and appropriate care in the pretransplant period.Allogeneic hematopoietic stem cell transplantation  has been used to treat TDT for over 40 years, demonstrating its capacity to cure an inherited globin disorder.21 A 2016 EBMT report analyzed over 1500 TDT patients with transplants after year 2000, of whom only 5% were adults. The mortality rate has improved in the adult cohort, but still approached 20%.12 This improvement was ascribed to better clinical conditions for adult patients compared to previous cohorts, resulting in an increased tolerance to conditioning regimen toxicity and allo-HSCT itself. It follows that adult TDT patients, who have received optimal transfusion and chelation therapy with lifelong control of iron toxicity, represent an appropriate population for gene therapy—a strategy that does not entail the risk of immunological complications associated with allo-HSCT.22Adult TDT patients remain a challenge for transplantation with a high mortality rate (>30%) in initial reports.24 Negative associations between older age or chronic GVHD and HRQL scores were reported.19 Furthermore, HRQL scores of patients living in high-income countries and who are cured of thalassemia after allo-HSCT have improved when compared with those of patients treated with blood transfusions and iron chelation therapy.24Patients living with TDT have impaired health-related quality of life (HRQL). Studies conducted over 1-2 years or longer, up to more than 20 years, after allogeneic transplantation have reported physical and emotional improvements in HRQL.26T87Q) that differentiates it from endogenous or post-transfusion hemoglobin . The first TDT patient was treated with the HPV569 vector in 2007 as part of the LG001 trial.27 The patient suffered from beta+/beta0 TDT . Transfusion independence associated with benign clonal dominance was achieved. However, after 9 years, a decline in Hb levels has been reported and the patient required occasional red blood cell transfusions.25 Since this first report, results from phase I/II trials using the BB305 vector were published. A myeloablative regimen containing a PK-adjusted busulfan dose was administered before beti-cel injection in 22 TDT patients; transfusion independence was reached for the majority of non-beta0/beta0 patients.28 Next, improvements in transduction procedures with increases in both vector copy number and the proportion of transduced CD34+ cells in the phase III HBG-207 trial (NCT02906202) resulted in transfusion independence in 20/22 non beta0/beta0 pediatric and adult TDT patients, with no severe adverse events related to the experimental product reported.29 The results of these clinical trials were the basis for the conditional approval of the aforementioned first-in-class GTMP for non-beta0/beta0 TDT patients, aged 12 years or older and without HLA-identical sibling donors. Results were less satisfactory in beta0/beta0 TDT patients treated in the HGB-204 trial. However, the results of the HGB-212 trial (NCT03207009) that evaluated an improved manufacturing process based on the same transduction technology—similar to the HGB-207 trial—demonstrated transfusion independence in 12/14 beta0/beta0 TDT patients.29 Longer-term data suggest a stable response as transfusion independence was maintained in all patients who achieved it, sustaining HbAT87Q levels for up to 7 years. This gives hope for a one-time curative treatment with a favorable long-term safety profile.30 Liver iron concentrations improved after successful gene therapy and several patients were able to stop chelation therapy. Indeed, any claim to cure the disease requires not only transfusion independence with no or mild anemia but also mitigation of dyserythropoiesis and restoration of iron homeostasis—as demonstrated with allo-HSCT, even when mixed chimerism is present.31 Patients treated with beti-cel for non-beta0/beta0 genotypes who reached transfusion independence also showed improvements in ineffective erythropoiesis markers such as plasma levels of soluble transferrin receptors, the hepcidin/ferritin ratio, and erythropoietin levels (in correlation with hemoglobin levels), and in myeloid/erythroid ratios assessed by bone marrow biopsy.32 These results need to be confirmed with a longer follow-up.Gene therapy recently emerged as an alternative curative option for children and young adults with thalassemia and without HLA-identical donors. It has the potential for phenotypic correction through either expression of a functional copy of the beta-globin gene in hematopoietic stem cells and their progeny or through enhanced expression of gamma chains that assemble with alpha chains to produce fetal hemoglobin.34 A smaller phase I clinical trial was conducted in the US (NCT01639690) and is mostly notable for being the only clinical trial that used a non-myeloablative-based conditioning regimen—all treated TDT patients failed to reach transfusion independence36 suggesting the need for sufficient myeloablation in preparation for engraftment of genetically modified autologous cells.Transfusion independence was also reached in 4 of 6 pediatric patients (but none of the 3 adults) mainly bearing beta0/beta0 mutations, who were treated with the GLOBE vector in another trial conducted in Italy (NCT02453477). The investigators used a beta-globin gene addition strategy, with a conditioning regimen of thiotepa and treosulfan and intrabone injection of the graft.37 The repair process with CRISPR-Cas9 involves the creation of insertions or deletions within the non-coding BCL11A erythroid lineage-specific enhancer on chromosome 2, thus downregulating BCL11A in erythroid precursors with no expected effect on other hematopoietic lineages.39 This non-coding change reactivates gamma-globin gene transcription and increases fetal hemoglobin protein concentrations in red blood cells. The aim is to replicate the natural expression of high concentrations of fetal hemoglobin in adulthood as in the hereditary persistence of fetal hemoglobin.Gene engineering technologies that resort to transduction and transgene supplementation are likely to be challenged by gene editing technologies in the near future. Initial gene editing strategies either using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technologies or zinc finger nucleases target the expression of BCL11A, a key regulator of the normal switch from fetal to adult globin production that normally occurs during the first few months of life.40 The industry-sponsored THALES trial targets a similar objective while using a different gene editing technology. So far, 3 patients who did not achieve transfusion independence have been reported.41Early results have been reported from the industry-sponsored CLIMB-Thal-111 trial evaluating the safety and efficacy of CTX001, an investigational GTMP made from autologous CRISPR-Cas9-modified CD34+ hematopoietic stem and progenitor cells. Accrual in the CLIMB-111 is ongoing for TDT patients aged 12 to 35 years. Fifteen patients have been infused with CTX001 and stopped transfusions within 2 months after treatment. They remain transfusion-free as of last follow-up as a result of high levels of HbF production.42 With the exception of academic clinical trials, the starting material is shipped from collection/processing facilities at the treating hospital to a central manufacturing organization for further processing. The PB CD34+ cells undergo selection, are genetically modified ex vivo and then formulated and cryopreserved as a cell suspension, which qualifies as a GTMP. Upon release by the qualified pharmacist, using the results of in-process quality control and potency assays, the drug product is shipped from the manufacturing site back to the treating hospital for reception, temporary storage, thawing, and infusion. Thus, the use of a central manufacturing organization—as opposed to point of care manufacturing—often involves shipping the starting material from/the final product back to the treating hospital over long distances. As with every autologous living drug, consistency in the on-demand process does not guarantee product harmonization, nor does it exclude manufacturing failures or the possibility of out-of-specification products. Data regarding the actual manufacturing costs are not available, but information regarding the manufacturing of other types of gene therapies suggest that they are far below the price requested by the manufacturer and represent only a minor fraction of the price tag.A commonality across all clinical trials is that the starting material for medicinal product manufacturing is autologous peripheral blood (PB) stem cells obtained through apheresis of efficiently mobilized patients (HPC-A). The mobilization protocol associates G-CSF and plerixafor (used “off label” in this context) and allows for the collection of “megadoses” of PB CD34+ cells.The results described above represent a remarkable achievement, and potentially offer new hope to patients affected with TDT as heralded by the conditional approval of the first-in-class GTMP. Nevertheless, recent developments in the field illustrate just how difficult it will be to provide broad access to such treatments.Similarly to other GTMPs, such as autologous CAR-T cells, beti-cel has been approved based on data generated from small registration studies with limited follow-up and without being compared to a standard of care, whether supportive care or allo-HSCT. This mandates Post-Authorization Safety Studies and Post-Authorization Efficacy Studies in order to fully define safety and efficacy profiles, as illustrated by the recent description of several adverse events.43 Of note, a few cases of secondary MDS/AML derived from host cells associated with graft rejection have been reported after allo-HSCT for SCD.44 The second case was AML and led to the temporary suspension of accrual in the HGB-206 and HGB-210 SCD trials.46 The vector was present in leukemic cells that were not exposed to busulfan and insertional mutagenesis was suspected. The Marketing Authorization Holder soon thereafter communicated that the investigation revealed an insertion of the lentiviral vector sequence into the VAMP4 gene. VAMP4 has no known role in AML or cancer, and its regulation/expression was not affected. These observation do not support direct causality between vector integration and the development of leukemia.47 Indeed, the risk of insertional mutagenesis of SIN-LV is considered low compared to gammaretroviral vectors that have demonstrated considerable risk of leukemogenesis due to their preferential insertion near transcriptional start sites and proto-oncogenes.48-50 The presence of the erythroid-specific BB305 promoter probably decreases the risk of oncogene activation in other lineages.51 No cases of insertional mutagenesis leading to secondary leukemia were reported in clinical trials using SIN-LV until very recently (August 2021), when a patient developed secondary MDS after gene addition therapy for cerebral adrenoleukodystrophy (ALD) via the vector Lenti-D containing a MNDU3 promoter for ALD protein expression.52 The risk of leukemogenesis after gene therapy may be influenced not only by vector design but also by the host condition and the underlying disease. The baseline risk of hematological malignancies was reportedly higher in SCD patients than in the general population,53 possibly in relation to the chronic inflammation and stress erythropoiesis encountered in the disease.46 The hypothesis that a preexisting clonal hematopoiesis of indeterminate potential may progress to overt leukemia after gene therapy ,55 raises the question of pretreatment screening in SCD patients. The use of hydroxyurea before and/or after gene therapy—while not associated with oncogenesis in the routine treatment of SCD patients—could be an additional risk factor. Until now, MDS/AML events or clonal dominance were not observed in thalassemia patients. Unlike SCD, thalassemia does not create a chronic inflammatory condition: no clear increased risk of MDS/AML has been identified, ineffective erythropoiesis encountered in TDT is partly suppressed by a regular transfusion regimen, and hydroxyurea is rarely used in TDT patients. At present, the benefit/risk ratio of treatment with beti-cel remains favorable for patients with TDT. This was also the conclusion of the EMA Pharmacovigilance Risk Assessment Committee evaluation performed after reporting of the 2 SCD cases.Two cases of secondary myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) were reported in two patients with sickle cell disease  included in the HGB-206 trial, which used the same BB305 vector than in the TDT HGB studies. An additional case of MDS was initially reported but later reconsidered as unrelated to the therapy. Further investigation led to the conclusion that the first case of MDS/AML, reported in 2020, likely resulted from busulfan exposure; monosomy 7 was present while no vector was detected in blast cells; monosomy 7 is frequently associated with myeloid malignancies induced by alkylating agents.56-58 For example, the direct cost of non-curative medical care for Italian TDT patients was calculated to be the equivalent of 31,833 USD/year in 2017.59 Worldwide, HSCT costs are highly variable depending on the country’s healthcare organization and complication rate. HSCT is nevertheless considered cost effective when compared to standard medical therapy since the cost amounts to nearly the same as the cumulated costs of standard medical therapy over 3 to 5 years. Gene therapy has also been proposed to be cost effective when compared to standard medical care,60 but it is argued that cost-comparison analyses of the curative options should be performed and subsequently a better comparator would be allo-HSCT. Only one paper compared US costs (not price) of HSCT and gene therapy (transduction step with academic point of care manufacturing)61 and concluded that gene therapy was 3 times more costly than HSCT. Following conditional marketing approval in Europe, bluebird bio—the manufacturer of Zynteglo (beti-cel)—asked for a price tag of 1 650 000 EUR or 1 900 000 USD while considering the savings in supportive care that 50 years of hypothetical thalassemia-free survival would represent.62Pricing and additional costs linked to supply chain complexity and patient care in the acute phase following treatment represent another hurdle to widespread adoption of these innovative therapies.63 the price of the medicinal product still appears prohibitive to public and private payers in many high-income countries, and is entirely unaffordable in low- to middle-income countries. Following conditional marketing authorization, payment or reimbursement conditions have not been approved in a single European country, preventing patients to receive this treatment. The German health system’s offer of a single payment of 950 000 USD with the condition of persistent clinical efficacy64 was refused by the company and—together with the lack of rapid decisions from several other major EU countries—triggered the discontinuation of its operations in Europe and the reorganization of its operations in the USA, as announced in a press release in August 2021.65 It is anticipated that bluebird bio will withdraw its marketing authorization for beti-cel from both the EU and the UK by early 2022.66 The U.S. Food and Drug Administration has accepted bluebird bio’s Biologics License Application for beti-cel for priority review.Beti-cel thus ranks among the most expensive therapies ever marketed. While bluebird bio offered to install annual payments over 5 years, and offered the condition of payment only upon demonstrated sustained clinical activity,A global framework for pricing negotiations for this new category of medicinal product is urgently needed. Claims from pharmaceutical companies of a treatment with lifelong efficacy will need to be further substantiated through the collection of reliable data in real-world conditions over extended periods of time. Global, transparent and open discussions must be conducted concerning what is and is not acceptable in relation to drug pricing, development costs, manufacturing, and savings associated with sparing prolonged or lifelong supportive care in order to overcome reimbursement issues and allow innovative treatments to reach the market and enter clinical practice. Hopefully, competition between companies, developing technologies, and large-scale manufacturing will improve the situation, however, this objective appears out of reach in the short term.61 Numerous health authorities recommend using cost-utility analyses for the economic evaluation of new treatments based on not just the quantity but also the QOL gained from healthcare interventions. An improvement in QOL after gene therapy is expected given the successful achievement of transfusion independence and lack of GVHD. Preliminary short-term results in children and adolescents who achieved transfusion independence in the HGB-207 and 212 studies demonstrated clinically meaningful improvement in child-reported PedsQL 4.0 total scores, especially in those with low baseline values.67 Until long-term data become fully available, the selection of patients who will benefit from receiving gene therapy will represent a significant challenge. To this end, the Italian Society for Thalassemia and Hemoglobinopathy (SITE) recently published an algorithm for patient selection and prioritization.22 In conclusion, while gene therapies appear to represent a major therapeutic advancement in the treatment of thalassemia, they raise the critical issues of long-term efficacy and safety, accessibility, and affordability. All patients treated with gene therapy in clinical trials must be rigorously followed in long-term studies, in order to allow for the evaluation of long-term disease control, the risks of secondary malignancies, and how the technology used in genetic engineering affects these risks.Finally, as TDT is a chronic disease, long-term comparisons are warranted for gene therapies with existing options such as allo-HSCT . Such ev


Deep learning is being increasingly applied for obtaining digital microscopy image data of cells. Well-defined annotated cell images have contributed to the development of the technology. Cell morphology is an inherent characteristic of each cell type. Moreover, the morphology of a cell changes during its lifetime because of cellular activity. Artificial intelligence (AI) capable of recognizing a mouse-induced pluripotent stem (miPS) cell cultured in a medium containing Lewis lung cancer (LLC) cell culture-conditioned medium (cm), miPS-LLCcm cell, which is a cancer stem cell (CSC) derived from miPS cell, would be suitable for basic and applied science. This study aims to clarify the limitation of AI models constructed using different datasets and the versatility improvement of AI models. The trained AI was used to segment CSC in phase-contrast images using conditional generative adversarial networks (CGAN). The dataset included blank cell images that were used for training the AI but they did not affect the quality of predicting CSC in phase contrast images compared with the dataset without the blank cell images. AI models trained using images of 1-day culture could predict CSC in images of 2-day culture; however, the quality of the CSC prediction was reduced. Convolutional neural network (CNN) classification indicated that miPS-LLCcm cell image classification was done based on cultivation day. By using a dataset that included images of each cell culture day, the prediction of CSC remains to be improved. This is useful because cells do not change the characteristics of stem cells owing to stem cell marker expression, even if the cell morphology changes during culture. A minor population of tumor cells promotes tumor growth. The cell labeled as cancer stem cell (CSC) depicts the analogy between the renewal of normal and tumor tissues through each activity of the tissue specific to the adult stem cell . Recent Cell morphology is associated with stem cell (SC) property. Non-SC fibroblasts change their morphology during the induction of stem cells by Yamanaka’s factors . Mouse iRecently, AI technology was applied in cell biology to learn novel skills of a trainee with expertise, such as, for the evaluation of human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) culture into bad and good conditions, as performed through inspection by a well-trained experimenter. Accordingly, a trained AI was used to classify the two groups using CNN . The indThe AI can segment cells with the same morphologic features from other cell types in cases where clear distinctions among the various types exist, such as flat and square shapes. So far, we studied the possibility of application of the AI to segment cells with the unrecognized feature of CSC morphology using CGAN . Althoug2 incubator . The culture reagents were purchased from FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan.Cells of miPS-LLCcm derived from iPS-MEF-Ng-20D-17 provided by the Riken BioResource Research Center through the National BioResource Project of the Ministry of Education, Culture, Sports, Science, and Technology/Japan Agency for Medical Research and Development, Japan , were cuWe observed cells grown in 96-well plates under a fluorescence microscope BZ-X800  equipped with a CFI Plan Fluor DL 10× lens . We acquired a GFP fluorescence image combined with a corresponding phase contrast image for each well. All images had a resolution of 1920 × 1440 pixels in each channel. Images were saved in tiff format.https://github.com/affinelayer/pix2pix-tensorflow), obtained on 17 May 2019, built in the Linux OS. We processed a pair of images obtained from microscopy having a resolution of 1920 × 1440 pixels to make 35 new image pair files, with each image having a resolution of 256 × 512 pixels. The paired images were used as input to the pix2pix. We calculated the F-measure value to evaluate the AI output against the target. The color images were transformed to black and white images; the images in the pair were overlaid to find an identical region; and the identical region was employed for the calculation of precision and recall. We defined the F-measure using Equation (1), as: R is the recall and P is the precision.We performed machine learning using a personal computer equipped with one CPU, 32 GB of RAM, and one GPU . We performed CGAN using pix2pix  ported tWe performed CNN classification in Chainer and PyTorch environments built in the Linux OS. Gradient-weighted class activation mapping (Grad-CAM), guided Grad-CAM, and Grad-CAM++ were used to obtain visual explanation of CNN classification ,15.Single-factor analysis of variance (ANOVA) was used for the evaluation of group distribution. Furthermore, Scheffe’s F test was performed to evaluate the differences between the two groups.As a CSC model, we used miPS-LLCcm cells . The celWe used 1000 images for each AI training. The AI depicted a CSC image as an “output” from the phase-contrast image; the “input” from the remainder of the datasets comprising the selected images for the 1-day and 2-day cultures are shown as examples in To examine the versatility of AI, we evaluated AI models using different kinds of test datasets that were not used to train the AI. Examples are shown in It could be beneficial for AI to have the capability of depicting CSC images from phase-contrast images taken during any cultivation period. We trained the AI using a dataset comprising a mixture of 1-day and 2-day culture images for deep learning. The AI model trained with the mixed dataset of 1000 images showed a mean F-measure value of 0.29 for a test dataset containing a mix of 1- and 2-day culture images as control a. The vaBecause AI models of 1-day and 1-day-selected culture images showed lower performance on depicting CSC images than 2-day culture images, we hypothesized that these AI might recognize the difference in datasets between 1- and 2-day culture images. Thus, we examined whether we could classify cell images from 1-day and 2-day culture by deep learning using CNN a. For trSubsequently, we explored the referred region in the image to discriminate between the datasets of 1-day and 2-day culture. We applied Grad-CAM and improved Grad-CAM++ d techniqWe further examined other datasets consisting of only 1-day culture images in which each dataset had a difference, to provide better explanation of CSC recognition by AI models. Dataset of the 1-day-center consisted only of images prepared from the center of large original images whereas whole images were used in the dataset of 1-day culture. This was previously shown to have a significant increase in the F-measure values against the dataset for the AI model of CGAN . ThereafWe investigated the property of AI models trained to recognize the morphology of cultured CSC expressing the Nanog-GFP reporter gene using CGAN. The AI models had the capability to depict CSC images with high accuracy through F-measure values. However, average F-measure values were relatively low. To improve the efficiency, we found several points that needed reminding for training the AI models. Blank images in the AI training dataset reduced the F-measure value. The F-measure values tended to reduce when an AI model was tested for cell images with a longer cultivation period than when using the images employed for training the AI model. By contrast, an AI model trained using cell images of a long cultivation period did not show such inefficiency to cell images of short cultivation. Therefore, an improved workflow for training AI consists of using a pool of cell images ranging from short to long cultivation periods, regardless of including blank images, for improving efficiency.The application of deep learning is emerging on cell morphology and structures . The perThe nuclear architecture of cells is recognized to undergo morphological changes when SC differentiates . For exaAI is expected in cancer diagnosis and the related studies. This study implied the importance of training datasets being used from several days of cultivation. Our approach to achieve an AI model could also be useful for CSCs other than miPS-LLCcm. Although we showed the usefulness of CGAN for deep learning of CSC morphology, the values evaluating AI models were low. To improve AI models, further studies are required in the analysis, using available technology by utilizing datasets consisting of only CSC images and non-CSC images. Because miPS-LLCcm cells grow crowded and often lie on top of other cells, it is difficult to identify their cell boundaries. It is interesting to train AI to not use datasets of a mixture of CSCs and non-CSCs, but rather, separated datasets at the individual cell level.We evaluated the versatility of AI models using CGAN trained to map undefined live CSC morphology. When the AI model was applied to live CSCs, it was efficient in using CSC images from various culture conditions. The visualization technology of deep-learning progress of CNN classification of CSC was powerful to obtain insights into how outputs of AI models are produced. However, it showed that objects used to differentiate classes were not always cells but also the instruments and microscopic effect of the view. The improved deep-learning workflow indicated in this study could be useful for the development of an AI model identifying undescribed morphological characteristics in CSCs.