To investigate differences in behavioral and psychological symptoms of dementia (BPSD) and caregiver distress caused between older adults with dementia living in the community and in nursing homes.A comparative cross-sectional study.Participants were recruited from outpatient clinics of a tertiary psychiatric hospital and dementia units of a nursing home in Guangzhou, China.Neuropsychiatric Inventory was used to assess symptoms and caregiver distress. Dementia severity was determined using the Clinical Dementia Rating.2 0.36–0.82). Group differences in clinically significant symptoms and moderate-to-severe caregiver distress showed at the stage of moderate-to-severe dementia.This study included 157 community and 112 nursing home residents with dementia. Clinically significant symptoms (item score ≥ 4) were found in 88.5% of the former and 75% of the latter. Caregivers of 79.6% of the former and 26.8% of the latter reported that at least one of these caused them moderate-to-severe distress (distress score ≥ 3). Among the community patients, anxiety was the most frequent “very severe” symptom, while sleep disorders and agitation caused the most frequent “very severe” caregiver distress. After controlling for dementia severity and medication use, family caregiving remained an independent risk predictor for clinically significant symptoms and moderate-to-severe caregiver distress. The prediction of caregiver distress based on symptom scores varied across caregiver types and individual symptoms (RTailored management strategies to relieve family caregivers’ BPSD-induced distress are needed, especially at the stage of moderate-to-severe dementia. An effective service system should be established for supporting family caregivers to cope with BPSD. Dementia affects approximately 55.2 million people and their families worldwide in 2019, and this is expected to rise to 139 million by 2050 , 5. One Family caregivers are mainly responsible for managing BPSD of people with dementia living at home –15, but By comparison with their counterparts in long-term care facilities, we may get a better appreciation of clinically significant and moderate-to-severe distressing BPSD and their risk predictors in community-dwelling older adults with dementia cared by family caregivers. This is necessary to generate research evidence to inform the design of tailored management strategies and support family caregivers to enable people with dementia to stay at home longer, especially in low- and middle-income countries. Previous studies have investigated differences in caregiver distress caused by BPSD between community-dwelling older adults and residents of long-term care facilities , 20–22. This study therefore aimed to explore differences in BPSD and caregiver distress in responding to individual symptoms between community-dwelling older adults with dementia and their nursing home counterparts after controlling for dementia severity and medication use, and explore risk predictors of clinically significant symptoms and moderate-to-severe caregiver distress caused in the target population.A comparative cross-sectional study.Between March 2019 and September 2019, community-dwelling older adults with dementia and their primary family caregivers were recruited from the outpatient clinics of a 1920-bed tertiary psychiatric hospital in Guangzhou  using convenience sampling. The hospital provides neuropsychiatric services for 800 thousand outpatients per year. The inclusion criteria for community-dwelling older adults with dementia were (1) aged ≥ 60 years, lived at home and with support from family members; (2) had a physician diagnosis of dementia, following expert assessments. The exclusion criteria for older adults with dementia were (1) having past or present comorbidity of another major psychotic disorder, such as schizophrenia and bipolar disorders; (2) having a terminal illness (with life expectancy < 6 months); or (3) having significant variations in dosage of psychotropic medications during the past four weeks. The inclusion criteria for family caregivers were as follows: (1) the primary family caregiver of the patient; (2) had been in the role for at least 4 h per day during the past 3 months; (3) was able to read and speak Mandarin, and cognitively intact. The family caregiver who refused to participate was excluded. Nursing home residents with dementia were recruited from dementia units of nursing homes in Guangzhou, China. Eligibility for nursing home residents with dementia and their caregivers  has been described elsewhere .Diagnosis of dementia was made using the Diagnostic and Statistical Manual of Mental Disorders, 4th edition . The diaSeverity of dementia was assessed using the Clinical Dementia Rating (CDR) . ClinicaThe Neuropsychiatric Inventory (NPI) was used to interview family caregivers. The NPI-Nursing Home Version (NPI-NH) was used to interview formal caregivers enrolled from nursing homes . Both NPBasic activities of daily living (BADL) were measured using the Barthel index (BI). We used a BI score of 0–20 to indicate total dependency, 21–60 for severe dependence, 61–90 for moderate dependence, and 91–99 for slight dependence .Clinical Dementia Rating (CDR) was rated by psychiatrists who worked in the tertiary psychiatric hospital and the nursing home from where the participants were enrolled. Primary caregivers were asked to complete the NPI-NH, and demographic information about care recipients was also collected through face-to-face interviews with the caregivers. The interviewers were three graduate students majoring in nursing, who received training in data collection, including all procedures and content of the assessments. Clinical data were obtained from medical records. Data about the actual use of medications were also collected based on the patients’ medical records. Medication use was classified according to the Anatomical Therapeutic Chemical classification, including anti-dementia drugs, antipsychotics, anxiolytics, anticonvulsants, and antidepressants.The sample size was calculated using a formula for calculating sample size in a prevalence study . The allP value of less than 0.05 was considered statistically significant.Statistical analysis used SPSS 25.0 . Continuous variables were described as mean and standard deviation (SD), and categorical variables as number (n) and percentage (%). Non-parametric analyses were used when the data showed a non-normal distribution. Between-group comparisons used the Chi-square test  for categorical variables and Mann–Whitney U tests for continuous variables. Linear regression was conducted with continuous caregiver distress scores as a dependent variable and NPI item scores as independent variables. The NPI item score was dichotomized into ≥ 4 and < 4 to identify clinically significant symptoms, and caregiver distress score was dichotomized into ≥ 3 and < 3 for moderate-to-severe caregiver distress. Multivariate logistic regression  was used to test whether the odds of a clinically significant symptom and moderate-to-severe caregiver distress differed by CDR, caregiver type , medication use , physical dependence, age, sex, and education. An additional independent variable for the latter was clinical significance of the symptoms (item score ≥ 4). As for controlling for the medication use, first, we compared medication use between the community-dwelling older adults with dementia and nursing home residents with dementia. And then, the multivariate logistic regressions above-mentioned were used in the full sample. A The Ethics Committee of N Hospital (No. NFEC-201511-K2) approved this study. Participants or their legal representatives were given a written and verbal explanation about the study, the benefits, and any risks. Written informed consent was obtained from all participants or their legal representatives. All personal information was kept confidential and anonymous as required.The study included 157 community-dwelling older adults with dementia  and 112 nursing home residents with dementia . Of those living in the community, 71 (45.5%) were cared for by their children, 64 (40.8%) by their spouse, and 22 (14%) by other relatives. The family caregivers spent an average of 12.2 ± 6.3 h per day providing supervision and caregiving. The demographic and clinical characteristics of the participants are shown in P > 0.05). Apathy  was the most common symptom in both groups  community patients and 104 (92.9%) nursing home patients had at least one symptom .The community patients had higher NPI item scores than the nursing home patients, except for euphoria, disinhibition, and eating disorders . ClinicaApathy was the most frequent clinically significant symptom in both the community (57.4%) and nursing home (39.3%) patients, followed by anxiety (47.8%), agitation (47.1%), and aberrant motor behaviors (47.1%) in the former, while aberrant motor behaviors (25.1%), irritability (24.1%), and agitation (20.6%) in the latter .2 = 74.72, P < 0.001). In total, 110 (70.1%) community patients and 11 (9.8%) nursing home patients had at least one symptom causing severe caregiver distress (distress score ≥ 4) .Overall, the caregivers of 125 (79.6%) community patients and 30 (26.8%) nursing home patients reported moderate-to-severe distress (distress score ≥ 3) caused by at least one symptom (χAgitation was the most frequent moderate-to-severe symptom in both the community (44.6%) and nursing home (12.5%) patients, followed by sleep disorders (38.2%), anxiety (37.6%), and irritability (34.4%) in the former, while irritability (11.6%), delusions (9.8%), and sleep disorders (8.0%) in the latter .After controlling for dementia severity, medication use, and symptom severity, family caregiving remained an independent risk predictor for moderate-to-severe caregiver distress caused by BPSD (OR 2.11∼18.16), especially eating disorders and anxiety .R2 0.63–0.82), except for apathy, euphoria, disinhibition, hallucination, and eating disorders (R2 = 0.37) and nursing home patients (R2 = 0.46). Item scores for euphoria (R2 = 0.36), disinhibition (R2 = 0.48), and hallucination (R2 = 0.59) did not highly predict caregiver distress in the community patients. Eating disorders scores did not highly predict caregiver distress in nursing home patients (R2 = 0.55).Linear regression analysis shows that caregiver distress was highly predicted by NPI item scores (isorders . The apaP > 0.05).Among participants with mild dementia (CDR = 1), there were no group differences in prevalence, clinical significance, and moderate-to-severe caregiver distress caused by the symptoms  and moderate-to-severely distressing  in community patients than their nursing home counterparts. However, there was no group difference in the prevalence of agitation (P > 0.05). Hallucinations , apathy , and aberrant motor behavior  caused more frequent moderate-to-severe distress in the caregivers of community patients, but there was no group difference in the prevalence and clinical significance (P > 0.05).Among participants with moderate dementia (CDR = 2), anxiety, depression, and agitation were more clinically significant  and more clinically significant  in the community patients, but there was no difference in the distress it caused (P > 0.05). Eating disorders and disinhibition were more clinically significant  and moderate-to-severely distressing  in community patients, but there was no group difference in the prevalence of these symptoms (P > 0.05).Among participants with severe dementia (CDR = 3), all symptoms except euphoria were more prevalent, clinically significant, and moderate-to-severely distressing in community patients than their nursing home counterparts . EuphoriIn this study, community-dwelling older adults with dementia showed more frequent clinically significant BPSD than nursing home patients, and these caused more severe caregiver distress. This was especially true for those with moderate-to-severe dementia. After controlling for dementia severity and medication use, family caregiving remained an independent risk predictor for presenting clinically significant symptoms and moderate-to-severe caregiver distress. The prediction of caregiver distress based on symptom severity varied across caregiver types. The strength of this study is that our findings were controlled for dementia severity and medication used by participants. Our findings add to the growing understanding of BPSD and caregiver distress they cause in low- and middle-income countries.Clinically significant symptoms were found in 88.5% of community-dwelling older adults with dementia and 75% of nursing home patients in this study. After controlling for dementia severity and medication use, we found that caregiver type remained an independent risk predictor for presenting clinically significant BPSD. One potential explanation is that BPSD rely on caregiver proxies to report, and family caregivers may have more opportunities to observe these symptoms. Moreover, family caregivers might be more involved in affective disturbances of care recipients, and the internalizing symptoms of dementia, such as anxiety and depression, might be over-reported by family caregivers because of their increased risks for stress, depression, and burden. Another explanation is that formal caregivers are more likely to have had some training in managing BPSD . TraininImpressively, up to 70% of the community-dwelling older adults with dementia had at least one symptom causing severe caregiver distress in this study. However, only 9.8% of the nursing home residents with dementia had at least one symptom causing severe caregiver distress. Family caregivers caring for community-dwelling older adults with dementia experienced more severe distress caused by symptoms than formal caregivers caring for nursing home residents with dementia. After controlling for symptom severity, family caregiving remaining an independent risk predictor for moderate-to-severe caregiver distress in this study, especially eating disorders and anxiety. One explanation is that family caregivers provide longer company for care recipients and undertake additional caregiving tasks than formal caregivers, while formal caregivers are more likely to work cooperatively to manage patients in their workplace than family caregivers. Another explanation is that there is currently insufficient quality and quantity of family based care services in China to meet the needs of community-dwelling older adults with dementia . Family Previous studies suggest that information on caregiver distress caused by individual symptoms is crucial to inform the design of tailored management strategies for supporting family caregivers , 15, 37.In this study, community-dwelling older adults with dementia had more than ten times the risk of presenting clinically significant anxiety and depression, which causing five to ten times the risk of moderate-to-severe distress to family caregivers. Affective disturbances should be taken into account in alleviating distress for family caregivers. We also found that the severity of euphoria and disinhibition did not strongly predict caregiver distress caused in community-dwelling older adults, while the severity of eating disorders did not strongly predict caregiver distress caused in nursing home residents with dementia. Euphoria and disinhibition, which have been classified as “Frontal” endophenotype , were thFrom a public health perspective, optimizing dementia care in the home may provide the maximum population-level benefit (such as decreasing the huge economic costs associated with transitions to other care settings) and is desirable given the preference of older people to remain in their familiar communities for as long as possible . The majThis study had several limitations. First, the cross-sectional design of this study does not allow for a test of causal relationships. Second, modest sample size and insufficient information on dementia subtypes limited further analysis. Third, our data were collected from family caregivers who voluntarily visited the outpatient clinic of a tertiary psychiatric hospital, which may have introduced referral bias. Fourth, the study did not investigate factors related to caregiver burden or mental health status (such as depression and anxiety). The above-mentioned factors might limit the generalizability of our findings. Future studies with a larger sample size should consider these factors.This study found that family caregivers experienced more severe distress caused by behavioral and psychological symptoms of dementia than formal caregivers; family caregiving was an independent risk predictor for clinically significant and moderate-to-severe distressing symptoms; the prediction of caregiver distress based on symptom scores varied by individual symptoms and caregiver types. Our findings highlight that tailored management strategies are needed to relieve family caregivers’ distress caused by BPSD, and that an effective support service system should be established to give family caregivers access to resources, information, and knowledge to improve their ability to cope with BPSD.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by The Ethics Committee of Nanfang Hospital (No. NFEC-201511-K2) approved this study. The patients/participants provided their written informed consent to participate in this study.XJL, JW, and RY gathered the data, completed the data analysis and interpretation, and took part in drafting of the manuscript. DR helped with acquisition of data and clinical diagnosis. XYL advised on the design of the study, secured funding, supervised the research, took responsibility for the accuracy of the data analysis and interpretation, and provided critical and substantive commentary to both the process and final product. All authors granted approval for submission and publication, contributed to revising the manuscript critically for important intellectual content, and were in agreement with the content of the manuscript.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.


During the COVID-19 pandemic, health systems rapidly introduced in-home telehealth to maintain access to care. Evidence is evolving regarding telehealth’s impact on health disparities. Our objective was to evaluate associations between socioeconomic factors and rurality with access to ambulatory care and telehealth use during the COVID-19 pandemic.We conducted a retrospective study at an academic medical centre in midwestern United States. We included established and new patients who received care during a one-year COVID-19 period vs pre-COVID-19 baseline cohorts. The primary outcome was the occurrence of in-person or telehealth visits during the pandemic. Multivariable analyses identified factors associated with having a health care provider visit during the COVID-19 vs pre-COVID-19 period, as well as having at least one telehealth visit during the COVID-19 period.All patient visit types were lower during the COVID-19 vs the pre-COVID-19 period. During the COVID-19 period, 125 855 of 255 742 established patients and 53 973 new patients had at least one health care provider visit, with 41.1% of established and 23.5% of new patients having at least one telehealth visit. Controlling for demographic and clinical characteristics, established patients had 30% lower odds of having any health care provider visit during COVID-19 vs pre-COVID-19 (adjusted odds ratio (aOR) = 0.71, 95% confidence interval (CI) = 0.698-0.71) period. Factors associated with lower odds of having a telehealth visit during COVID-19 period for established patients included older age, self-pay or other insurance vs commercial insurance, Black or Asian vs White race and non-English preferred languages. Female patients, patients with Medicare or Medicaid coverage, and those living in lower income zip codes were more likely to have a telehealth visit. Living in a zip code with higher average internet access was associated with telehealth use but living in a rural zip code was not. Factors affecting telehealth visit during the COVID-19 period for new patients were similar, although new patients living in more rural areas had a higher odds of telehealth use.Healthcare inequities existed during the COVID-19 pandemic, despite the availability of in-home telehealth. Patient-level solutions targeted at improving digital literacy, interpretive services, as well as increasing access to stable high-speed internet are needed to promote equitable health care access. The United States (US) declared a public health emergency due to the COVID-19 pandemic on January 13, 2020. Subsequently, the Center for Medicare and Medicaid Services (CMS), followed by other insurance payers, expanded access to and increased reimbursement for telehealth services. Specifically, the 2020 Coronavirus Preparedness and Response Supplement Appropriations Act enacted in March 2020 allowed for reimbursement of home telehealth visits by video or telephone. The goals of expanding telehealth coverage were to mitigate gaps and prevent surges in demand for patient care  in efforPrior to the COVID-19 pandemic, telehealth was primarily used in special situations, particularly in rural areas and with military veterans to address health care needs in underserved populations . In thesSeveral studies have evaluated disparities in telehealth use during the early stages of the COVID-19 pandemic. The most consistent finding was that older, non-white patients who have Medicaid or are uninsured and who live in rural areas were less likely to have a telehealth visit compared to white, English-speaking patients with commercial insurance living in non-rural areas ,12,18. AWe aimed to evaluate socioeconomic factors and the geospatial distribution associated with access to care and telehealth use during the COVID-19 pandemic in a large population of rural and urban patients receiving care through a US academic health system. We hypothesized that telemedicine would contribute to the efforts to maintain access to outpatient care, including in remote areas, which would have been otherwise negatively impacted by the COVID-19 pandemic. However, this access to care may be muted in patients from socioeconomic backgrounds known to face health care disparities.This retrospective, cross-sectional study included patients treated by primary care and speciality providers in clinics owned and operated by the University of Nebraska Medical Center (UNMC), Omaha, Nebraska, in the United States. UNMC is a large midwestern academic tertiary health care centre in the United States with over a million outpatient visits annually. Telehealth was provided via a video platform embedded in the health system’s electronic health record (EHR). Telephone visits were used when video visits were not feasible.The study protocol was approved by the medical centre’s Institutional Review Board. We collected deidentified data from all in-person and telehealth ambulatory provider visits from the EHR system from March 16, 2017, through March 15, 2021. We used the one-year COVID-19 period defined as March 16, 2020, to March 15, 2021, as the study period of interest. COVID-19 period data were compared to a baseline pre-COVID-19 period from March 16, 2019, to March 15, 2020. To determine whether patients were established with a medical centre provider during these time periods, we also identified corresponding establishment timeframes which ran for two years prior to the start of each period of interest .Dependent variables included the occurrence of overall ambulatory provider visits and by in-person vs telehealth (including video and phone) delivery. We created summary variables for counts of in-person and telehealth visits, after which patients were categorized as having all in-person visits or one or more telehealth visit.Independent variables identified for each period of interest included demographic data and diagnosis of diabetes (a common comorbidity for adjustment). For established patients, we based the patient’s age on the age at the beginning of the period of interest, while we pulled other baseline characteristics ) from the patient’s last visit in the respective establishment period. An exception was the diagnosis of diabetes, which was based on having a documented ICD-10 code for type 1 or type 2 diabetes at any time during the respective establishment period. For new patients, we used baseline characteristics from their first visit in the period of interest. Due to the nature of the data warehouse, patient residential zip codes were associated with a patient’s address at the time of the data extraction May 2021). For data not available at the patient level , we assigned patients the value associated with their zip code as a proxy, using American Community Survey 2019 five-year estimates [21. For d2 tests and independent samples t tests or Wilcoxon Rank Sum tests (when data was highly skewed) to assess the differences in categorical and continuous variables, respectively, between groups of interest when the two groups were independent . We provide only descriptive statistics when groups were not independent . No steps were made to impute missing data. We thus calculated percentages for all descriptive statistics using the non-missing data within each variable as the denominator.For univariable statistics, we used χP-values of less than 0.05 were considered to be statistically significant. Statistical analyses were performed using SAS version 9.4 .We conducted multivariable regression analyses to identify 1) the odds of established patients having a visit during the COVID-19 period relative to the pre-COVID-19 period, controlling for patient characteristics, and 2) patient characteristics associated with having at least one telehealth visit vs all in-person visits during the COVID-19 period (with separate models for new and established patients). We used logistic regressions when patients only contributed a single observation . For outcomes where a patient could contribute multiple observations , we utilized generalized estimating equations to account for the correlation within patient. For all models, we presented adjusted odds ratios (aOR) with 95% confidence intervals (95% CIs), which were Bonferroni adjusted based on the number of possible pairwise comparisons that could be made within each variable. We further summarized patient-level data at the zip code level for each period of interest. Rates were calculated and the percentage of established patients who had a visit in the associated period of interest, and the percentage of patients seen during COVID-19 with a telehealth visit for established and new patients, separately. Zip codes with less than five patients in the denominator were excluded from mapping to avoid extreme percentages. We associated summary statistics for each zip code with the centroid for that zip code, and inverse distance weighting interpolation was used to generate an estimated surface of percentages across the entire state. The mapping was done using ArcGIS Pro software version 2.7.0 .A total of 128 598 out of 243 248 (52.9%) established patients seen and non-deceased at the end of each establishment timeframe had at least one ambulatory provider visit during the pre-COVID-19 period; 125 855 out of 255 742 (49.2%) had at least one ambulatory provider visit during the COVID-19 period. Approximately 33.6% (65 068 out of 193 666) of all patients seen in the pre-COVID-19 period were new, while only 30.0% (53 973 out of 179 828) of all patients seen in the COVID-19 period were new.Baseline characteristics of the patient cohorts are reported in In multivariable regression analyses, established patients were less likely to have any ambulatory provider visit  in the COVID-19 period compared to the pre-COVID-19 period , controlling for demographic and clinical characteristics .The percent of established patients with at least one telehealth visit increased from 0.4% in the pre-COVID-19 period to 41.1% in the COVID-19 period. Similarly, 1% of new patients had a telehealth visit during the pre-COVID-19 period vs 23.5% of new patients during the COVID-19 period  improving digital literacy, b) interpretive language services (including inclusion of multi-language instructions and websites), and c) access to stable high-speed internet. These findings should prompt strategies and policy changes to allow for a more equitable health care system.


Alexithymia, that is the inability to recognize and describe one’s own emotions, is a transdiagnostic feature across eating disorders (EDs) and it has been associated to a prolonged stress exposure.Therefore, we evaluated whether alexithymia affects the hypothalamus-pituitary-adrenal (HPA) axis functioning in patients with anorexia nervosa (AN) or bulimia nervosa (BN).Twenty-six women with AN and 26 with BN participated in the study. Alexithymia was evaluated by the Toronto Alexithymia Scale–20 and eating-related psychopathology was measured by the Eating Disorder Inventory-2. The activity of the HPA axis was assessed by the salivary cortisol awakening response (CAR). Group differences in saliva CAR were tested by repeated measures 3-way ANOVA with diagnosis and alexithymia as between-subject factors.2=1.24, p=0.26). Alexithymia was associated with more severe eating-related psychopathology in AN women but not in BN women. A significant reduction in the magnitude of CAR occurred in alexithymic patients with BN compared to non-alexithymic patients with BN , but not in alexithymic women with AN .The prevalence of alexithymia did not differ significantly between the two diagnostic groups (cThese results confirm the presence of a more severe eating-related psychopathology in alexithymic individuals with AN and show, for the first time, an association between alexithymia and a dampened basal activity of the HPA axis in BN.No significant relationships.


Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the main concern is whether reinfections are possible, and which are the associated risk factors. This study aims to describe the clinical and molecular characteristics of 24 sequence-confirmed reinfection SARS-CoV-2 cases over 1 year in Barcelona .Patients with > 45 days between two positive PCR tests regardless of symptoms and negative tests between episodes were initially considered as suspected reinfection cases from November 2020 to May 2021. Whole-genome sequencing (WGS) was performed to confirm genetic differences between consensus sequences and for phylogenetic studies based on PANGOLIN nomenclature. Reinfections were confirmed by the number of mutations, change in lineage, or epidemiological criteria.From 39 reported suspected reinfection cases, complete viral genomes could be sequenced from both episodes of 24 patients, all were confirmed as true reinfections. With a median age of 44 years (interquartile range [IQR] 32–65), 66% were women and 58% were healthcare workers (HCWs). The median days between episodes were 122 (IQR 72–199), occurring one-third within 3 months. Reinfection episodes were frequently asymptomatic and less severe than primary infections. The absence of seroconversion was associated with symptomatic reinfections. Only one case was reinfected with a variant of concern (VOC).Severe acute respiratory syndrome coronavirus 2 reinfections can occur in a shorter time than previously reported and are mainly found in immunocompetent patients. Surveillance through WGS is useful to identify viral mutations associated with immune evasion. In December 2019, an outbreak of severe acute respiratory disease caused by a new coronavirus was emerged in Wuhan, China. The disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and named Coronavirus Disease-19 (COVID-19), was finally declared as a global pandemic by the World Health Organization on 11 March 2020. SARS-CoV-2 is an enveloped, single-stranded, positive-sensed RNA virus. Its 30 kb genome encodes for four major structural proteins, which are as follows: Spike (S), Envelope (E), Membrane (M), and Nucleocapsid protein (N); 16 non-structural proteins (nsp1–16) are encoded by the open reading frame (ORF) 1ab and six accessory proteins  .Since the beginning of the SARS-CoV-2 pandemic, more than 413 million people have been infected with more than 5 million deaths. One of the main concerns for public health is whether reinfection is possible despite the immune response elicited after the primary infection. Large studies suggest that this is an uncommon event, as natural immunity decreases the risk for reinfection . Only a This study is aimed to describe the characteristics of the first 24 sequence-confirmed cases of reinfection detected from March 2020 to February 2021 in a tertiary hospital laboratory in Barcelona, Spain.From November 2020 to May 2021, upper and lower respiratory specimens were received for the laboratory confirmation of SARS-CoV-2 from children and adults accomplishing the World Health Organization case definition criteria of SARS-CoV-2 infection , who werA suspected case of SARS-CoV-2 reinfection was defined as a patient with two SARS-CoV-2 positive PCR tests separated at least 45 days  , regardlCases were confirmed as true reinfections when at least one of the following criteria was accomplished: (a) a change of lineage in the second episode versus the first one; (b) the number of acquired nucleotide mutations was similar to or higher than the expected to happen during the time period between episodes according to the evolution rate estimated by Nextstrain (2 mutations per month approximately) ; or (c) Suspected reinfection cases were not collected by systematic surveillance but reported by hospital staff to the laboratory.® SARS-CoV-2 Test, Roche Diagnostics; and Xpert Xpress SARS-CoV-2 test, Cepheid), or transcription-mediated amplification (TMA)-based assays , depending on the availability of the products over time. All assays were performed following the instructions of manufacturers. For RT-PCR assays requiring nucleic acids extraction, NUCLISENS easyMAG  and Microlab STARlet System  were used. Clinical SARS-CoV-2-positive specimens were kept frozen for further studies.Detection of SARS-CoV-2 was carried out by non-commercial , commercial real-time reverse transcription-polymerase chain reaction (RT-PCR) assays  or ELISA , following the instructions of manufacturers. Assays were selected depending on the products’ availability.1 with the V3 primer pools  for complete genome multiplex, overlapping amplification. Library preparation was performed with KAPA HyperPrep Kit  or Illumina DNA Prep  depending on the product’s availability. Briefly, cDNA synthesis was performed with SuperScript IV reverse transcriptase (Invitrogen) and further PCR amplification for the two pools of ARTIC V3 primers with Q5 Hot Start High-Fidelity DNA Polymerase . PCR products from each sample were individually indexed using the SeqCap Adapter Kit A/B  when using KAPA HyperPrep; or IDT® for Illumina® DNA/RNA UD Indexes Set A-D (384 IDX)  when using Illumina DNA Prep. Finally, the products were added to the final pooled library tube, which, together with a 5% of PhiX internal DNA control , were loaded in a MiSeq Reagent Kit (600 cycles) v3  and sequenced using the MiSeq platform .Whole-genome sequencing of SARS-CoV-2 was performed following the ARTIC Network protocolEPI_ISL_2284948, EPI_ISL_6595267-6595311, and EPI_ISL_6596214-6596215.FastQ files generated in the MiSeq output were uploaded to BaseSpace Sequence Hub  and analyzed with the DRAGEN COVID Lineage app for the Kmer-based detection of SARS-CoV-2, mapping of the passing filters’ reads to the reference genome (NC_045512 in GenBank), variant calling generation of a consensus genome sequence with a coverage threshold at least of 20×. Finally, Pangolin (version of 19 May 2021) was also run on those consensus sequences to determine the viral lineage. Mutations were reported using MEGA v6.0 software , in compRandom complete SARS-CoV-2 genomes sequenced at our hospital available at GISAID database  were selSocio-demographic, clinical, and epidemiological characteristics for each episode were retrospectively reviewed from electronic medical records. Days between episodes were calculated according to the onset of symptoms that occurred before a positive test result.Institutional Review Board approval (PR(AG)259/2020) was obtained from the hospital’s Clinical Research Ethics Committee.Frequencies and percentages were used to describe categorical data. For continuous data, median and interquartile ranges (IQR) were calculated. For analysis of categorical data, chi-squared or Fisher’s exact test was performed. All computations were made in Excel and the R language . R: A laA total of 503,825 samples were received from November 2020 to May 2021, of which 54,101 (11%) were laboratory confirmed for SARS-CoV-2. Overall, 39 cases of suspected reinfections were reported to the laboratory (0.07% of positive samples). Samples of both episodes were recovered and were suitable for sequencing in 24 cases which all were confirmed as true reinfections according to the criteria above. The main characteristics of these 24 patients with COVID-19 reinfection are summarized in The median age was 44 years old (IQR 32–65). More than half of the cases happened in healthcare workers (HCWs) either from the hospital or nursing homes, whose median age was 38 (IQR 30–46), different from non-HCW cases, 68 (IQR 45–78). Among HCWs, 12 of 14 were women, compared to 4 of 10 non-HCWs. Comorbidities were present in 10 cases, and two were immunosuppressed.Cases were confirmed as true reinfections due to a higher number of observed mutations than expected by intra-host evolution , the change of lineage , or due to clinical and epidemiologic criteria (presence of symptoms or recent contact with an infected person) (P20 and P22). Demographic, clinical, and molecular features of 24 sequenced suspected reinfections are presented in n = 16), followed by screening (n = 5) and contact tracing (n = 3). Second episodes were detected mostly by screening (n = 13), followed by clinical suspicion (n = 6) and contact tracing (n = 5).The time between episodes for each case and its distribution over time is represented in p = 0.02). Upon the second episode, all tested patients (16/24) developed or maintained specific antibodies.In the first episode, 19 (79%) patients manifested symptoms and 6 (25%) required hospitalization. After the first infection, 13 patients were detected to have specific anti-SARS-CoV-2 149 IgG antibodies, 6 were seronegative, and 5 were not tested for antibodies. In the second episode, 14 (58%) patients manifested symptoms, of which two (P16 and P18) required hospitalization and one was (P18) died. These two hospitalized cases presented symptoms in the first episode and did not seroconvert after it. Regarding the relation between symptoms and the presence of specific antibodies, among 13 cases that seroconverted after the first episode, five were symptomatic during the second, while all six cases without seroconversion had symptoms at reinfection. The presence of symptomatology in the reinfection episode was more frequent if there was no seroconversion following the first episode  and B.1.177 . No relevant mutations were found in the two hospitalized patients.The most observed lineages were B.1 , with 60% being men. Despite HCWs could be overrepresented in this study, the main characteristics of the patients in the present study reflect the well-known populations at high risk for severe illness as the elderly  and at iThe median days between episodes  were similar to the reported in other studies . There iThe presence of symptoms was more common during the first episode (80%) than during the reinfection (50%). Until June 2020, testing was limited to severe cases and symptomatic HCWs, which could have skewed our results. However, when focusing on cases whose first episode happened after June 2020 , the same pattern is repeated, where the second episodes were more frequently asymptomatic. This decrease in the proportion of symptomatic cases was also observed in the SARS-CoV-2 immunity and reinfection evaluation (SIREN) study , where oThe second episode was found to most commonly manifest with lower severity than the first one, as previously reported . HoweverThe high proportion of asymptomatic patients in the second episode could be underestimating the reinfection events, as they are only identified through contact tracing or screening strategies. Implementing systematic surveillance and other strategies as serial testing in high-risk populations would allow the identification of a higher number of asymptomatic cases contributing to transmission control.Regarding seroconversion, it stands out that 13 patients had a positive antibody test after the first episode that did not prevent them from reinfection. On the other hand, negative sera tests after the first episode were more frequent to be found in symptomatic cases rather than in asymptomatic ones, suggesting that seroconversion could prevent further symptomatic episodes, along with reducing the risk of reinfection, as it is already described .Due to the presence of anti-SARS-CoV-2 IgG antibodies during the reinfection episode, five patients were neither placed under isolation precautions nor initiated with contact tracing, as the positive PCR test was clinically considered as persistent viral shedding  without transmission capacity. This could pose a risk of onward transmission and nosocomial outbreaks, highlighting that serological tests should not be used to estimate the transmissibility period. Thus, collecting information about secondary transmission from reinfected cases could bring light on their transmission capacity and its impact on public health.A high proportion of asymptomatic reinfections were identified through routine screening or contact tracing. A positive result for SARS-CoV-2 in an asymptomatic patient with a previous positive test could be wrongly interpreted as persistent viral shedding . Long viral shedding has already been reported, as well as intra-host viral evolution, but mainly in immunocompromised patients . OtherwiDespite the short time found between episodes in our study, none of them was classified as persistent viral shedding or reactivation. Thus, in a context of increased circulation of VOCs , these results suggest considering a suspected case of reinfection for those presenting with a SARS-CoV-2 positive test and compatible symptoms in even less than 3 months after their first episode to proper implement prevention and control measures.Observed variants in both episodes were similar to the sequences that circulated in the community at that moment , providiOther mutations, such as those observed in nsp3, nsp6, and ORF10, are important to be further studied to clarify their implication in the virus’ fitness or in reinfection. The role of ORF10 is yet to be described, so it is difficult to hypothesize the reason why we found an association with the presence of symptomatology in reinfections. No relevant mutations related to immune evasion were found in the two hospitalized patients.Finally, there is a lack of evidence on whether natural immunity to a non-VOC will have a good neutralizing capacity against VOCs. VOCs have proved to be more transmittable, which increases the concern of what effects these variants may have on public health, as they may lead to an increase in reinfections, and thus, in an extension in time of this pandemic. Hence, this article reinforces the need for continuous monitoring and sequencing of reinfections.In addition, though no chronic active infections are reported in this manuscript, some cases have been observed in our hospital setting, mostly in immunocompromised patients (data not shown), and there are many publications reporting them. It is important to discern between reinfections and chronic infections, as the procedures applied are different in these cases, and it is also basic to differentiate when the virus is transmissible or not. In fact, there are some reports that postulate that these chronic infections are the origin of the emerging variants of SARS-CoV-2, such as the VOCs. Thus, monitoring these cases and studying those mutations occurring at the consensus level and those occurring at a low frequency are essential.The findings in this study must be interpreted with caution as there are some limitations. First, the characteristics of the reinfected cases could be biased, as case identification in the present study was not performed by systematic surveillance. HCWs could be overrepresented as they have been a priority population for testing as well as hospitalized patients, as mild cases were mostly diagnosed by rapid tests performed in Primary Care Centers and these samples did not arrive at the hospital laboratory. Second, over 39 suspected reinfections, only 24 could be sequenced and confirmed, as WGS is suitable in cases with a moderate to high viral load.Reinfections can occur in a shorter time than previously reported, mostly in immunocompetent patients, and these episodes are more feasible to be less symptomatic and less severe than the first ones. This suggests that immune response was insufficient to prevent reinfection but modulated the clinical course in these cases. Moreover, reinfections are rare, but since they are usually asymptomatic, they may be underestimated and pose a risk for ongoing transmission. Thus, efforts should be addressed to strengthen reinfection SARS-CoV-2 surveillance. Performing WGS of all cases will allow us to rapidly identify key mutations and variants of special interest due to immune evasion or enhanced transmission.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.The studies involving human participants were reviewed and approved by Comité de Ética de Investigación con medicamentos [Vall d’Hebron Institute of Research (VHIR)]. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.MP, BB-B, CA, JE, MC, JQ, AA, and TP conceived the study. DG-C, AR, CCam, MGC, and SC-C participated in the design and implementation of the study. MP, CA, DG-C, CCam, and SC-C optimized the methodology of whole-genome sequencing. MM, CCas, KG-C, RV-M, and RM-M performed the whole-genome sequencing. AG-S, CA, and MP did the bioinformatic analyses. JE reviewed serological test results. OP-B, RZ, and BB-B reviewed medical records. MP and BB-B did the analysis of the results and drafted the manuscript that was critically reviewed by AA, MC, JQ, TP, and CA. All authors approved the final manuscript.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.


Prevention of nosocomial severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) transmission in acute-care hospitals represents a major challenge. One important reason is that diagnosis in asymptomatic or presymptomatic patients and healthcare workers (HCWs) is often delayed. Also, understaffing during the pandemic may decrease adherence to standard hygiene measures. Before the SARS-CoV-2 vaccine became available, we observed several wards with nosocomial COVID-19 outbreaks, whereas others were spared. We sought to identify ward-level risk factors associated with nosocomial COVID-19 outbreaks.The coronavirus disease 2019 (COVID-19) pandemic made its way from private, public, and work spaces into healthcare systems all over the world, imposing an unexpected burden on global health care. COVID-19 outbreaks in acute-care hospitals are a frequent problem, increasing morbidity and mortality among patients and leading to staff shortages.P ≤ .05 was considered statistically significant. We used SPSS Statistics for Windows version 20.0 software for all statistical analyses .We conducted a matched case–control study in our 700-bed tertiary-care center during the second wave of the COVID-19 pandemic. Infection prevention and control (IPC) measures consisted of the use of personal protective equipment (PPE), social distancing, placing confirmed cases in cohorts, and visitor restrictions. Routine testing of asymptomatic HCWs was not implemented. We defined nosocomial SARS-CoV-2 outbreaks as the occurrence of ≥2 patients with nosocomial infection within a 14-day period on the same ward. Nosocomial infection was defined as a positive SARS-CoV-2 test on day 5 or later of hospital admission. Wards with at least 1 nosocomial outbreak between July and December 2020 were defined as outbreak wards. Wards without outbreaks served as controls. Intensive care units and designated COVID-19 wards were excluded. Ward matching was done 1:1 for approximate number of beds (±10) and time of the outbreak by choosing the same period of investigation on control and outbreak wards. The beginning of an outbreak was defined as the day of the first positively tested nosocomial COVID-19 patient on the ward; the same day was chosen as reference date for the control ward  and younger ages of nurses . The only factor that was significantly different between outbreak and control wards was the percentage of positively tested HCWs immediately before the outbreak or reference date . We detected no difference in the percentage of infected HCWs in the earlier period (prior to 2 weeks before the outbreak). We detected no association with factors reflecting structural characteristics, workload, patient turnover, or work experience , and the median number of nurses was 30 . In total, 40 patients acquired nosocomial COVID-19, with a median of 4 patients per outbreak ward . Compared to control wards, outbreak wards demonstrated trends toward higher numbers of beds per room (2.22 vs 1.97; 7–31, andIn this matched case–control study, the only factor associated with occurrence of nosocomial COVID-19 outbreaks was an increased number of infected HCWs shortly before the outbreak.This finding supports the hypothesis that infected HCWs are an important source of nosocomial COVID-19. Because affected patients were highly dependent on nursing care, close contact between HCWs and patients was hypothesized to be the driving force for transmission. Similar findings have been reported by others. Our study, using a different approach by comparing ward-level factors between outbreak and non-outbreak wards, adds to the mounting evidence that HCWs are indeed the main source of nosocomial COVID-19 among patients. These findings corroborate the importance of adherence to IPC measures, not only in hospitals but also in public and private environments. Furthermore, vaccination or repetitive testing of asymptomatic HCWs might be other useful measures to efficiently prevent nosocomial SARS-CoV-2 infection.Our data agree with those of other studies. In a recent investigation of nosocomial COVID-19 outbreaks using whole-genome sequencing, the investigators concluded that SARS-CoV-2 transmission occurred most likely from HCW to patients.Our study has limitations. We lacked whole-genome sequencing data for SARS-CoV-2 isolates. Also, we were not able to perform multivariable analyses because of the limited sample size.In conclusion, our data support findings from other studies suggesting that infected HCWs are the main source of nosocomial COVID-19 cases, at least in a prevaccination era. Further studies will be needed to conclusively determine whether vaccination of HCWs and patients will reduce the rate of nosocomial COVID-19.


Public health vaccination recommendations for COVID-19 primary series and boosters in previously infected individuals differ worldwide. As infection with SARS-CoV-2 is often asymptomatic, it remains to be determined if vaccine immunogenicity is comparable in all previously infected subjects. This study presents detailed immunological evidence to clarify the requirements for one- or two-dose primary vaccination series for naturally primed individuals. The main objective was to evaluate the immune response to COVID-19 mRNA vaccination to establish the most appropriate vaccination regimen to induce robust immune responses in individuals with prior SARS-CoV-2 infection. The main outcome measure was a functional immunity score (zero to three) before and after vaccination, based on anti-RBD IgG levels, serum capacity to neutralize live virus and IFN-γ secretion capacity in response to SARS-CoV-2 peptide pools. One point was attributed for each of these three functional assays with response above the positivity threshold. The immunity score was compared based on subjects’ symptoms at diagnosis and/or serostatus prior to vaccination. None of the naïve participants (n=14) showed a maximal immunity score of three following one dose of vaccine compared to 84% of the previously infected participants (n=55). All recovered individuals who did not have an immunity score of three were seronegative prior to vaccination, and 67% had not reported symptoms resulting from their initial infection. Following one dose of vaccine, their immune responses were comparable to naïve individuals, with significantly weaker responses than individuals who were symptomatic during infection. These results indicate that the absence of symptoms during initial infection and negative serostatus prior to vaccination predict the strength of immune responses to COVID-19 mRNA vaccine. Altogether, these findings highlight the importance of administering the complete two-dose primary regimen and following boosters of mRNA vaccines to individuals who experienced asymptomatic SARS-CoV-2 infection. As of March 2022, more than 450 million people have been infected by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), leading to over six million deaths from Coronavirus Infectious Disease 2019 (COVID-19) . The ranUp to one year after their infection, recovered individuals present strong and slowly decaying humoral and cellular responses –13. ImpoIn this report, we used three biomarkers of immunity to characterize an effective immune response to SARS-CoV-2. We assessed (1) circulating anti-RBD IgG levels, (2) functional serum capacity to neutralize live SARS-CoV-2 virus and (3) IFN-γ secretion by memory T cells in response to mega pools of SARS-CoV-2 peptides in a cohort of naïve and previously infected individuals before and after COVID-19 mRNA vaccination. We compared the impact of symptoms at the time of primary infection and serology at enrollment (prior to vaccination) on the immune responses to one and two vaccine doses.Our cohort consisted of 55 previously infected health care workers (HCWs) who were recruited following a PCR-confirmed SARS-CoV-2 infection as part of the RECOVER study (n=569) , and 14 Blood samples were collected into serum separation tubes  and acid–citrate–dextrose tubes . They were shipped to the Rare Pediatric Disease (RaPID) biobank at the Sainte-Justine University Hospital Research Center, where serum and peripheral blood mononuclear cells (PBMCs) were isolated according to standard operation procedures (SOPs) using SepMate™ tubes  for PBMCs isolation. Serum was cryopreserved at -80°C, and PBMCs were cryopreserved in complete RPMI (Gibco) with 10% DMSO and stored in liquid nitrogen until used.Exposure was defined as vaccination with Pfizer BioNTech BNT162b2 mRNA vaccine. Vaccine administration was not part of the study itself, as participants received their vaccines through routine public health programs. The primary outcome was the multi-functionality of the immune response developed after each vaccine dose, represented as an immunity score from zero to three. One point was attributed for each of the following immune assays above positive threshold: anti-RBD IgG levels by ELISA, plasma capacity to neutralize live SARS-CoV-2  by micro-neutralization assay, and IFN-γ secretion by PBMCs in response to SARS-CoV-2 mega pool of Spike peptides, determined by ELISpot.490 ratio greater than one with the experimentally determined cut-off, which corresponds to the mean of 10 negative controls plus three times the standard deviation of the negative controls for each experiment. This anti-RBD IgG ELISA was validated on the Canadian National Microbiology Laboratory SARS-CoV-2 National Serology Panel (An ELISA was performed on serum samples to detect specific IgG antibodies for the Receptor Binding Domain (anti-RBD) of SARS-CoV-2 Spike Glycoprotein as previously described , 25. Sergy Panel , 26. Abs® CRL-1586™). Neutralizing antibody titers were defined as the reciprocal of the serum dilution that completely neutralized the infectivity of 100 TCID50 of SARS-CoV-2, determined by the absence of cytopathic effect on cells after four days , isolated from a clinical sample in March 2020 in Québec City, Canada. Two-fold dilutions of heat-inactivated serum were prepared, starting with 1:20 dilution. Equal volumes of serum and virus were mixed and incubated for one hour at room temperature. The residual infectivity of those mixtures was assessed in quadruplicate wells of African green monkey kidney E6 cell line . Culture media and phytohemagglutinin  were used as negative and positive controls, respectively. The positive threshold response was defined as more than 25 spot-forming units (SFU) per million cells.Cell-mediated immune (CMI) response was estimated by ELISpot. Frozen PBMCs were rapidly thawed and rested overnight. They were then stimulated with 1 µg/mL of spike glycoprotein (S), nucleocapsid protein (NCAP) or membrane protein (VME1) mega pools of SARS-CoV-2 peptides from the ancestral Wuhan-1 like strain  as described elsewhere . Spots wThawed and rested PBMCs were stimulated for 20 hours with 1 µg/mL of the CD4-Spike peptides mega pool developed and synthesized by the Sette laboratory , 31. Conth-75th percentile]. Mann-Whitney or Kruskal-Wallis unpaired nonparametric tests were used to evaluate statistical significance between groups for variables that did not follow a normal distribution. Simple linear regression is presented with Pearson correlation (R2) and P-value (p). Significance was set as *P < .05; **P < .01; ***P < .001; ****P < .0001.All statistical tests were performed using Prism 9, version 9.2.0  and data were displayed as median , 1.5 [1.0-2.2] vs. 0.7 [0.5-1.5], P<.0001) in contrast to subjects who did not experience symptoms. Further, antecedent infection led to long-lasting humoral immune responses in most HCWs  , while serum neutralization capability was mainly absent or low. Following one vaccine dose, recovered individuals demonstrated a striking increase in their anti-RBD IgG levels, neutralization capacity and cell-mediated immune response  (6 PBMCs) compared to recovered HCWs   and IFN-γ responses to Spike antigens  , neutralizing antibody titers  and IFN-γ secreting cells  of recovered HCWs were seronegative at enrollment in the study . We thus+ T cells, as assessed by the expression of activation markers, was the highest  for naïve subjects  following one dose of the vaccine, compared to seropositive symptomatic subjects. Notably, this subgroup of asymptomatic seronegative individuals was the only group to demonstrate an immune response following one dose of vaccine that was similar to naïve individuals. In fact, prior to vaccination, these individuals lacked immunological markers of previous antigen encounter, as did SARS-CoV-2 naïve subjects . Waning immunity has been reported six months after a two-dose primary series in naïve individuals , 36. In Limitations of this study are mainly related to the fact that results were obtained from a cohort of primarily middle-aged Caucasian females infected with SARS-CoV-2 eight months before vaccination. The immunization strategy in our province led to an important delay of sixteen weeks between the first and the second doses in these previously infected individuals, compared to ten weeks in uninfected individuals, which should be taken into consideration when interpreting the results. Indeed, an extended vaccination regimen was shown to favor better immune memory . It therThis report reveals the critical importance of the impact of an individual’s symptomatology at the time of infection and serostatus at the time of vaccination on the capacity to mount adequate immune protection, including cell-mediated responses. In contrast to individuals who were symptomatic during infection, we demonstrated that a third of the individuals who recovered from an asymptomatic infection responded like naïve individuals. We recommend that the two-dose primary series, and subsequent boosters, be offered to all previously infected individuals who were asymptomatic, in order to elicit a global and sustained adaptive immune response following mRNA vaccination.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Research Ethics Board (REB) at the Sainte-Justine University Hospital and Research Center. The patients/participants provided their written informed consent to participate in this study.HD, CQ: Conceptualized the project, designed the study and experiments, and obtained funding. SN, BB, JuC, HR, M-EH: Performed the experiments. SN, BB, HD, MB: Analyzed the data. SN, BB, HD: Interpreted the results and prepared the figures. KA, DM: Performed the clinical follow-up of the study. SN, BB, HD: Wrote the manuscript. CQ, VG, MC, PS, GS, JaC, YL, MB, GB: Edited the manuscript and provided critical input. All authors contributed to the article and approved the submitted version.This work was funded by the Canadian Institutes of Health Research (CIHR) (VR2172712) and the Public Health Agency of Canada, through the Vaccine Surveillance Reference Group and the COVID-19 Immunity Task Force. This work was also supported by NIH contract 75N93019C00065 .We want to thank Valérie Villeneuve, Jocelyne Ayotte, and the Rare Pediatric Disease (RaPID) Biobank personnel at the Sainte-Justine University Hospital and Research Center for their technical help and expertise. Special thanks to Fazia Tadount and the team at the Vaccine Study Center for coordinating the RECOVER studies, and to Zineb Laghdir for administrative support for the RECOVER studies. We are grateful to the nurses and research coordinators at each University Hospital for their involvement in this study and the healthcare workers who agreed to participate in this research. We also thank Alessandro Sette and Daniela Weiskopf for providing the mega pool of peptides utilized in the AIM assays.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.


Mammalian ovaries contain many oocytes that are enclosed in different follicles, commonly one oocyte in one follicle. Poly-ovular follicles are those with more than one oocyte within a single follicle. This type of follicle has been observed in many mammals, with an especially high incidence in canines, representing an intriguing condition. The quality of oocytes from poly-ovular follicles is heterogeneous, as different oocyte morphologies have been observed. Oocyte meiotic potential has been a key factor in determining the success rate of the in vitro maturation process in canines because of the mixed population of oocytes collected from follicles at different stages of development and at random phases of the estrus cycle. Several oocytes may be derived from poly-ovular follicles without knowledge of their meiotic capacity in vitro. Therefore, to improve in vitro maturation and reproductive biotechnologies in canines, evaluating the maturity potential of oocytes according to follicle type and estrous cycle is necessary. Our study demonstrated that the efficiency of in vitro maturation of oocytes retrieved from poly-ovular or mono-ovular follicles depends on the follicle type according to the phases of the estrous cycle during collection, which is important to consider when improving in vitro maturation protocols in this species.2 for 72 h. After culturing, the meiotic development of each oocyte was evaluated using epifluorescence microscopy. Meiotic stages were classified into germinal vesicle (GV), germinal vesicle breakdown (GVBD), first metaphase (MI), and second metaphase (MII). Data were evaluated using an analysis of variance. Oocytes from poly-ovular follicles at all phases exhibited a higher (p < 0.05) percentage of oocytes arrested at the GV stage than those from mono-ovular follicles, showing the highest rate of GV in small antral follicles during anestrus. In contrast, there were no differences in MII rates (p < 0.05) in oocytes from mono-ovular and poly-ovular follicles during the estrus and diestrus phases in all sizes evaluated, with the highest MII rate in estrus. These results suggest that oocytes from poly-ovular follicles can resume meiosis at a slower rate than those from mono-ovular follicles; however, the maturation in vitro of such oocytes is possible. Furthermore, the relationship between the maturation capacity of oocytes from both poly-ovular and mono-ovular follicles depends on the ovarian cycle and follicular development.Poly-ovular follicles are defined as those with more than one oocyte present in single follicles. The occurrence frequency of this follicle type is higher in canines than that in other species. This study aimed to evaluate the in vitro meiotic maturation of dog oocytes from this follicle type in comparison to those from mono-ovular follicles of various sizes  considering different phases of the estrus cycle . Canine oocytes were obtained separately from the poly-ovular and mono-ovular antral follicles from the ovaries of adult females. In each experimental replicate, cumulus-oocyte complexes (COCs) from poly-ovular and mono-ovular follicles were incubated in supplemented TCM-199 at 38.5 °C and 5% CO Poly-ovular follicles contain more than one oocyte per follicle. Although infrequently occurring in most mammals, this type of follicle has a high incidence in the ovaries of canine species ,2,3.During follicular development, the oogonia show intercellular cytoplasmic bridges that form nests during growth in the developing ovary . Nest brThe significance of these follicles in canine reproduction is important due to the high number of occurrences. According to a previous study, oocytes in poly-ovular follicles start their development at a similar size to those in mono-ovular follicles, but differences appear later, suggesting some developmental lag in oocytes from poly-ovular follicles .Morphological analysis has revealed poly-ovular follicles containing oocytes that are discordant in maturity . Thus, oTo date, the meiotic competence of dog oocytes from poly-ovular follicles remains unknown. The in vitro maturation (IVM) process depends greatly on the prior period inside the follicles under the influence of intraovarian factors and hormonal conditions. The canine oocytes used for maturation in culture protocols are usually obtained by the mincing method . Although mincing of ovarian tissue may yield high numbers of oocytes, the population of oocytes recovered in this way are at random stages of development, regardless of the follicular condition (mono- or poly-ovular). Since oocyte meiotic development is a limiting factor for IVM efficiency in canines, many oocytes used for IVM may be derived from poly-ovular follicles; however, nothing is known about the ability of these oocytes to mature in culture. Therefore, this study aimed to evaluate the meiotic maturation of dog oocytes from poly-ovular follicles compared to those from mono-ovular follicles, specifically examining the follicle development stage and the estrus cycle.w/v NaCl) at 37 °C with 100 IU/mL penicillin G and 0.1% streptomycin sulfate . All samples were transported to the laboratory within 20–30 min.Ovaries were obtained from mixed-breed non-pregnant female dogs between 1 to 4 years of age that underwent elective ovariohysterectomy at local veterinary clinics near our laboratory. The ovaries were placed into saline solution , proestrus (n = 11), estrus (n = 17), and diestrus (n = 31)—were dissected free of ovarian tissue under a stereomicroscope .At the laboratory, each ovary was washed in phosphate-buffered saline . Individual antral follicles from ovaries at different phases of the estrus cycle—anestrus  with a progesterone canine kit . The sensitivity of the assay was 0.19 ng/mL.The phases of the estrus cycle of each donor were assessed by evaluating the presence or absence of follicles and corpus luteum (CL) on the ovarian surface  and confAfter careful dissection of the follicles, the cumulus–oocyte complexes (COCs) were collected with a narrow-bore Pasteur pipette from the mono-ovular A,B and p2 for 72 h as previously described [COCs from each follicular size retrieved from poly-ovular and mono-ovular follicles were selected for IVM according to oocyte diameter (>100 mm), measured with a micrometer disc ; uniform ooplasm; and a compact cumulus cell mass surrounding the oocyte, as previously described . The expescribed ,19. In eSince dog oocyte maturation is influenced by in vitro culture conditions, the same conditions were used for all oocytes subjected to IVM.®; Vector Laboratories Inc., Burlingame, CA, USA) sandwiched between paraffin strings covered with a cover slip. The meiotic development of each oocyte was evaluated using an inverted epifluorescence Olympus IX71 microscope (UV emission 480 nm) equipped with an IX2-RFA lamp and a ProgRes-Capture Pro camera .After IVM culturing, the COCs were removed from the medium and placed into disposable dishes with PBS at room temperature (21–22 °C). With a fine-tipped pipette, the oocytes were denuded of the cumulus cells by gentle pipetting under a stereoscopic microscope . Denuded oocytes were fixed in 100 μL of paraformaldehyde solution (4%) for 20 min and washed twice in PBS for 5 min each. Afterward, the oocytes were mounted on slides and incubated in 1 μg/L 4′-6-diamidino-2-phenylindole solution  for 5 min and washed twice in PBS solution for 5 min each to remove excess staining . The oocMeiotic stages were classified into germinal vesicle (GV), germinal vesicle breakdown (GVBD), and first (MI) and second (MII) metaphases, as previously described  Figure . OocytesFor each assay, samples were divided into groups according to their reproductive status and follicle type. To establish whether there was a difference between the meiotic development of oocytes from different follicle types (poly-ovular or mono-ovular follicles) and ovarian phases, the data were arcsine transformed and evaluated by ANOVA, followed by Duncan’s test for determining the differences between each group.The analyses were performed using the InfoStat program . The model included the main effects of follicle type, phase of the estrus cycle, and their interaction for each stage of nuclear development.p value less than 0.05 was considered statistically significant.A Poly-ovular follicles were present in all the examined ovaries; however, mono-ovular follicles were the predominant type found in the ovaries at different sizes and phases of the estrus cycle. The number of oocytes per poly-ovular follicle ranged from two to eight. However, most contained approximately three to four oocytes per follicle. Some morphological differences were observed in the oocytes from the same follicle, including oocyte size (50 μm to >100 μm), cytoplasm darkness, and integrity of the cumulus cells A,B. As tp < 0.05) than oocytes from mono-ovular follicles  and reached a minor percentage of the MI stage compared to oocytes from mono-ovular follicles of the same size during anestrus . The rate of oocytes arrested at the GV stage from medium poly-ovular follicles (33.3%) decreased at anestrus (p < 0.05) compared to those from small follicles. This rate of GV was higher (p < 0.05) than that of oocytes from mono-ovular follicles (20.9%), but without differences in the MII stage between both types of follicles .The meiotic development of oocytes from mono-ovular and poly-ovular follicles throughout the different phases of the estrus cycle is presented in p < 0.05) in the percentage of oocytes arrested at the GV stage between oocytes from mono- and poly-ovular medium antral follicles , increasing (p < 0.05) final maturation with size. During estrus, oocytes from medium and large poly-ovular follicles showed significant differences in the GV stage (p < 0.05). The lowest rate (p < 0.05) of oocytes arrested at the GV stage was observed in oocytes from medium (8%) or large (7.6%) mono-ovular follicles at the estrus phase, while the highest rates of oocytes at the MII stage were those from mono-ovular and poly-ovular follicles of medium  and large  sizes, without differences (p > 0.05) in the final meiotic status (MII) between oocytes from mono-ovular or poly-ovular follicles.At proestrus, there were differences (p < 0.05) between oocytes in poly- and mono-ovular follicles arrested at the GV stage in small  and medium  antral follicles. Although more (p < 0.05) oocytes from mono-ovular follicles reached the MI stage in comparison to oocytes from poly-ovular follicles, oocytes from both types of follicles did not differ in reaching the final maturation stage at MII in small (8.0% and 7.1%) or medium (6.0 and 6.1%) follicles.The diestrus phase showed differences  increases , which iDuring proestrus, a direct relationship between follicular size and oocyte competence was also observed after IVM in poly-ovular follicles, with the oocytes harvested from small follicles being less competent in reaching the MII stage than those from medium follicles. This difference was not observed in oocytes from mono-ovular follicles. In other species, there is some retardation in the developmental capacity of poly-ovular follicles in acquiring the ability to grow to more advanced stages . The minThe gene expression patterns of the oocyte and follicular microenvironment in which the oocyte grows and matures are factors that contribute to oocyte quality and meiotic competence . ChangesThe highest rate of oocytes at the MII stage was observed in the estrus phase in both poly-ovular and mono-ovular follicles. Although the percentage of oocytes arrested at the GV stage and those that reached the MI stage in the estrus phase were different in oocytes from mono-ovular and poly-ovular follicles, the ability to reach the final stage of maturation at MII did not differ. In addition, no differences were found in the final meiotic maturation stage between both follicular types during the diestrus period. In dogs, the estrus and diestrus phases are preceded by a surge in luteinizing hormone (LH) which initiates many biochemical processes in the ovary, promoting an acute increase in progesterone levels . The proDuring diestrus, the rate of meiotic development decreased compared to that during the estrus phase, and there were no differences in MII percentage in oocytes from either poly-ovular or mono-ovular follicles. In diestrus, the predominant structures in the canine ovaries are CLs, and according to a previous study in bovines, the presence of a CL in the ovary exerts a negative effect on oocyte maturation because the CL receives the highest rate of blood flow compared to other ovarian cells .The effect of the estrus cycle on the in vitro meiotic maturation of canine oocytes has been described in other reports, with controversial results. According to the data reported in a meta-analysis approach , more ooThis study demonstrated for the first time in canines that although oocytes from poly-ovular follicles can resume meiosis at a slower rate than those from mono-ovular follicles, they can nevertheless grow, and in vitro maturation of such oocytes is possible. Furthermore, the relationship between the maturation capacity of oocytes from poly-ovular and mono-ovular follicles depends on the ovarian cycle and follicular size. Understanding the oocyte development process in each type of follicle will collaborate for improving IVM protocols in this species, a prerequisite for further advancements in the field of reproductive biotechnology.


The implantation depth (ID) is a critical condition for optimal hemodynamic and clinical outcomes in transcatheter aortic valve replacement (TAVR). The recently recommended cusp-overlap technique (COT) offers optimized fluoroscopic projections facilitating a precise ID. This single-center observational study aimed to investigate short-term clinical performance, safety, and efficacy outcomes in patients undergoing TAVR with self-expandable prostheses and application of COT in a real-world setting.From September 2020 to April 2021, a total of 170 patients underwent TAVR with self-expandable devices and the application of COT, while 589 patients were treated from January 2016 to August 2020 with a conventional three-cusp coplanar view approach. The final ID and 30-day outcomes were compared after 1:1 propensity score matching, resulting in 150 patients in both cohorts.p = 0.007) with an improvement of ID symmetry of less than 2 mm difference below the annular plane . The rate of new permanent pacemaker implantation (PPI) following TAVR was effectively reduced . While the fluoroscopy time decreased , the dose area product increased in the COT group . Patients implanted with COT had a shorter length of in-hospital stay .The mean ID was significantly reduced in the COT cohort (−4.2 ± 2.7 vs. −4.9 ± 2.3 mm; Transcatheter aortic valve replacement using the cusp-overlap deployment technique is associated with an optimized implantation depth, leading to fewer permanent conduction disturbances. However, our in-depth analysis showed for the first time an increase of radiation dose due to extreme angulations of the gantry to obtain the cusp-overlap view. Transcatheter aortic valve replacement (TAVR) is a fast-growing section in interventional cardiology. In the last decade, TAVR has become a safe and effective alternative to surgical valve replacement (SAVR) to treat symptomatic severe aortic valve stenosis across all surgical risk categories –3. OptimIn 2020, the manufacturer of the self-expandable THV CoreValve Evolut  introduced new best practice recommendations for valve deployment, including the cusp-overlap technique (COT). This is a series of procedural steps designed to provide optimized angiographic projections for TAVR with self-expandable devices . ApplicaTherefore, this single-center observational study aimed to investigate short-term clinical performance, safety, and efficacy outcomes in patients undergoing transfemoral, self-expandable TAVR with newer-generation CoreValve Evolut THV regarding COT during valve deployment in a real-world setting.From 1530 consecutive patients who underwent transfemoral TAVR with newer-generation self-expandable CoreValve Evolut system  from January 2016 to April 2021 at the Heart Center Düsseldorf, 759 patients with completed datasets were included in the analysis . Most ofn = 170; 22.4%). The control group (Non-COT) treated with former manufacturer’s recommendations without COT but with a conventional three-cusp coplanar view and a target ID of 3–5 mm from January 2016 to August 2020  was analyzed retrospectively. To erase potential confounders of the treatment outcome relationship, we performed a 1:1 propensity-score matched analysis resulting in a final study cohort of 150 COT and 150 Non-COT patients .Multislice computed tomography was routinely performed as native and contrast-enhanced, electrocardiogram gated images. Pre-procedural MSCT data were transferred to a dedicated workstation for 3-dimensional volume-rendered reconstruction . Annular plane projection in the three-cusp view was routinely predicted from MSCT reconstruction and optimal angulation of the cusp-overlap view was generated by overlapping the right coronary cusp (RCC) and the left coronary cusp (LCC) on the MSCT annular plane toward right anterior oblique (RAO) and caudal angulation , 11.Transcatheter aortic valve replacement procedures were conducted according to current guidelines and under local anesthesia. A total of four experienced operators were involved in this trial. In 2020, Medtronic issued new best practice advice for the CoreValve Evolut system, including cusp-overlap view with isolation of NCC for optimization of the ID during valve deployment. If extreme angulations of more than 30° RAO and/or 30°caudal were suggested for cusp-overlap view, a “near” cusp-overlap view with less extreme angulation was performed. Before final release, a three-cusp view was established to check for complete valve expansion and implantation depth in relation to the left coronary cusp.The final ID of device implantation was determined angiographically in the three-cusp view after complete clearing of parallax in both COT and Non-COT groups for better visual differentiation of ID below NCC and LCC compared to the cusp overlap view . Distanct-test or Mann–Whitney U-test depending on the variable distribution. Categorical variables are presented as frequencies and percentages and comparisons were made using chi-square or Fisher exact test. All statistical tests were 2-tailed, and a value of p < 0.05 was considered statistically significant. To account for the differences in baseline characteristics of COT and Non-COT group, we performed 1:1 propensity score matched analysis using logistic regression and the nearest neighbor method with a caliper of 0.1 because of the observational nature of this study and the comparison of two non-contemporary cohorts with different group sizes. Covariates were chosen according to baseline differences between both cohorts listed in Continuous data are described as mean + standard deviation (SD) for normal distribution and comparisons were performed using unpaired Student’s All statistical analyses were conducted using SPSS version 23.0  and figures created with GraphPad Prism version 8.4 .n = 759), patients undergoing TAVR with COT were younger , predominantly male  and had a lower incidence of coronary artery disease  as well as lower surgical risk  (p = 0.005). A total of 1:1 propensity score matching successfully eliminated any major differences between the two cohorts (In the unmatched cohorts (= 0.008) . COT pat cohorts .After pre-procedural MSCT planning to determine the optimal cusp-overlap projection angle, we achieved a projected cusp-overlap gantry view on CT reconstruction in 121 patients (80.7%) of patients and a near cusp-overlap projection in the remaining 29 cases (19.3%).p < 0.001) (p = 0.023)  (p < 0.001) . COT patients had less need of contrast agent volume   with a r= 0.023) . Patient= 0.005) . COT res< 0.001) . Additiop = 0.007) without a significant difference in the achievement of the target ID of 3 mm in the COT group and 3–5 mm in the Non-COT group  (p = 0.003) (r > 0.8) because we used a standardized measurement technique at our heart center for the analysis of ID after TAVR. Functional improvement was observed in both groups without significant differences concerning mean pressure gradients or paravalvular leakage assessed by TTE during 30-day follow-up  . In COT = 0.003) . Both thollow-up .p = 0.028) (p = 0.289).The rate of new PPI following TAVR was markedly reduced in the COT group  . The mosp = 0.027). Patients implanted with COT had a shorter length of ICU stay  and a shorter length of total in-hospital stay compared to Non-COT patients    . All prop = 0.364), but finally did after 1:1 propensity score matching . All procedural data and 30-day procedural outcome events of the unmatched study cohorts are shown in The outcome results between the unmatched cohorts are in large parts similar to those between the matched cohorts and do not differ in the main results, except for new left bundle branch block following TAVR. This parameter did not reach statistical significance in unmatched cohorts  Optimization of implantation depth by both reducing the mean ID and improving the symmetry of ID between NCC and LCC,(2) Significant reduction of permanent conduction disturbances with the need for new pacemaker implantation following TAVR,(3) Consistent quality of hemodynamic outcome regarding valve pressure gradients and paravalvular leakage, and(4) Increase of radiation dose due to extreme angulations.After pre-procedural MSCT planning to determine the optimal cusp-overlap projection angle, we achieved a projected cusp-overlap gantry view on CT reconstruction in 81.9% of patients and a near cusp-overlap projection in the remaining cases. Finally, no increase in adverse events during 30-day follow-up could be observed compared to TAVR procedures without the usage of COT and a three-cusp coplanar view during valve deployment instead.To increase the accuracy of ID with Evolut CoreValve THV, we have performed COT according to the manufacturer’s recommendations since September 2020. In the three-cusp coplanar view, the device is often poorly aligned and there is no clear view of the native annulus relative to the conduction system due to the foreshortening of the LVOT . COT vieAlthough balloon-expandable valves exert higher radial forces, rates of new PPI are higher after implantation of self-expandable THV due to differences in the design of the prosthetic frame and technique of implantation. TAVR with supra-annular, newer-generation CoreValve Evolut prostheses have been associated with the need for new PPI in 14.7 to 26.7% of patients within 30 days after the intervention .The proximity of the aortic valve and the cardiac conduction system is one of the reasons for the occurrence of new conduction disturbances following TAVR , 17. TheOur single-center experience using COT for valve deployment showed that only 8% of patients without preexisting pacemaker required new PPI compared to 16.8% in Non-COT group, and the incidence of new-onset LBBB could also be reduced, what is in accordance with former studies –9. BecauA potential limitation of the COT is the implanter’s concern that a shallow THV implantation may lead to a higher rate of valve embolism with upward displacement (“pop-outs”), potentially resulting in increased procedural complexity and patient morbidity , 24. In The reasons for the reduction in length of in-hospital stay in the COT cohort are multifactorial, but one of them is very likely the reduced post-procedural conduction disturbances and therefore the decreased need for a temporary or permanent pacemaker. The clinical impact of new PPI following TAVR has been somehow controversial, but meta-analyses have suggested an increased risk of all-cause mortality at 1 year in patients receiving a new permanent pacemaker and a higher rate of heart failure rehospitalizations . CertainSimilar to our results, there are already former studies examining the effect of COT on conduction disturbances following TAVR with observation of a significant reduction in both LBBB and PPI rate after TAVR –9. MendiAt least in our hands, the price for optimized ID is a higher rate of resheathing due to a more accurate achievement of a target ID of exactly 3 mm than before with a range between 3 and 5 mm. Thus, higher rates of repositioning did not result in less device success or increased mortality of the COT group as described before during multiple resheathing . Thus, tAlthough even the length of fluoroscopy time could be reduced, the dose area product is higher in the COT group, what can be explained by more extreme angulations during cusp-overlap view with an automatic increment of radiation energy that is exposed to the patients’ body surface by the X-ray tube assembly .A significant reduction of contrast medium was observed in the COT cohort. This is the consequence of the need for less contrast agent usage to visualize the NCC only (2–4 ml) during most valve positioning up to the point of no return. A larger amount of contrast agent is only necessary for the final assessment of valve position before deployment, when, as a second angulation, a three-cusp view is obtained for position control and ID measurement. In contrast, during valve positioning in three-cusp only view, more contrast agent and longer fluoroscopy time were administered to delineate all three cusps.Several limitations should be considered when interpreting this study. This is a single-center observational study with a limited number of patients. Two non-contemporary groups were compared to assess differences between COT and 3-cusps coplanar view only technique with four different operators performing TAVR procedure with different levels of practical knowledge and experience. There may exist temporal, confounders and selection bias due to high rate of excluded patients with incomplete dataset that was not accounted for in our analysis. Therefore, larger multicenter randomized trials should be conducted to verify the usefulness of COT during THV deployment.Transcatheter aortic valve replacement with self-expandable valves using the cusp-overlap deployment technique is associated with an optimized implantation depth, leading to fewer permanent conduction disturbances and a shortened length of in-hospital stay. Any prevention of conduction disturbances can potentially reduce rehospitalizations and late mortality rate, whereas the procedural and functional outcome is not remarkably influenced by COT. Thus, COT should be regularly performed for TAVR with a self-expandable prosthesis to achieve optimized ID, especially as TAVR is expanded to the low-risk population with higher life expectancy.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 the Institutional Ethics Committee of the Heinrich-Heine University of Düsseldorf (4080). The patients/participants provided their written informed consent to participate in this study.OM: conception and design, investigation, analysis and interpretation of data, drafting of the manuscript, project administration, and final approval of the manuscript. KP, SB, NB, SA, AP, RW, PH, and CJ: analysis and interpretation of data, revision of the manuscript for important intellectual content, and final approval of the manuscript. KK: analysis and interpretation of data, data curation, revision of the manuscript for important intellectual content, and final approval of the manuscript. MK: conception and design, validation, drafting of the manuscript, and final approval of the manuscript. VV and TZ: conception and design, investigation, drafting of the manuscript, project administration, and final approval of the manuscript. All authors contributed to the article and approved the submitted version.