This study aimed to assess the associations of caesarean delivery (CD) with risk of wheezing diseases and changes of immune cells in children.The cross-sectional study was conducted between May, 2020 and April, 2021.via face-to-face inquiry and physical examination by clinicians.The study was conducted in Shanghai Children’s Medical Center, Shanghai, China. A total of 2079 children with a mean age of 36.97 ± 40.27 months and their guardians were included in the present study Logistic regression was applied to estimate odds ratio (ORs) and 95% confidence intervals (CIs) for the association between CD and first episode of wheezing (FEW) or asthma. Models were adjusted for premature or full-term delivery, exclusive breastfeeding (at least 4 months) or not.Among the 2079 children, 987 children (47.47%) were born by CD and 1092 (52.53%) by vaginal delivery (VD). Children delivered by caesarean had significantly lower gestational age (P<0.01) compared with those who delivered vaginally. Our results also showed that CD was related to increased risk of FEW by the age of 3 and increased tendency to develop asthma by the age of 4 . The subgroup analysis revealed that the negative effects of CD on asthma were more obvious in children without exclusive breastfeeding  or without postnatal smoking exposure . Furthermore, compared with children born through VD, a significant change of the T cells (increased proportion of CD4+ T cells and decreased number and proportion of CD8+ T cells) were observed before the age of one in the CD group. However, the changes were insignificant in children over 1 year old.This study showed age-dependent associations of CD with asthma and FEW in offspring. Moreover, CD appeared to have an effect on the cellular immunity in infants, the disorder of which may contribute to the development of asthma in children. In recent decades, the prevalence of caesarean delivery (CD) has risen dramatically in many middle-income and high-income countries. The occurrence of CD has been estimated to be 41.6% in China . EvidencThe incidence of the wheezing diseases in children has risen dramatically over past decades . ChildreConsidering factors above, the aim of this study was to assess the age-dependent associations of caesarean delivery with risk of wheezing diseases and changes of immune cells after birth.The cross-sectional study was conducted in Shanghai Children’s Medical Center, a 1000-bed tertiary teaching hospital in Shanghai, China. Patients who were hospitalized in the department of respiratory medicine were enrolled between May, 2020 and April, 2021. The study was approved by the ethics committee of Shanghai Children’s Medical Center and conducted according to Declaration of Helsinki guidelines.After being admitted to the department of respiratory medicine, all patients were inquired with detailed information of wheezing diseases, birth status, family history and recorded by clinicians. Flow cytometry of immunocyte was conducted on all the patients. The exclusion criteria were any of the following: 1) the guardians refused to sign the obtaining informed consent, 2) information was seriously absent (the results of wheeze and asthma).Definition), current status , previous or current other allergic diseases, test of immune cells.The collected data included baseline , birth status , environmental exposure , family history , previous wheezing diseases  and positive pulmonary function  increased ≥ 12%) were diagnosed as having asthma.https://www.bdbiosciences.com/zh-cn) was used for flow cytometry (FC) of TBNK . It contains FITC-labeled CD3, clone SK7; PE-labeled CD16, clone B73.1, and CD56, clone NCAM16.2; PerCP-Cy™5.5†–labeled CD45, clone 2D1 (HLe-1); PE-Cy™7–labeled CD4, clone SK3; APC-labeled CD19, clone SJ25C1; and APC-Cy7‡–labeled CD8, clone SK1. For conducting FC of regulatory T cells(Treg), activated T cells, and activated monocytes of peripheral blood, the following antibodies were used: FITC-labeled CD25, PerCP-labeled CD3, APC-labeled CD4, APC-H7-labeled CD4, V450-labeled HLA-DR, V500-labeled CD44 , and PE-labeled CD127 (Beekman Coulter).We collected 2 milliliter(mL) peripheral blood from children and placed the samples in EDTA-coated anticoagulant tube. BD Multitest™ 6-color TBNK reagent . Firstly, we added 20 microliters (ul) of reagent to the BD Trucount™ tubes. Secondly, 50ul blood was added into the reagent. After mixing at low speed, cells were incubated in dark for 15 minutes. Then, 450ul 1xBD FACS lysing solution were added into the system, and mixed at low speed. Incubation in dark for another 15 minutes was then required. Finally, FC was performed on a BD FACSCanto II cytometer. The procedure of FC for Treg, activated T cells, and activated monocytes of peripheral blood: Firstly, the certain amounts of antibodies (according to their instruction) were mixed up with 100ul peripheral blood, then incubated in dark for 15 minutes at room temperature (20–25°C). Secondly, 1ml FAC™ lysing solution (diluted 10 times with deionized water) was added into the system, and incubated in dark for 15 minutes at room temperature (20–25°C). Supernatant was discarded after centrifugation at 1500rpm for 5min. After adding 2ml PBS into the sediment and mixing well, the sample was centrifuged at 1500rpm for 5min and was resuspended by the PBS. Finally, FC was performed on a BD FACSCanto II cytometer.The absolute number (cells/µL) of positive cells in the sample can be determined by comparing cellular events to bead events. Absolute counts of cells are calculated by BD FACSCanto clinical software using the following formula: cell population absolute count test volume = (events in cell population/events in absolute count bead region)*(beads of each test/test volume).Two thousand and seventy-nine patients were divided into two groups according to the delivery mode. We classify all the population by age in the following analysis. Most of the children were preschoolers . Categorical variables were analyzed by the χ2 test or the Fisher’s exact test, as appropriate. Logistic regression was applied to estimate odds ratio (ORs) and 95% confidence intervals (CIs) for the association between CD and FEW or asthma. The effects of CD on the development of FEW and asthma was assessed by Kaplan–Meier curves and quantified by means of Cox proportional hazards regression . Models were adjusted for premature or full-term delivery and breastfeeding status. Other variables were evaluated but not included in the final model because they were not significant (P>0.1). Prespecified subgroup analyses were conducted to assess whether the observed association of CD and FEW or asthma was affected by sex (girl or boy), father’s allergic diseases (yes or no), mother’s allergic diseases (yes or no), exposure to smoke (yes or no), breastfeeding (exclusive breastfeeding more than 4 months or not), and full-term birth (premature or full-term birth). A Pvia CD and 1092 children (52.53%) were delivered via VD. The descriptive statistics of included children were presented in We invited the 2079 guardians and their children to participate in the present study. The acquisition method was face-to-face inquiry and conduction of physical examination by clinicians, and the response rate was 100%. The mean age of the children included in our study was 36.97 ± 40.27 months old. Among the 2079 children, 987 children (47.47%) were delivered All data of FEW were valid. Children were divided into 6 groups by age . Each group had 1373, 1035, 800, 540, 374, 302 children respectively. The risk of FEW in the first 1, 2, 3, 4, 5, and 6 years after birth were evaluated. Univariate and multivariate logistic regression analyses suggested that CD increased the risk of FEW in the first 3 years  Figure 1interaction > 0.05).Subgroup analyses were conducted to explore the potential influencing factors for the effects of delivery mode in the first 3 years. The detailed information of the subgroup analyses was shown in First of all, twelve children without clear information for asthma were omitted. And children were divided into 4 groups by age . Each group had 797, 537, 371, 299 children respectively. The risk of asthma in the first 3, 4, 5, and 6 years after birth was evaluated. Univariate and multivariate logistic regression analyses between asthma and delivery mode suggested that CD increased the risk of asthma in the first 4 years  Figure 1interaction> 0.05). The detailed information of the subgroup analyses was shown in Subgroup analyses were conducted to explore the potential influencing factors for the effects of delivery mode on the risk of asthma in the first 4 years. CD was found to be associated with higher risks of asthma in boys , in children who had a mother without allergic diseases , in children without smoke exposure , in children without exclusive breastfeeding , and in full-term children . However, there was no significant interaction between CD and above factors for asthma  during this hospitalization. Children were divided into 6 groups by age . Each group had 184, 280, 351, 424, 467, 481 children respectively. About 1697 guardians approved to conduct the flow cytometry (FC) for immune cells, however, a few people refused to complete the FC. The results of cytology were shown in In this observational study, we revealed significant age-dependent associations of CD with the risk of asthma and FEW. Subgroups analyses further showed that these associations might be different in different age group. No significant age-dependent association was observed for LOS for wheezing diseases. Significant difference of CD4+ and CD8+ T cells in early life (before the age of 1) was found between CD and VD groups.Several studies had examined the association of CD with asthma , 14, 15,Interestingly, the results of subgroup analysis showed that CD was associated with higher risks of asthma in some specific population, such as boys, children without smoke exposure, and children without exclusive breastfeeding. However, the P for interaction was insignificant among them, which indicated that these factors might not truly modify the effects of CD on asthma or FEW. However, some P values were close to 0.05, suggesting that we need to further find evidence from more population.Our results showed that CD may affect the cellular immunity in infants and the profiles of the T cells. Increased proportion of CD4+ T cells and decreased number and proportion of CD8+ T cells were observed in CD group. The T cells in those less than one is not in the same children that then go on to develop wheeze later. One previous study showed a similar results that pre-labor cesarean section might contribute to the increasing ratio of CD4+ T cells . One preThis study had a number of strengths. This is the first study to explore the age-dependent associations of CD with risk of asthma and FEW. Furthermore, our study found that the changes of CD4+ T cells and CD8+ T cells in the early life of offsprings may be the potential mechanism for the age-dependent association of CD with asthma.Our study has several limitations. Firstly. We divided all of the participants by age, however, we did not observe the same group of people at different age, which may lead to bias. Secondly, since this is a retrospective cohort study, recall bias might occur when the medical history was collected from the guardians. However, this bias is nondifferential, because it was not expected to be associated with future asthma. Furthermore, though we adjusted for several confounders, our results may still be subject to residual confounding due to unrecognized or unmeasured confounders.The results of our study suggested that CD is associated with increased risks of FEW only in the first 3 years and asthma in the first 4 years. However, these findings need to be confirmed in other populations and settings. The significant differences of CD4+ and CD8+ T cells in infants were found between CD and VD groups, which suggested future studies could focus on the role of CD4+ or CD8+ T cells in CD associated wheeze or asthma in early life.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The study design was approved by the ethics committee of Shanghai Children’s Medical Center, Shanghai Jiaotong University School of Medicine and conducted according to the Declaration of Helsinki guidelines (SCMCIRB-W2021055). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.JL, SY and BD conceptualized and designed the study, supervised data collection, carried out the initial analyses, drafted the initial manuscript. JZ, LZ and JW designed the data collection instruments and collected data. JC, MT, BZ, HW, YD and XQ coordinated and supervised data collection, assisted in the statistical analysis and carried out the initial analyses. SL and YH coordinated and supervised data collection, and critically reviewed the manuscript for important intellectual content. YY, LBZ and LX conceptualized and designed the study, supervised data collection, reviewed and revised the manuscript. All authors read and approved the final manuscript.This work was supported by the Science and Technology Innovation-Biomedical Supporting Program of Shanghai Science and Technology Committee [No.19441904400]; the Science and Technology Innovation-Biomedical Supporting Program of Shanghai Science and Technology Committee [No.19441909000]; Program for artificial intelligence innovation and development of Shanghai Municipal Commission of Economy and Informatization [No.2020-RGZN-02048].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.


An international online patient community, Carenity, conducted a patient study in two independent waves among adults affected by non-communicable diseases (NCDs) in Europe and in the United States of America (USA). The study aimed to assess the real time impact of the coronavirus disease 2019 (COVID-19) on the medical conditions of patients with NCDs, their access to health care, and their adaptation to daily life as well as to describe their sources of information on COVID-19 and their needs for specific information and support. During the pandemic, 50% of the patients reported a worsening of their medical condition, and 17% developed a new disease. Additionally, 26% of the respondents reported an impact of the pandemic on regular/long-term treatment intake. 54% of the patients felt very or completely socially isolated and reported a strong impact of the COVID-19 pandemic on their stress level and state of mind, with higher levels observed in the USA compared to Europe. 59% of the respondents wished to have received additional information regarding the risks associated to their medical condition during the pandemic. Television was the most used source of information, whereas physicians were the most trusted one. This study describes the substantial impact of the COVID-19 pandemic on NCD patients. Non-communicable diseases (NCDs), also known as chronic diseases, result from genetic, physiological, environmental, and behavioral factors. These long-term and sometimes life-long pathologies include cardiovascular diseases, cancers, chronic respiratory diseases, diabetes as well as mental health and neurological disorders . Since 2In December 2019, a novel coronavirus infection caused by SARS-CoV-2 first emerged in China; following its worldwide spread, the World Health Organization (WHO) declared the coronavirus disease 2019 (COVID-19) as a pandemic on 11 March 2020. Between March and May to June 2020, governments of most countries worldwide declared health emergency states and imposed confinement restrictions (lockdowns) to tackle the pandemic and avoid an overflow of intensive care units in hospitals. With the rapid spread of COVID-19 across the world, broad and sudden disruptions to health services occurred, mostly due to restrictions in public transport hindering access to health care facilities for patients, insufficient Personal Protective Equipment (PPE), insufficient clinical staff because of redeployment to COVID-19 units as well as the unavailability of essential medicines and services . Those dThe objective of this study, performed in two independent waves, was to understand how patients with NCDs—a population that is particularly vulnerable in the context of the pandemic and lockdown—have been affected during the COVID-19 outbreak in Europe and in the USA. To do so, this study aims to evaluate: (1) the impact of COVID-19 and lockdowns on different aspects of NCD patients’ lives , and (2) patients’ expectations towards information on COVID-19 .Carenity, an international online patient community, conducted a study based on an online survey among adult patients affected by NCDs. The study aimed to understand the impact of the COVID-19 pandemic on patients with NCDs in terms of the evolution of their medical condition and access to physicians and treatments, and to describe their sources of information regarding COVID-19 and their needs for specific information and support. This study was conducted in two waves: a first wave following the first strict lockdown and a second one some months later, while most countries were still under sanitary restrictions due to COVID-19 infection (strict or soft lockdown depending on the ongoing stage of the pandemic).The study population included registered Carenity members. Carenity is a leading digital patient platform with 400,000 patients and caregivers worldwide (88% patients/12% caregivers). Through Carenity communities, members can share experiences and find health-related information on their condition as well as contribute to medical research by participating in online studies to highlight their patient experience.Eligible patients were defined as adult patients (18 years old or older) living in Belgium, France, Germany, Italy, Spain, the UK, or the USA, having at least self-reported a diagnosed NCD and who were members of the Carenity community. The NCDs included metabolic diseases, chronic respiratory diseases, mental health diseases, cardiovascular diseases, and cancers. Because of the small sample size of the respondents in Belgium  and theiPatients meeting the inclusion criteria completed an initial survey (wave 1) and/or a second survey (wave 2) to self-report the impact of the COVID-19 pandemic and lockdown on their care pathway. The two survey periods were enrolled independently, and patients who participated in wave 1 could self-select, but were not required, to complete the wave 2 survey. The study population was defined by all patients who completed the questionnaire (wave 1 and/or wave 2).This study was conducted in accordance with the Declaration of Helsinki and following the principles of good clinical practice. Prior to data collection, all patients gave their informed online consent , which allowed for data collection and publication. The participants’ privacy and confidentiality was guaranteed following the USA health care personal data protection law  as well as European laws and regulations . Ethical review and approval were waived for this study, as the aim of the study is to determine the patients’ insights from their experiences, and informed consent was given by all patients prior to answering the survey.Carenity has setup a barometer (the 2 independent waves) to measure the impact of COVID-19 on access to health care and quality of life for chronic patients in real time. Members of the Carenity NCDs communities were asked to complete the same online survey at two distinct periods. In the countries involved in this study, a strict lockdown was in place for several weeks between March and June 2021, depending on the country; from 22 June 2020 to 14 August, patients reported experience during this first lockdown in Europe and in the USA (data from wave 1). The data regarding the impact of the second lockdown were collected in real time (prospectively) from 2 November 2020 to 29 December 2020 (data from wave 2). During the second survey sample (wave 2), not all of the countries included in this study had a further strict lockdown at that point in time; however, all of the maintained strict sanitary restrictions concerning displacements and physical and social distancing were still in practice in those countries. During the second wave, many EU countries were considering a stricter lockdown, which began to be applied during the yearend holidays. The collected data of both waves were analyzed in two separated cohorts; patients could participate in both waves, but this was not mandatory. For comparison purposes, the same survey including 39 closed-ended questions was used for both waves. The questionnaire was validated to ensure compliance for each country involved as well as by native speakers, as it was programmed in local languages for the different Carenity worldwide platforms. The collected data included self-reported sociodemographic and medical profiles as well as information about the patients’ health-related quality of life (HR-QoL) during the COVID-19 pandemic and lockdown and their needs towards the pandemic. The following notions were especially evaluated: disability  , social n (%). Multivariate analysis included subgroup analyses by country. A multiple correspondence analysis was also performed using the statistical software R (version 3.5.2) for the clustering analysis, and internal validation measures were used to assess the quality of the clustering. The resulting clusters were thus analyzed through a hierarchical cluster analysis. The p-values were calculated with a confidence interval of 95%. A p value < 0.05 was considered statistically significant.Descriptive univariate and multivariate analyses were performed using the statistical software R ). For quantitative data, the following indicators were calculated: mean, range , frequency—Overall, 22,372 members of the Carenity NCDs communities were invited to participate in the study after the first lockdown (wave 1) from 22 June 2020 to 14 August 2020 and 23,412 after the second lockdown (wave 2) from 2 November 2020 to 29 December 2020 . Among tAs few differences were observed between results from wave 1 and wave 2, the results of wave 1 are presented in the article, and only the differences found between both waves are highlighted for clarity.n = 1602/2489, 64%) and the mean age was 53 years , with 66% (n = 1649/2489) of the patients being under 60 years old. During the lockdown, 80% (n = 1957/2489) of respondents were living in Europe (25% (n = 615/2489) in France or Belgium, 18% (n = 458/2489) in the UK, 14% (n = 354/2489) in Spain, 13% (n = 313/2489) in Italy, 10% (n = 247/2489) in Germany, and 20% (n = 502/2489) in the USA. Overall, respondents were predominantly living in small cities , followed by mid-sized cities  or metropolitan cities . During the pandemic, most participants  were residing in a house with a yard while 33% (n = 814/2489) lived in an apartment, 5% (n = 125/2489) in a house without a yard, and 3% (n = 63/2489) in a studio apartment. Including children, most households housed two persons  during the pandemic and 24% (n = 598/2489) of households included more than three persons. Sociodemographic profiles from both surveys showed a very good degree of consistency between the NCD population in both waves.In wave 1, a total of 2489 patients with NCDs living in six different countries answered the survey for the Carenity community. n = 1042/2489) answered metabolic diseases, 37% (n = 930/2489) respiratory diseases, 24% (n = 603/2489) cardiovascular diseases, and 23% (n = 577/2489) mental disorders. Among the most frequent conditions, patients mentioned type 2 diabetes , arterial hypertension , asthma , COPD , or depression . Compared to wave 1, a decrease in the number of cited respiratory diseases  and an increased in the number of respondents with mental disorders  was observed during wave 2. The noted increase in the proportion of mental disorders between the first and second lockdowns (from 23% to 30% of respondents) showed great disparity among the countries: an increase of 21% took place between the first and second lockdowns in the UK , 11% in France/Belgium , and 9% in Germany , whereas mostly no change was observed in the USA  or in Spain .n = 1132/2489) of respondents considered that their chronic disease was disabling , with a mean disability level of 5.5 . The second wave did not show any relevant difference compared to the first wave concerning this parameter.Overall, 45% (n = 2242/2489) of the respondents were currently taking regular/long-term treatments for their primary chronic illness.In total, 90% (n = 1033/2489) of participants declared having experienced depression symptoms and 46% (n = 1138/2489) experienced anxiety. The proportion of respondents with depression symptoms increased slightly before the second lockdown ; the proportion of patients with anxiety was stable .Before the first lockdown caused by the COVID-19 pandemic, 41% (n = 612/2489) respondents had a COVID-19 test. Of those tested, 4% (n = 109/2489) had a positive result. Not surprisingly, those numbers increased during the second lockdown: among the 35% (n = 825/2342) of tested patients, 8% (n = 176/2342) revealed to be positive for COVID-19.During the first lockdown, 25% of respondents  in the UK, metabolic diseases were the most cited answer in France/Belgium , in the USA , in Germany , and in Spain   .n = 1242/2489, 50%) experienced a worsening of their medical condition during the lockdowns, the main reasons reported being a reduction or stoppage of physical activities  followed by the normal evolution of their chronic illness  and the fact that they did not consult their doctor . Moreover, 17% of respondents (n = 412/2489) developed a new disease during the first lockdown. Among those participants, 83% (n = 340/412) contacted an HCP about it: their general practitioner (for 51% of patients (n = 210/412)), their specialist  , or a healthcare professional in a hospital emergency center . Compared to the first lockdown, more specialists  and pharmacists  were contacted by the respondents because of a new condition that had developed during the second lockdown.One out of two respondents (n = 2296/2489) of participants implemented solutions to take care of their medical condition during the first lockdown. Most of them  followed recommendations from health authorities, while 31% (n = 764/2489) of respondents scheduled a teleconsultation to avoid having to go to the hospital or the doctor’s office, and 16% (n = 408/2489) used an app on their smartphone, an alarm, or a pill organizer to remind them to take their medications. The percentage of patients using teleconsultation decreased to 23% (n = 536/2342) during the second wave. Additionally, 10% (n = 240/2489) of participants mentioned that an informal  carer had helped them. Between the first and the second lockdowns, a reduction in the compliance of the health authority recommendations was observed .In total, 92% (n = 1601/2489) of respondents adopted a negative behavior during the lockdowns: 39% (n = 967/2489) of them reduced or stopped physical activities, 22% increased or started unhealthy nutrition (n = 539/2489), 16% increased or started taking medicine to sleep at night because of insomnia (n = 403/2489), 15% increased or started taking medicines for anxiety or depression (n = 367/2489) while some patients started or increased smoking  or alcohol . On the contrary, half of respondents (n = 1231/2489) adopted at least one positive behavior during the lockdowns: 21% (n = 513/2489) practiced breathing exercises, 20% (n = 492/2489) increased or started healthy nutrition, 17% (n = 424/2489) increased or started physical activity inside or outside, and 12% (n = 299/2489) practiced yoga or meditation (n = 891/2480) adopted simultaneously negative and positive behaviors, for instance, the 5% (n = 47/891) of participants who increased/started healthy nutrition and reduced/stopped sports and physical activities during the same period.As shown in ditation b. In totn = 1330/2489) of respondents mentioned that they felt very or completely socially isolated during the lockdown  .During the first lockdown, 54% (olated”) a; the men = 1346/2489) of respondents mentioned that both parameters had been strongly impacted by the lockdown , with a mean stress level of 6.3 out of 10.0 (range: 0–10) .When asked about their stress level and state of mind, 54% (e: 0–10) b. A trenp-value < 0.05)  compared to European countries ; similar outcomes have been observed in the impact of the pandemic on the stress level and the state of mind of patients ( < 0.05) . This di < 0.05) .p-value < 0.05). This result was observed in both waves.Among the patients who experienced depressive symptoms before the pandemic, these symptoms worsened during the lockdowns . Similarly, in respondents who previously had anxiety, this symptom increased during lockdown . Younger patients (≤40 years) tended to experience a worsening of their depressive symptoms and anxiety compared to the older ones (>50 years) (n = 475/2489) reported having developed a mental health problem during the first lockdown and 23% (n = 536/2489) during the second lockdown. Patients from the USA tended to be much more affected than patients living in France/Belgium .In addition to depression and anxiety symptoms, 19% of respondents (n = 958/2489) of respondents said they were seeing their physician less than usual, whereas 16% (n = 393/2489) reported having consulted them more than usual. Compared to the first lockdown, the proportion of patients who consulted their physician less than usual decreased from 38% (n = 958/2489) to 23% (n = 546/2342) during the second lockdown.During the first lockdown, patients with NCDs had to change the frequency of their visits to their physician: 38% (n = 749/2489) declared having had difficulties in finding an available physician during the first lockdown. Moreover, medical visits or procedures were strongly impacted, as 52% of respondents (n = 1298/2489) reported that long-planned medical consultations or surgeries had been canceled or rescheduled since the start of the pandemic. However, between both waves of the study, the proportion of cancelled or postponed medical consultations or procedures decreased from 52% (n = 1298/2489) to 32% (n = 740/2342).Overall, almost 30% of respondents (n = 572/2242) of respondents under a regular/long-term treatment reported an impact of the pandemic on their regular/long-term treatment intake: 6% (n = 136/2242) completely stopped some of their treatments and 10% temporarily stopped some of them (n = 230/2242), while 11% (n = 242/2242) took them more regularly than they usually did (n = 407/2242) reported having difficulties in finding their treatment at the pharmacy during the lockdown.During the first lockdown, 26%  of them feeling frustrated most of the time. Considering the access to specific information related to their chronic illness in the context of the pandemic, 31% of patients (n = 776/2489) did not receive any illness information, and 25% of them (n = 614/2489) reported receiving insufficient information. Of those participants, 59% (n = 822/1390) wished they had received additional information regarding the risks associated to their medical condition, while 32% (n = 445/1390) requested advice on how to deal with the end of the lockdown, and 29% (n = 408/1390) needed more information on available psychological support. During both waves, patients living in the USA tended to be more satisfied regarding the access to specific information related to their primary chronic illness compared to patients from Europe  or in the media , respondents also expressed their needs in terms of solutions and services to better cope with the COVID-19 pandemic. Patients wished to have had unrestricted access to their family or close friends during the lockdown , access to monthly consultations with their specialists , or easier access to telemedicine .Beyond the need for more reliable and complete information from health authorities (n = 1512/2489) as a source of information, it was poorly trusted . On the contrary, physicians were the most trusted source of information  but could be used more often as they were as the fifth most used source by the patients. This outcome did not significantly change between both lockdown situations.As shown on p-value < 0.05). In comparison to Cluster 3 (older patients), respondents in Clusters 1 and 2 suffered more depressive and anxiety symptoms before lockdown and had self-reported disabling medical conditions, felt isolated and stressed, had a worsening of their medical condition, had seen their physician more than usual, were very affected by the cancellation of their consultations or surgeries, and developed a new disease  more frequently) . Moreover, during the first lockdown, respondents in Cluster 2 were those who were the least satisfied with the access to specific information related to their chronic pathology (63% unsatisfaction vs. 49% in Cluster 1 and 55% in Cluster 3). During the second lockdown, no major differences were observed in these three clusters, except that patients from Cluster 2 reported mental diseases as primary chronic illness instead of respiratory diseases and cancers.To identify the groups of patients with similar patterns of responses to the questions, a clustering analysis on the categorical data was performed through a multivariate analysis. Based on the answers collected through the questionnaire, three different clusters emerged for each wave: younger highly impacted patients, middle-aged moderately impacted patients, and less impacted older patients  . A majority of the patients adopted negative behaviors such as reducing or stopping physical activities, starting or increasing unhealthy nutrition, and increasing smoking or alcohol use. Similar negative behaviors have been already reported in numerous European countries ,23,24,25Social isolation was mostly enforced through governmental policies on social distancing; in our study, social isolation affected a lot of patients (54%) living with NCDs, especially in the UK and USA, and many patients wished to have unrestricted access to their family and close friends during lockdown. It should be noted, though that if social isolation had been largely overlooked in general, specific initiatives were developed to address this issue, for instance, the UK Campaign to End Loneliness . NeverthAdditionally, the COVID-19 pandemic had a strong impact on patients’ access to HCPs and their treatments. Most medical practices as well as health care and rehabilitation services suddenly closed or scaled down non-urgent visits while hospitals were overwhelmed with severe COVID-19 cases, lacking staff, PPE, and essential medicines. Additionally, confinement restrictions strongly limited the access of NCD patients to health care and medication, which increased their uncertainty. Especially in the early days of the lockdown, this situation, which led NCD patients unattended, forced them to change their access to care. In our study, respondents reported seeing their physician less than usual, having difficulties finding an available physician, and trouble finding their treatment at the pharmacy during the first lockdown. Subsequently, patients often modified the intake frequency of their regular or long-term treatment, which might have decreased their treatment compliance and increased the risk of iatrogenesis. This was also shown in other surveys . LimitedSoon after the start of the pandemic, triaging was the most widely adopted strategy worldwide to ensure high risk NCD patients continuing access to critical monitoring services and treatments without increasing the risk of COVID-19 contagion at hospitals . For thiTo better cope with their chronic diseases during the pandemic, patients needed more specific information, especially on how COVID-19 infection might impact their current pathology in terms of the associated risks and appropriate care management during lockdown. At the beginning of the pandemic especially, patients had difficulties distinguishing reliable medical facts and misinformation among the huge amount of available information. During the pandemic, governmental agencies such as the WHO were providing relevant information on patients with NCDs and the COVID-19 pandemic ,11. In oAs rehabilitation care services as well as health and social care at home were the most commonly disrupted services worldwide , NCD patThis pandemic therefore stressed the long-needed support of informal carers/caregivers and the need for integrated care supported by the WHO and the European healthcare system ,48. PatiDifferences in the health care systems could also explain the differences observed. Health coverage in Europe is universal. Having different structures of interactions between insurers, providers, and patients, all European healthcare systems aim to provide care to everyone on the grounds of free access, equality and equity, and fairness: no matter how much patients earn, patients receive a basic package. Unlike Europe, not all American citizens have access to publicly-funded insurance; government funds are available for certain Native American tribes, military families, and veterans. There is also national health insurance, called Medicare, which covers people over 65 and some people with disability status, people with end-stage renal disease, and amyotrophic lateral sclerosis . These dThis study presents several limitations, mostly inherent to its design. The following bias should be mentioned: selection bias , self-reported perceptions and symptoms, and recall bias, especially concerning the retrospective aspect of the study (wave 1). However, as the answering of the questionnaire was done directly by the patients in absence of their physician, the desirability bias was greatly limited. Moreover, the high similarity of the results obtained in both waves validates our study concept. Finally, belonging to a patient community likely reflects the need to self-assess and compare the levels of the disease. Poorly controlled, highly symptomatic, and heavily treated patients may then be overrepresented, and our findings should be extrapolated only cautiously. However, the majority of people use the Internet. In 2019, it was estimated that 86.7 percent of people living in developed countries used the internet . MoreoveDuring the pandemic, healthcare services disruptions coupled with newly adopted negative behaviors put patients living with NCDs at high risk for the worsening of their chronic condition or developing multimorbidities. The burden of NCDs, which increased during the pandemic, has recurrent health and economic consequences for the patients’ ecosystems. Hopefully, lessons from the COVID-19 pandemic will be learned, and emergency plans will be put in place to protect the NCD vulnerable population against upcoming outbreaks. NCDs were included for the first time in the United Nations 2030 agenda for sustainable development as a health priority to reinforce WHO engagement by 2030; the aim is to reduce premature mortality from NCDs by one third through prevention and treatment as well as to promote mental health and well-being . Our stu


This paper summarizes the MRI imaging findings of primary central nervous system lymphoma (PCNSL) in the posterior cranial fossa to improve the accuracy of PCNSL diagnosis in the posterior cranial fossa.This study retrospectively analyzed the MRI imaging manifestations of 15 PCNSL posterior cranial fossa cases confirmed by puncture or surgical pathology from June 2017 to May 2018, including their occurrence sites, the number of lesions, MRI plain and enhanced manifestations, and diffusion-weighted imaging (DWI) and magnetic resonance spectroscopy. Imaging (MRS) performance.-3mm2/s. MRS examination showed obvious Lip peak in two cases.A total of 15 cases were enrolled, including 10 cases of single lesion and five cases of multiple lesions. The total number of lesions was 25, which were in the cerebellar hemisphere and cerebellar vermis, midbrain, fourth ventricle, and pontine cerebellum. The lesions were round, irregular, nodular, patchy, with low or medium signals on T1WI, equal or slightly higher signals on T2WI, and enhanced with 25 meningiomas-like gray matter signals. All of them were significantly strengthened. “Acupoint sign” and “umbilical depression sign” were seen in eight lesions. There were 17 massive and nodular enhancements, four striped enhancements, three patchy enhancements, and one circular enhancement. five cases of DWI showed homogeneous high signal, two cases showed uneven high signal, and 3 cases showed medium signal. The ADC value of tumor parenchyma in 10 patients was (0.62±0.095)×10PCNSL in posterior cranial fossa has certain characteristics. DWI, ADC value and MRS are helpful to improve the correct diagnosis rate of PCNSL. Primary central nervous system lymphoma (PCNSL) refers to non-Hodgkin’s lymphoma that only occurs in the central nervous system and no tumors are found elsewhere in the body.4Tallies entropy, also called non-extended entropy, is an extension of Shannon entropy.ip is the gray probability. Let the infrared image to be processed have N gray levels, and the histogram of the probability distribution of each gray level is represented as {1,p2,...,pNp}. Take the threshold t to divide the infrared image into two parts: target and background. The target probability is  and the background probability is . According to equation (1), the Tallies entropy of the target and background are obtained asAmong them: q is the undetermined coefficient, which describes the non-extensiveness of its entropy value; The magnitude of Tallies entropy changes according to the value of the threshold t. The total entropy value of the background and the target isq(t)S), that is,The process of finding the optimal threshold t to solve for the maximum value of Traditional one-dimensional Tallies entropy threshold segmentation is susceptible to noise interference, especially for infrared images with low signal to noise.According to the probability values of the target pixel and the background pixel, the Tallies entropy values of the target and background are obtained asIn summary, the two-dimensional Tallies entropy isThe imaging data of 15 patients with posterior cranial fossa PCNSL confirmed by pathology from June 2017 to May 2018 were collected in our hospital. There were 11 males and 4 females, aged 6 to 78 years, with an average age of (49.8±7.2) years. All patients had no history of congenital or acquired immune dysfunction, and had not been treated with hormone therapy before MRI, and secondary lymphoma was excluded by bone marrow cytology or other imaging tests (PET-CT). Fifteen patients were confirmed as PCNSL by surgery or stereotactic biopsy.US GE750 3.0T superconducting magnetic resonance scanner and head and neck combined 8-channel coil was used. The scan sequences included T1WI, T2WI, and enhanced T1WI in axial, coronal, and sagittal positions. Ten patients underwent diffusion-weighted imaging (DWI) and four underwent magnetic resonance spectroscopy (MRS). The layer thickness is 6mm, the layer spacing is 1mm, the field of view (FOV) is 230mm×230mm, and the matrix is 384×384. T2WI: TR6000ms, TE90ms; T1WI: TR200ms, TE2.5ms; FLAIR: TR3500ms, TE90ms; DWI: TR3100ms, TE80ms. The enhanced scan uses a TSE sequence, the injection contrast agent is Gd-DTPA (0.1 mmol/Kg body weight), and the injection flow rate is 2 mL/s. Scan parameters: TR200ms, TE2.5ms, layer thickness 1mm, layer spacing 0. The DWI scan uses an EPI sequence, with b values of 0 and 1000s/mm2, TR3200ms, TE80ms, and FOV 230mm×230mm. MRS examination uses multi-voxel scanning, TR1600ms, TE125ms, FOV230mm×230mm, and the acquisition time is 8min. MRS post-processing was performed on the workstation to obtain the distribution map of each metabolite.Two senior MRI physicians independently analyzed and measured the images, and observed the number, size, morphology, signal, and peritumoral edema of the lesions. When the opinions were not consistent, the two physicians discussed together. The statistical analysis software 20.0 was used to perform the Mann-Whitney U test on the ADC values of the solid part of the tumor and the contralateral brain parenchyma. P <0.05 was considered statistically significant.The manifestations included 11 cases of headache, 12 cases of dizziness, eight cases of malignant vomiting, six cases of unstable walking and ataxia. Only one case was diagnosed as lymphoma before operation, five cases were misdiagnosed as metastases, four cases were gliomas, one case was medulloblastoma, one case was inflammatory lesions, and three cases could not be characterized. Of the 15 cases, 12 were diffuse large B-cell lymphomas and three were Burkitt lymphomas.Of the 15 patients, 10 were single lesions, which occurred only under the curtain , Fig.2, The horizontal axis T1WI , T2WI F, and enhThe plain scan axis T2WI  and enhaThe first axial enhancement T1WI , Fig.2 sTen patients underwent DWI. Five patients showed homogeneous high signal, two patients showed uneven high signal, and three patients showed moderate signal. The ADC picture showed equal or low signal. The ADC value of tumor parenchyma in 10 patients was (0.62±0.095)×10-3mm2/s, while the ADC value of contralateral brain parenchyma was (0.73±0.038)×10-3mm2/s, and the difference was statistically significant. (P <0.05). Four cases underwent MRS examination, two cases showed a Lip peak at 1.33 ppm, and the other two cases did not see a Lip peak.Metastatic tumors: similarities can be single or multiple, often accompanied by significant enhancement. For enhanced MRI images, those with larger metastatic tumors often have necrosis in the center. On imaging, the tumor has a circular enhancement, which is nodular, and lymphoma is relatively rare. Glioma: Most gliomas have long T1 and long T2 signals, showing obvious infiltrative growth. Among them, oligodendroglioma may have calcification, while central nervous system lymphoma calcification is rare. The most malignant glioblastomas are more solitary, and the tumor occupying effect is more obvious, but the enhancement is more irregular, and the edema around the tumor is more severe. Meningiomas are mostly located near the skull base or convex meningeal attachment site, most of them are clear-shaped, round, with some features of adjacent axial tumors such as hyperplasia or destruction of the skull, white matter collapse signs, and rare in lymphomas. On imaging, meningiomas usually show obvious uniform enhancement, and some of them show “meningeal tail sign”. Brain abscess: Most of them have a history of fever, and the imaging is typically a ring-shaped enhancement of the abscess wall, which can be distinguished from lymphoma according to the history of infection and the age of the typical abscess formation.Due to the dense composition of tumor cells in PCNSL, less cytoplasm, high cytoplasmic ratio, and small extracellular space in the tumor, the diffusion of water molecules in PCNSL is often limited.Experiments showed that the focus of this group of cases is mainly located in the cerebellar hemisphere, and a few are in the cerebellar vermis, the four ventricles and the brain stem. It is generally believed that the incidence of rare sites of PCNSL is more common in people with immunodeficiency, such as patients with AIDS or patients who have been taking immunosuppressants for a long time after organ transplantation.In this group of patients, mass-type PCNSL showed high or homogeneous signals on DWI, while non-massive lesions were mostly moderate signals. This group of 10 patients underwent a DWI scan. The measured ADC value of the parenchymal part of the tumor was (0.62±0.095)×10-3mm2/s, which was statistically different from the ADC value of the contralateral normal brain parenchyma. The PCNSL in the posterior cranial fossa needs to be distinguished from other subcancerous tumors. This study was a retrospective analysis. The equipment used in most cases was backward, and there was a lack of comparative studies on magnetic resonance spectroscopy (MRS), perfusion imaging (PWI), and diffusion imaging (DWI), etc. These functional imaging methods play an important role in the diagnosis and differential diagnosis of PCNSL. Therefore, further analysis should be made from these aspects in future studies. Differences in MRI images of complications of PCNSL were found, but not confirmed in terms of treatment. Only after large data analysis can its clinical application value be proved, so it will be further studied from this aspect.Although PCNSL is rare in the posterior cranial fossa, it still has certain characteristics. The predominant sites are the cerebellar hemisphere and cerebellar vermiform. After enhancement, in addition to the typical “acuminate sign” and “umbilical concavity sign”, it can also show nodular, striped, and patchy enhancement. DWI, ADC value and MRS can help improve the correct diagnosis rate of PCNSL. However, there are still many clinical manifestations of PCNSL in the posterior cranial fossa. The imaging findings are atypical, and early diagnosis is extremely difficult. Ultimately, they need to be confirmed by stereotactic biopsy or surgical pathology.GL: Conceived the study, literature review, participated in its design, coordination, analyzed the data, helped to draft the manuscript and also the responsible and accountable for the accuracy or integrity of the work. YL & XL: Helped in design, data collection, article drafting & critical revision. ZZ: Takes the responsibility and is accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.


Understanding the ongoing evolution of SARS-CoV-2 is essential to control and ultimately end the pandemic. We analyzed more than 300,000 SARS-CoV-2 genomes available as of January 2021 and demonstrate adaptive evolution of the virus that affects, primarily, multiple sites in the spike and nucleocapsid protein. Selection appears to act on combinations of mutations in these and other SARS-CoV-2 genes. Evolution of the virus is accompanied by ongoing adaptive diversification within and between geographic regions. This diversification could substantially prolong the pandemic and the vaccination campaign, in which variant-specific vaccines are likely to be required. Understanding the trends in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution is paramount to control the COVID-19 pandemic. We analyzed more than 300,000 high-quality genome sequences of SARS-CoV-2 variants available as of January 2021. The results show that the ongoing evolution of SARS-CoV-2 during the pandemic is characterized primarily by purifying selection, but a small set of sites appear to evolve under positive selection. The receptor-binding domain of the spike protein and the region of the nucleocapsid protein associated with nuclear localization signals (NLS) are enriched with positively selected amino acid replacements. These replacements form a strongly connected network of apparent epistatic interactions and are signatures of major partitions in the SARS-CoV-2 phylogeny. Virus diversity within each geographic region has been steadily growing for the entirety of the pandemic, but analysis of the phylogenetic distances between pairs of regions reveals four distinct periods based on global partitioning of the tree and the emergence of key mutations. The initial period of rapid diversification into region-specific phylogenies that ended in February 2020 was followed by a major extinction event and global homogenization concomitant with the spread of D614G in the spike protein, ending in March 2020. The NLS-associated variants across multiple partitions rose to global prominence in March to July, during a period of stasis in terms of interregional diversity. Finally, beginning in July 2020, multiple mutations, some of which have since been demonstrated to enable antibody evasion, began to emerge associated with ongoing regional diversification, which might be indicative of speciation. High mutation rates of RNA viruses enable adaptation to hosts at a staggering pace –4. Never59During the current coronavirus pandemic (COVID-19), understanding the degree and dynamics of the diversification of severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) and identification of sites subject to positive selection are essential for establishing practicable, proportionate public health responses, from guidelines on isolation and quarantine to vaccination . To inveTo investigate the evolution of SARS-CoV-2, we analyzed all SARS-Cov-2 genomes, as of January 8, 2021, in the Gisaid  databaseSeveral methods for coronavirus phylogenetic tree inference have been tested , 15. TheTherefore, building on the available techniques, we utilized a “divide and conquer” approach which is not subject to these artifacts and furthermore can be employed for datasets that cannot be structured by sequencing date, including metagenomic analyses. This approach leverages two ideas. First, for any alignment, a diverse representative subset of sequences can be used to establish a deep topology, the tree “skeleton,” that corresponds to a maximum likelihood solution over the entire alignment. Second, deep branches in an unrooted tree are primarily determined by common substitutions relative to consensus. In other words, rare substitutions are unlikely to affect deep splits or branch lengths. We adopted the following steps to resolve the global phylogeny for SARS-CoV-2  with the most common substitutions relative to consensus was constructed. Sequences redundant over this narrow alignment were removed. Sets of diverse—based on the hamming distances over this reduced alignment—representative sequences were then achieved, and all subtrees generated from each diverse subset were aggregated to constrain a single, composite tree.This composite tree reflects the correct tree skeleton and could be used to constrain the global topology; however, due to numerous sequencing errors in this dataset, another intermediate step was taken. A second reduced alignment was constructed in which nearly invariant sites, which may represent sequencing errors and should not be used to infer tree topology, were omitted. As before, sequences redundant over this reduced alignment were removed, and a tree was then constructed from this alignment, constrained to maintain the topology of the composite tree wherever possible. This tree reflects the correct topology of the global tree but has incorrect branch lengths. Finally, the global tree with the correct branch lengths A was conk means, returned five categories , of which four correspond to partitions 5 and 8, and the divergent clades v1 and v2. These findings indicate that an alternative tree with a comparable likelihood but a dramatically different coarse-grain topology, most likely, cannot be constructed from this MSA.We identified eight principal partitions within this tree, in general agreement with other work –19, alon20SI Appendix, Table S1), some of which are shared by two or more partitions and appear independently many times, consistent with other reports . Examination of the relative frequencies of all 12 possible nucleotide substitutions indicated a significant genome-wide excess of C to U mutations, approximately threefold higher than any other nucleotide substitution, with the exception of G to U, as well as some region-specific trends. Specifically, G to U mutations increase steadily in frequency throughout the second half of the genome, and the distribution of nucleotide substitutions over the polyprotein is dramatically different from other open reading frames (ORFs) .Each of the eight partitions and three variant clades can be characterized by a specific amino acid replacement signature , general reports . The rec reports  are enriSI Appendix, Fig. S4) and is enriched in mutations uniformly across the genome, primarily among high-frequency sites. This pattern suggests a mechanistic bias of the errors made by the coronavirus RNA-dependent RNA polymerase (RdRP). Evidently, such a bias that increases the likelihood of observing multiple, independent substitutions in the NCN context complicates the detection of selection pressures. However, only two of the nine contexts with an excess of nonsynonymous events are NCN , suggesting that at least some of these repeated, nonsynonymous mutations are driven by other mechanisms. Thus, we excluded all synonymous substitutions and nonsynonymous substitutions with the NCN context from further consideration in the determination of candidate sites evolving under positive selection.Motivated by the observation of the mutational biases, we compared the trinucleotide contexts of synonymous and nonsynonymous substitutions as well as the contexts of low- and high-frequency substitutions. The contexts of high-frequency events, both synonymous and nonsynonymous, were found to be dramatically different from the background frequencies. The NCN context  harbors substantially more events than other contexts , we obtained both a maximum likelihood estimate of dN/dS across 10 subalignments and an approximation computed from the global ancestral reconstruction  across most of the genome , with a reasonable agreement between the two methods. This estimate is compatible with previous demonstrations of purifying selection affecting about 50% of the sites surveyed or more . When a moving average with increasing window size was computed, only a weak positive correlation was observed between the numbers of synonymous and nonsynonymous substitutions . Most sites in the virus genome are highly conserved, the sites with most substitutions tend to reside in conserved neighborhoods, and the local fraction of sites that harbor at least one mutation strongly correlates with the moving average . Together, these observations indicate that SARS-CoV-2 genomes are subject to diverse site-specific and regional selection pressures, but we did not detect regions of substantially elevated mutation or recombination, in general agreement with other studies  in the respective genomic regions. Contrary to what one would expect under the hotspot scenario, we found that codons with many synonymous substitutions tend to harbor few nonsynonymous substitutions, and vice versa . The positively selected residues form a cooccurrence network that likely reflects epistatic interactions  that do not include 614D.Spike D614G appears to boost the infectivity of the virus, possibly by increasing the binding affinity between the spike protein and the cell surface receptor of SARS-CoV-2, ACE2 . ConclusAdditionally, apart from the selective advantage of a single replacement, it should be emphasized that D614G (but not G614D) is a central hub of the epistatic network D. Concei38SI Appendix, Table S4, List 1). Most of the mutations in the VOCs were not phylogenetically informative, having emerged independently multiple times in more than one partition, and not all VOC sequences clustered within the tree. The notable exceptions are P.2, which corresponds to v1, and B.1.1.7, which corresponds to v2. Additionally, although the mutations that characterize B.1.351 do not form a clade, they are largely or exclusively represented within partition 6 and correspond to the emergent variant vAfrica defined below through trends in the metadata.In addition to 614G, 31 spike mutations, most within the RBD, are signature mutations for divergent clades v1 to v3; emergent variants vAfrica or vOceania (see below); or established variants B.1.1.7, B.1.1.7_E484K, B.1.258_delta, B.1.351, B.1.429, P.1, or P.2 42 , 6 are in the N protein .Evolution of beta coronaviruses with high case fatality rates, including SARS-CoV-2, was accompanied by accumulation of positive charges in the N protein that might enhance its transport to the nucleus . ThirteeThe replacements R(agg)203K(aaa) and G(gga)204R(cga) occur via three adjacent nucleotide substitutions. R(agg)203K(aaa) resolves to two independent mutations in the ancestral reconstruction: first, R(agg)203K(aag), then K(aag)203K(aaa). Furthermore, the rapid rise of 220V  in a European cohort during the summer of 2020 might be related to a transmission advantage of the variant carrying this substitution . These sSI Appendix, Fig. S10).In addition to the many mutations of interest in the N and S proteins, Orf3a|Q57H is a signature mutation for partitions 6, 7, and v1. Q57H is the fourth most common positively selected mutation. Although not considered a candidate for positive selection in our analysis due to its NCN context, ORF8 S84L is a hub in the larger epistatic network including all strongly associated residues  likely underrepresent the extent of epistatic interactions occurring during SARS-CoV-2 evolution. The early evolutionary events that shaped the epistatic network likely laid the foundation for the diversification of the virus relevant to virulence, immune evasion, and transmission. As discussed above, these early mutations (including S|G614D) might provide only a modest selective advantage in isolation but exert a much greater effect through multiple epistatic interactions.Epistasis in RNA virus evolution, as demonstrated for influenza, can constrain the evolutionary landscape and promote compensatory variation in coupled sites, providing an adaptive advantage which would otherwise impose a prohibitive fitness cost –68. BecaSI Appendix, Table S4, List 2), and this pair is in the top 25% of cooccurring pairs in our network, ranked by lowest probability of random cooccurrence.The epistatic network will continue to evolve through the entirety of the pandemic, and, indeed, all emerging variants at the time of this writing are defined not by a single mutation but by an ensemble of signature mutations. Moreover, in addition to the apparent widespread intraprotein epistasis, there seem to exist multiple epistatic interactions between the N and S proteins. In particular, S|N501Y and N|S235F are both signature mutations for variants B.1.1.7 and B.1.1.7_E484K . The growing ensemble of signature mutations that appear to be subject to positive selection, and the existence of a robust network of putative epistatic interactions including these signatures, suggest that ongoing virus diversification is driven by host adaptation rather than occurring simply by neutral drift.As with early founder mutations, when a new variant emerges with multiple signature mutations, it is unclear which, if any, confer a fitness advantage. Although it is natural to focus on substitutions within the RBD, we emphasize that all emergent variants contain substitutions in in the vicinity of known NLS motifs. In fact, the most statistically significant signature mutation (based on the Kullback−Leibler divergence) for vAfrica  is N|T205I. As we suggest for S|D614G, these variant signature mutations are likely to exert a greater influence through multiple epistatic interactions than in isolation, and each signature mutation can be a member of multiple epistatic ensembles beyond the group of signature mutations within which it was originally identified. Indeed, signature mutations are shared among defined variants, and we find evidence for an additional 18 putative epistatic interactions between variant signature mutations and other events throughout the tree which are not identified as signature mutations for any defined variant , indicating a sufficiently low level of noise in the data for subsequent analyses. Examination of the global distribution of each of the major SARS-CoV-2 partitions  indicates dramatic regional differences and distinct temporal dynamics  within that region. This unabated intraregional diversification is further evidence of a large repertoire of host-adaptive mutations of SARS-CoV-2 evolving within the human population.We first demonstrated a strong correlation between the sequencing date of SARS-CoV-2 genomes and the distance to the tree root . The second one is a partition-level measure. For every pair of regions over each time window, we computed the Hellinger distance of the 11-group frequency distribution between all pairs of regions over each time window , and calculated the ratio of interregional and intraregional values . However, this did not result in a global selective sweep that would involve the extinction of partitions 1 to 7. Instead, multiple NLS-associated mutations rose to prominence across different partitions, becoming globally dominant by September . To resolve this trend, at least two principal variants, N|203K/204R in partition 8 and N|220V in partition 5, have to be considered, and we identified six key amino acid replacements of interest for this period .The extinction of the earliest partitions, 1 and 2, corresponds to the advent of S|D614G, which became fixed in all other partitions and was globally ubiquitous by June 2020 D. PartitSI Appendix, Fig. S19), signatures associated with the rise of partition 6 were identified in both regions . Neither of these two groups of sequences  form topologically distinct clades; however, due to the conserved mutational signatures, we considered both groups to represent distinct emerging variants, vOceania and vAfrica. The signature for vAfrica is consistent with variant B.1.351. Additionally, two divergent clades within partition 8 and one clade within partition 3 emerged.In the next phase of the pandemic, partition 8 dramatically fell from dominance in two regions, Africa and Oceania, replaced by partitions 6 and 7. Although we did not find a distinct mutational signature associated with the rise of partition 7 in Oceania  requires the consideration of at least three variants and includes 59 signature mutations. Clade v1 appeared first in Europe in April 2020; v2 also appeared in Europe, in September 2020; and v3 appeared in Asia and North America, in April 2020 . As discussed above, many VOC mutations appeared multiple times in more than one partition, and few of these mutations are phylogenetically informative. However, some key mutations show clear-cut epidemiological trends, which can be regionally defined. The region where a key mutation first emerged is not always the region where it first rises to prominence.The most prominent is clade v2 with a signature consistent with variant B.1.1.7. Altogether, resolving this trend of emerging substitutions in the RBD  began to rise to prominence in Oceania in November. S|484K first rose to prominence in South America in September 2020, while appearing in sequences from Europe and North America as early as July. Also, notably, although S|477N initially appeared in February/March 2020 in Europe, Oceania, and North America, it dramatically rose to prominence in Oceania in April, about 3 mo before this mutation became prominent elsewhere. S|477N is a signature mutation for v1 stemming from partition 3; however, the sequences from Oceania bearing this mutation from summer 2020 are in partition 8. The dramatic diversity of signature mutations among these variants decreases the likelihood of future selective sweeps  and increases the likelihood of repeat epidemics.SI Appendix, Figs. S2 and S3). Using our SARS-CoV-2 MSA, we surveyed 10 regions from ORF1ab , with one region in N demonstrating variability in nearly one-third of all isolates. It can be expected that most targets within the polyprotein will remain subject to the fewest polymorphism-induced false negatives even as the virus continues to diversify.The ongoing diversification of SARS-CoV2 poses problems for both testing and vaccination. Substitutions in the E protein have already been demonstrated to interfere with a common PCR assay . Generalm ORF1ab , N(4), am ORF1ab  for subsOf the nine primary vaccines/candidates , three aVirus evolution during a pandemic is a fast-moving target, so that, unavoidably, aspects of this analysis will be outdated by the time of publication. Nevertheless, several trends revealed here appear general and robust. Although it is difficult to ascertain positive selection for individual sites, the overall adaptive character of SARS-CoV-2 evolution involving multiple amino acid replacements appears to be beyond reasonable doubt. As expected, there are multiple positively selected sites in the S protein, but, more surprisingly, N protein includes several sites that appear to be strongly selected as well. The involvement of these adaptive substitutions in the nuclear localization of the N protein appears likely. Importantly, some of the mutations, for which positive selection was inferred, cooccur on multiple occasions and seem to form a robust epistatic network. Most likely, the effect of positive selection is manifested primarily at the level of epistatic interactions.Clearly, despite the dramatic reduction in global travel , the evoSI Appendix for detailed methods. Genomes available as of January 8, 2021 were retrieved from the Gisaid . To assess the contribution of trinucleotide mutagenic contexts, mutations were divided into four classes: synonymous vs. nonsynonymous substitutions and substitutions with high vs. low frequency of independent occurrence . This reconstruction was used to estimate dN/dS for all regions encoding mature proteins, in addition to Phylogenetic Analysis by Maximum Likelihood (PAML)  .https://simplemaps.com/data/world-cities) .Metadata were assigned according to Gisaid, with location abbreviations matched to latitude/longitude of a representative city for each location using simplemaps (-cities) . Date anSupplementary File


The identification of the VOs was achieved bycalculating the matrix similarity at different confidence levels.The strategy demonstrated the capacity to efficiently recognize thepresence of adulteration and the type of VO adulterant in mixtures.Thus, the method was applied to 20 commercial EOs, and VOs were detectedand then identified in four samples.Essential oils (EOs)are valuable products commonly employed inthe food industry and intensively studied as biopreservatives forthe extension of food shelf-life. Unfortunately, EOs might be counterfeitto increase industrial profits. Among the possible adulterants, vegetableoils (VOs) must be considered for their characteristics and low costs.We aimed to apply nuclear magnetic resonance (NMR) spectroscopy forthe detection and identification of VOs in mixtures with EOs. Thisinnovative strategy is based on comparing the peak area ratio matricesof characteristic VO  The market of EOs is expected to grow inthe near future due to the increased consumer interest in “green”and natural products. Indeed, in the last decades, more and more consumersperceive organic foods as safer than conventional foods that may containadded chemical preservatives or pesticide residues.5 As a consequence, the consumption of organic foods hasincreased significantly. On the other hand, not adding food preservativesmay result in a shorter food shelf-life due to microbial growth. Conventionalmethods of microorganism growth inhibition, such as thermal processing,cannot be applied to certain foods due to the loss of essential nutrientsand sensorial properties. For these reasons, in the food industry,the application of EOs as natural inhibitors or biopreservatives withantioxidant activity against food-borne pathogens has been extensivelystudied.9 Moreover, EOs have exhibited pronounced effectiveness to kill pathogensand pests. The high content of lipophilic aromatic hydrocarbons providesinsecticidal, ovicidal, fungicidal, and nematocidal effects to theEOs. For this reason, EOs display marked and promising features fortheir employment as biopesticides in agriculture.10Throughouthistory, essential oils (EOs) have received great interestfor their curative effects, and since ancient times, humans have beenextracting them from aromatic plants. EOs have been employed for differentpurposes due to their potential in the pharmaceutical, food, cosmetic,and perfume industries. As a matter of fact, EOs exhibit several beneficialproperties such as antioxidant, anti-inflammatory, antibacterial,and antiviral activities.Generally, the employed EOs in most industrial fieldsare extractedfrom aromatic plants by steam distillation or cold squeezing, dependingon the type of plant material. Overall, the extraction yields of EOsare rather low, resulting in high production costs. Thus, the economicpotential and high market demand of EOs have led to a surge of adulterationpractices of these valuable products. Besides EO devaluation due tothis illicit practice, counterfeiting these products is potentiallydangerous for the consumers. Indeed, EO activity and efficacy arereduced leading to an unsuitable effect in the field of application.Moreover, the fact that the actual composition is undeclared couldresult in allergic reactions or undesirable toxic effects in consumers.For these reasons, the quality and authentication control of EOs arecurrently extremely important issues.11 The counterfeiting of EOs withVOs is one of the most common processes due to their low cost, highavailability, and difficulty in identifying their presence. Indeed,the physicochemical features of EOs are not altered by the additionof VOs since the color, refractive index, and density are similar.12 In addition, even gas chromatography (GC), themost employed technique for EO quality control, is lacking in theability to detect the occurrence of adulteration by VOs.13 Therefore, the development of alternative techniquesfor quality control is a challenging subject of investigation andit has been the topic of several recent studies.18 Among the recent studies on EO adulteration, themost applied technique is infrared (IR) spectroscopy coupled withmultivariate analysis.20 However, even though the detection and quantification of an adulterantcan be easily achieved, the precise identification of the VO is oftendifficult due to the signal overlap of similar molecules.21 As an example, Truzzi et al. applied IR spectroscopyin combination with chemometrics to detect and quantify VOs in EOsbut was unable to discriminate the identity of the VOs employed foradulteration.20 Indeed, VOs are composedof triglycerides with different ratios of various fatty acids thatidentify the plant origin of the oil, but the spectral absorbancein IR is almost the same. Nuclear magnetic resonance (NMR) spectroscopyrepresents a valid and promising alternative to GC and IR spectroscopysince simultaneous quantification and identification of the type ofthe adulterant VO can be achieved. NMR demonstrated to be successfulfor quality control of food products since it is based on the recordingof the nuclei electron cloud, which is different for each atom andmolecule.25 Moreover, as opposed to IR spectroscopy, in NMR spectroscopy, theemployment of multivariate analyses might be avoided when target signalsare not overlapped and clearly assigned. Thus, a direct and punctualresult can be obtained by simply integrating the peaks of interestwithout any manipulation of the raw data, with the exception of deconvolutionwhen required. On the contrary, chemometrics approaches require datapreprocessing and are based on probabilistic foundations.26Nowadays, common adulterationsare achieved by the addition ofsynthetic compounds, less valuable EOs, or vegetable oils (VOs) toreach the optimal terpene composition and increase the industrialprofit.13C NMR. NMR alone or in combination with chemometrics was alreadyexploited to discriminate VOs.29 However, the discrimination andidentification here described are based on a novel method withoutthe application of any multivariate analysis, which could be employedin the quality control of not only EOs but also on other food matriceswhere vegetable oils are present. For this purpose, binary mixturesof EOs  and VOs  were preparedand analyzed. The purity of the EOs was ensured prior to the analysisby 1H and 13C NMR to avoid possible interferencesin the analysis.Therefore, the aim of the present studywas to develop a methodfor the simultaneous detection and identification of VOs used as adulterantsin binary mixtures with EOs by applying high-resolution 30 On the other hand, citronella is an emerging food additiveand a fully recognized pest repellent. Almond, corn, peanut, sunflower,soybean, and colza seed oils and wheat germ oil were selected as adulterantsfor their widespread use and affordability.Lavender, citronella, rosemary, and orangeEOs were selected asmodels for their extensive use and potential application as food ingredients,biopreservatives, and biopesticides. In particular, lavender, rosemary,and orange are commonly used as flavoring agents in foods and exhibitedstrong antimicrobial and antioxidant activities.13C NMR spectra acquisition of the binary mixtures,the identification of the VOs was carried out by comparing the ratioof peak areas of characteristic signals of the main fatty acids withthose of pure VO used as standards. Furthermore, commercial samplesof EOs were provided and analyzed to detect and identify possibleadulterants.After 22.1d  and tetramethylsilane (TMS) forinternal referencing were purchased from Sigma-Aldrich .Commercial samples of almond, corn, peanut, sunflower, soybean, andcolza seed oils and wheat germ oil were purchased from a local marketplace. Cymbopogon nardus (L.) Rendle (citronella), Lavandula angustifolia Mill. (lavender), and Salvia rosmarinus L. (rosemary) EOs were purchasedfrom Erbamea , while Citrus sinensis (L.) Osbeck (orange) EO was kindly donated by L’Aromoteca. One sample of each EO was provided, and all of themwere certified as 100% pure.Chloroform-Mentha arvensis L. (two samples), Lavandula angustifolia Mill., Thymus vulgaris L. (two samples), Thymbra capitata (L.) Cav., Cymbopogonmartinii (Roxb.) Wats, Mentha piperita L. (two samples), Eucalyptus globulus Labill., Origanum vulgare L. (twosamples), Ocimum basilicum L., Juniperus communis L., Citrus limon (L.) Osbeck (two samples), Syzygium aromaticum (L.) Merr. & L.M.Perry, Salvia officinalis L., Chamaemelum nobile (L.) All., Salvia rosmarinus L.Furthermore, 20 commercial samplesof EOs were obtained from online shops: 2.23 solution was added.Lavender, citronella,rosemary, and orange EOs were mixed with almond, corn, peanut, sunflower,soybean, and colza seed oils and wheat germ oil to obtain binary mixtures.Starting from a stock solution at an adulterant concentration of 50%w/w, other dilutions  wereobtained by adding an increasing amount of EOs. For each EO–VOcombination, two dilutions were selected and a total of 56 binarymixtures were obtained for the NMR experiments. The selected dilutionsfor each pair of EO–VO assured that for all of the seven adulterantsat least one mixture at each percent concentration was analyzed. About50 μL of pure seed oils, pure EOs, or binary mixtures were transferredinto a Wilmad NMR tube, 5 mm, Ultra-Imperial grade, L 7 in., 528-PPpurchased from Sigma-Aldrich , and 550 μL of 13mM TMS CDClThe commercial EOspurchased from online shops were prepared in the same manner.2.31H and 13C spectra of VOs,pure EOs, binary mixtures, and unknown EOs were acquired with a BrukerFT-NMR Avance III HD 600 MHz spectrometer . Allof the experiments were performed at 298 K and nonspinning.One-dimensional 1H NMR experiments were acquired using Bruker sequence“zg30”; the acquisition parameters were as follows:time domain (number of data points), 131 072; dummy scans,2; number of scans, 32; acquisition time, 4.96 s; delay time, 5 s;pulse width, 13 μs; spectral width, 22 ppm (13 204 Hz),FID resolution 0.201480 Hz; and digitization mode, baseopt. Totalacquisition time was 5 min and 20 s. 13C NMR experimentswere performed by optimizing a sequence modified to reduce the ringingeffect and to completely avoid the 1H–13C coupling and NOE during relaxation. After measuring each carbonT1, delay time (D1) equal to 5 × T1MAX (the longestrelaxation time) was set to assure the complete relaxation of 13C nuclei. All of these experimental conditions were usedto make the carbon integral suitable for quantitative purposes. Theexperiments were acquired using Bruker sequence “zgpg_pisp_f2.fas”(details are in the Supporting Information), and the acquisition parameters were consequently modified andset as follows: time domain (number of data points), 65 536;dummy scans, 0; number of scans, 256; acquisition time, 0.98 s; delaytime, 10 s; spectral width, 220.87 ppm (33 333.332 Hz); FIDresolution, 1.017 Hz; and digitization mode, baseopt; to use thissequence as inverse-gated, the proton decoupling power (PLW13) duringrecycle delay and experiment time was set to 0 db. The total acquisitiontime was 47 min.1H NMR experiment was performed onthe commercial EOs that resulted as adulterated. Spectral acquisitionswere carried out with the standard Bruker “ledbpgp2s”pulse program using a longitudinal eddy current (LED) bipolar gradientpulse pair and two spoil gradients. Sixteen gradient steps were usedfor the diffusion dimension from 2 to 98% of gradient amplitude, where65.7 G/cm was the maximum gradient intensity. The acquisition parameterswere set as follows: number of scans, 32; pulse field gradient length, 1 ms; gradient strength (gpz6), 100%; LED delay (d21),5 ms; and diffusion time , 60 ms. Total acquisitiontime was 16 min.To confirm the adulteration with VOs, a two-dimensionaldiffusion-orderedspectroscopy (DOSY) After loading the sample into the probe, 5min was required toachieve thermal equilibrium. Afterward, the magnetic field was locked,the probe head was tuned and matched, and, finally, the sample wasshimmed. To assure the highest reproducibility, all of these procedureswere automatically executed.The baseline correction, the phasing,and the integration of NMRspectra were performed on TopSpin 3.5 software .13C NMR spectra with literature data.The assignments for the major fattyacid peaks were carried outby a comparison of 2.4Fourteen well-definedand characteristic signals present in all pure VOs (used as standards)and in the binary mixtures composed of EOs and adulterants were identifiedand integrated. Peak deconvolutions were carried out when required.These 14 resonances related to palmitic, oleic, and linoleic fattyacids were present in all of the seven VOs considered. The intensityof these signals is different in each VO since the relative abundanceof each fatty acid varies considerably depending on the plant origin.In particular, the signals of the terminal and carbonylic carbonsand carbons involved in double bonds in palmitic, linoleic, and oleicacid chains were selected. For each pure VO and each binary mixture,the chemical fingerprint was obtained by calculating a square ratiomatrix. Specifically, the integrals of the 14 peaks in each spectrumwere calculated and exported as absolute values to generate a squareand symmetrical table composed of 14 rows and 14 columns. Thus, theintegrals of the signals at lower chemical shifts were placed bothin the first row and column and so on for all of the other signals.Then, in each cell of the table, the ratio of the corresponding peakintegrals was calculated to complete the 14 × 14 matrix (196cells). Finally, the matrices were represented as heatmaps, wherethe ratio of the absolute values was depicted in the grayscale rangefrom black to white for lower and higher values, respectively.The identification of an adulterant oil in the binary mixtures withthe EOs was achieved by comparing its 14 × 14 peak ratio matrixwith those of the standard pure VOs using MATLAB software . In particular,for each cell, the following equation was appliedThen,the number of ratios of the total , whichexhibited an error of ≤25 or 10%, depending on the consideredconfidence level , were counted as acceptable.The percentage of likeness was calculated with the following equationTo simplify the calculations,the similarity was computed by consideringthe whole square matrices, even though the useful information is includedin half of each matrix. The same procedure was carried out in thecommercial samples that resulted in adulteration.31H NMR spectra, glycerol exhibited twosignals at 4.12 and 4.27 ppm, which corresponded to 2H in both positions sn-1′ and sn-3′, respectively,while the glycerol carbons were detected at 62.4 and 69.2 ppm forC1/C3 and C2, respectively.In the present work, the detectionand identification of adulteratingVOs in the counterfeiting of EOs were achieved using high-resolutionNMR. Both proton and carbon NMR spectra were employed to detect thepresence of VOs by focusing on the glycerol backbone signals of triglycerides.In particular, in 13C NMR was preferred over 1H NMRdue to the ease of interpretation. Indeed, 1H NMR of VOsexhibited several overlapping signals with poor spectral resolution and sn-2 positions: high-frequency and low-frequency groups. The C1 signalsat lower field were assigned to palmitic and linoleic acids, whereC1 of the saturated fatty acid (P1) was detected at a higher frequencyin accordance with the Vlahov report.35These14 peaks can be grouped into three different regions dependingon the chemical shifts that represent characterizing carbons of thefatty acids. Specifically, the most deshielded resonances representedthe carbonylic carbons of fatty acids of triglycerides. These signalscould be divided into two subgroups according to the resonances ofthe fatty acids in sn-2 position, with respect to that in sn-1 (or 3).An opposite trend was noticed for the unsaturated carbons fartherfrom the ester group.36In the mid-region ranging from 128 to 130 ppm, typical resonancesof the olefinic carbons were present and these signals could be differentiatedbased on the proximity to the ester group. As a matter of fact, theclosest unsaturated carbon to the glycerol backbone  shifted upfield compared to the closest unsaturated carbon tothe methyl moiety . Moreover, the chemical shiftsof olefinic carbons were further influenced by the fatty acid positionin the triglyceride. The carbon employed in a double bond closest to the carboxyl group resulted and shifted upfield in the 3 groupsof ω-6 acids have been reported to be shielded with respectto CH3 groups of saturated acids.37Finally,the signals at low frequencies were attributed to theterminal carbons. The resonances of terminal carbons (L18 and O18)belonging to different fatty acids could easily be distinguished sincethey are strongly affected by the chain length and the potential proximityto the last double bond. Indeed, the terminal CHThe above-listed carbon signals, whose intensity considerablyvarieddepending on the VO, were used to generate the chemical fingerprintfor each adulterant oil. Since each VO exhibits a characteristic fattyacid composition, not only the signal intensities but also the ratiosbetween the signals are constant. Therefore, the chemical fingerprintsof pure VOs, used as standards, were obtained by integrating the 14selected signals and calculating the ratio between each peak area.Thus, an identifier 14 × 14 matrix was attained for each VO.The ratio matrices were represented as heatmaps for convention, wherethe lower and higher values of the ratio were displayed in black andwhite, respectively 3.1H and 13C NMR spectrato avoid interferences in the analysis. 1H NMR was preferredfor purity assessment due to its higher sensitivity, related to thegreater isotopic abundance of 1H. All of the spectra didnot show any trace of the signals related to the glycerol backbone.Thus, 56 binary mixtures composed of lavender, citronella, rosemary,and orange EOs were mixed with the VOs and their spectra were acquired.In particular, for each EO–VO pair, two dilutions were analyzed,and for each VO, all of the range of percentages was assured. Concentrationsfrom 0.8 to 50% w/w were tested as this is the most common range foradulteration. Indeed, concentrations above 50% would lead to visibledetection of the VO, while concentrations lower than 0.8% would notbe advantageous for profits. The glycerol backbone signals were clearlyvisible in the 1H NMR spectra even at the minimum concentrationof VO in the mixtures. In the binary mixtures at 0.8% w/w, the signal-to-noiseratio was equal to 130, suggesting that the presence of adulterantoils could be detected at lower concentrations . Regarding 13C NMR spectra, the signal-to-noiseratio was decisively lower at the same concentration, equaling to5. This value is slightly higher than the acceptable ratio for detectionlimit in analytical methods.38 Thus, asexpected due to the poor isotopic abundance of 13C, carbonNMR could not provide reliable information at lower concentrationsof adulterant oils .Prior to the preparation of the binary mixtures, the absence oftriglycerides in lavender, citronella, rosemary, and orange EOs wasverified by examining their The14 selected carbon signals for the development of the identificationstrategy were well resolved and separated from the characteristicterpene peaks of the EOs in question 4.sn-2′signals disappeared in soybean, wheat germ, and corn oils in mixtures,as well as L12, L10, and L9 sn-2′ resonancesin almond oil, according to the fatty acid composition of each seedoil. The mixtures of sunflower oil showed a lack of L12 and L10 sn-2′ signals due to overlap with the more intense sn-1′(3) signals. Moreover, in peanut and colza oils,which are rich in oleic acid, the resonances belonging to L1 and L10 sn-1′(3) disappeared, probably due to the preferredesterification of linoleic acid in the sn-2′position of triglycerides, resulting in more intense signals stilldetectable at a concentration of 3%. Indeed, generally, polyunsaturatedfatty acids occur in the sn-2′ position ofVO triglycerides.41 Finally, P1 sn-1′(3) wasonly missing in sunflower and almond oils where it is less abundant.31 At lower concentrations (1.5%), further signalsdisappeared.All of the 14 typicalcarbon signals of the VOs were detectablein the dilution range from 6 to 50%. At a concentration of 3% w/w,some peaks disappeared. In particular, the O9 For all 56 binary mixtures, the 14 resonances relatedto the threemain fatty acids were integrated and the 14 × 14 ratio matriceswere computed. Thus, the chemical fingerprint of the adulterant oilin each blend was generated and compared to those of standard pureVOs. The comparison and identification of the VO were attempted inall of the mixtures, and the percentage of similarity was calculated.In particular, the percentage of similarity between ratio matriceswas calculated by considering the number of the peak area ratios ofmixtures that matched with a peak area ratio difference lower than25 or 10% compared to the standard VOs. Therefore, a similarity of100% means that all 182 ratios of the matrix exhibited a difference lower than 25 or 10% with the standard VO.In the case of binary mixtures at a concentration of 3%, the matriceswere reduced and the peak area ratios of the missing resonances wereeliminated. However, due to the lack of most signals, the recognitionwas not achieved on the samples below 1.5% of adulterants, and forthis reason, the results were not reported.viceversa. By further increasing the confidence level to 95%and reducing the acceptable error to 5% (data not shown), the correctidentification of the VO gave rise to a decreased percentage of likenesswith the exception of the binary mixtures with 50 and 25% adulterantconcentrations. In this case, the weakening of recognition is ascribedto a poor signal-to-noise ratio and imprecise signal deconvolutionsat low adulterant percentages. As a consequence, by decreasing themargin of error to 5%, several peak area ratios did not match withthe standard VO, leading to lower similarity percentages.As shown in 28 On the contrary, Zamora et al. argued that a better differentiationof VOs is obtained by considering minor compositional components,which emerged by fractionating the whole oil by column chromatography.42 However, all of these outcomes were attainedby applying principal component analysis on 1H and 13C NMR resonance intensities of VOs.These results demonstrated the capability of the developed methodto recognize the adulterant oil in EO mixtures. As a matter of fact,the correct identification was achieved for all of the prepared mixturesin the concentration range of 3–50% w/w of the adulterant inthe tested confidence levels, without any difference with respectto the EO in analysis. A confidence level of 75% was demonstratedto be less selective for the determination of the adulterant but moresensitive and accurate at low adulterant concentrations. On the contrary,the 90% confidence level was more precise in identifying which VOwas present but less sensitive to the presence of the adulterant ingeneral. The identification of VOs has already been tested exploitingthe potential of NMR analysis. Popescu et al. achieved a good differentiationof VOs, demonstrating that linoleic, oleic, linolenic, and free fattyacids were the most important discriminant variables.The advantagesof our approach relied on obtaining successful resultswithout requiring any data preprocessing or multivariate analyses,which are based on probabilistic foundations. Moreover, our promisingoutcomes were achieved by merely considering the signals of threecommon fatty acids in pure VOs without any pretreatment. Therefore,the feasibility and the strength of the novel developed method canbe affirmed.Thymbra capitata (thyme), Cymbopogon martinii , Mentha arvensis (mint), and Origanumvulgare (oregano) resulted in counterfeit since typicalsignals of the glycerol backbone were identified in both 1H and 13C NMR spectra. The recognition method revealedthat thyme was adulterated with sunflower, mint with soybean, andpalmarosa and oregano with corn with a certainty of recognition thatranged from 96.7 to 100% and 63.7 to 92.3% at the confidence levelsof 75 and 90%, respectively . Asan example, in The validated method was then applied to commercialsamples ofEOs purchased on the internet to detect possible adulterations. Theanalysis of these samples was carried out in triplicate to assurethe results. Among the 20 EOs, derived from different plant speciesand brands, the samples of ectively 5. To assD) of thecompounds in the sample. Specifically, triglycerides diffused moreslowly, while terpenes in the EO, being the lightest compounds inthe mixture, displayed a higher D and diffused morerapidly. In the case of adulterated palmarosa, three more groups ofcompounds could be identified. Indeed, within terpenes and triglycerides,molecules with medium molecular weight exhibited D equal to 2.5 × 10–5, 3.1 × 10–5, and 3.5 × 10–5 cm2/s. Being thatcorn is the richest source of tocopherols among the VOs, these signalsmight be related to them in their free form or esterified with fattyacids.45 The identity of these signalswas also confirmed by performing a DOSY experiment on our corn oilsample, demonstrating that these signals belong to the VO. Even thoughsunflower has been reported as a phytosterol source, no signals weredetected, probably due to a lower concentration of the adulterantin the commercial thyme with respect to palmarosa.The signals of the glycerol backbone(2H at 4.282 ppm and 2H at4.130 ppm) were used to identify triglycerides in the oils. For boththe adulterated EOs, DOSY signals can be divided into two main groupsbased on the diffusion rate coefficients (In conclusion,this newly developed strategy demonstrated feasibilityand efficiency to identify and recognize VOs used as adulterants inEOs. In particular, the detection of the presence of VOs could beachieved from the minimum concentration of 0.8% w/w of the adulterantboth in proton and carbon spectra. The VO’s identificationmethod was shown to be effective in all of the cases by acceptinga margin of error of 25%, without employing multivariate analyses.The simplicity of the proposed approach could be exploited in frauddetection of different food matrices containing VOs. To the best ofour knowledge, this method could represent a valid alternative toother conventional techniques, such as chromatographic methods orIR spectroscopy. Further studies will be carried out to improve theapplication of NMR spectroscopy in the quantification of VOs in thesevaluable products in the range of 0.8–50% w/w of adulterantcontent, being one of the most exploited adulteration practices.