The predictability of clinical outcomes in hypertension in specific patient groups, especially underrepresented populations is the key to rational treatment. This study aimed to investigate the impact of baseline characteristics of <65-year-old hypertensive women with an increased risk of cardiovascular events, managed with standard- or intensive-approach, on their clinical outcomes and serious adverse events (SAEs). Baseline characteristics of <65-year-old hypertensive women (n = 1247) in SPRINT, a multicenter randomized trial to compare standard and intensive antihypertensive treatment, were analyzed with Cox-regression method to determine potential predictors of the clinical outcomes and SAEs. The primary outcome was the composite of myocardial infarction (MI), non-MI acute coronary syndrome, stroke, heart failure, or cardiovascular death. The primary outcome occurred in 3.1% and SAEs in 27.6% of the population. The treatment groups were similar in terms of the primary outcome, SAEs, or their individual components. The primary outcome occurred significantly more in current smokers vs. nonsmokers . The subjects who were on aspirin in the intensive-group were significantly more likely to develop the primary outcome  and MI  compared with those not using aspirin. The risk of overall SAEs was significantly higher in blacks vs. nonblacks , in current-smokers vs. nonsmokers , and those with vs. without chronic kidney disease (CKD), . The likelihood of SAEs significantly increased with age . Smoking, aspirin, CKD, black race, and age seemed as important baseline characteristics in follow-up of <65-year-old hypertensive women, also depending on therapeutic strategy. Clinicians are expected to consider these critical parameters for effective antihypertensive management that promotes better outcomes in this middle-aged female population.  Sex-related variations exist in cardiovascular (CV) anatomy and physiology, predisposing women and men at differential CV risks . Being more vulnerable, hypertensive women have poorer CV outcomes than do men, especially in the presence of CV risk factors [3–5], despite the higher rates of awareness, antihypertensive prescription, controlled disease, and drug compliance among women . Moreover, the observed sex-related differences in CV outcome was reported to be independent from the response to antihypertensive medications [7]. Aging further contributes to sex-related differences in developing CV disease and outcomes [8]. In fact, female predominance in hypertension takes place after the age of 65 years [6]. This is accompanied by less-controlled and more severe disease in elderly hypertensive women than their younger counterparts [9]. Hypertensive patients usually have a number of CV disease risk factors, either modifiable or relatively fixed. Targeting modifiable risk factors, total CV risk assessment contributes both to improved blood pressure (BP) control and reduced CV disease burden . There is substantial evidence from clinical studies, where these risk factors are addressed by pre-determination of specific subgroups, helping physicians to achieve rational management of diseases in various patient subsets. Indeed, while it might not be possible to cover all clinical scenarios in a hypertension trial; underrepresentation of key demographic patient strata, like age or sex, is likely to have an impact on the generalization of the study results, e.g. predictability of clinical outcomes in such populations [12–14]. Representation of women in study populations was reported to vary from 35.2% to 59% in key hypertension trials among which the lowest rate belonged to the recent Systolic Blood Pressure Intervention Trial (SPRINT) [9]. In brief, SPRINT was a multicenter (n = 102) randomized clinical trial where 9,361 hypertensive patients at increased CV risk were assigned to either receive standard- (a target systolic BP [SBP] of <140 mmHg) or intensive-treatment (a target SBP of <120 mmHg) [15]. A recent review discussed low enrollment and fewer adverse event rates in SPRINT, implying gaps of evidence about optimal management of hypertension in women [5]. This might be more pronounced in younger women where the absolute CV risk could be regarded as low. Nevertheless, this population could also have relatively increased risk if markedly abnormal CV risk factors are present [11]. A better understanding of the key characteristics of such underrepresented populations could help to predict clinical outcomes in hypertension, and hence, contribute to the rational management of the disease. This study aimed to investigate the impact of baseline characteristics of <65-year-old hypertensive women, managed with standard or intensive approach, on their clinical outcomes and serious adverse events (SAEs) in SPRINT.The rationale and design of the study and eligibility of patients with their baseline characteristics in SPRINT was previously described [16]. Patients were regarded at increased CV risk if they had one or more of the following: history of clinical or subclinical CV disease, chronic kidney disease (CKD), 10-year Framingham risk score of ≥15%, or age ≥75 years. Subjects were excluded if they had diabetes mellitus, history of stroke, polycystic kidney disease, symptomatic heart failure or reduced ejection fraction <35%, estimated glomerular filtration rate (eGFR) <20 mL/min/1.73 m2 or end-stage renal disease, or were (or planned to be) pregnant [15]. In this subanalysis of the trial, we evaluated data of female population aged below 65 years old (range: 48–64 years). The primary outcome of the SPRINT was the first occurrence of the composite of myocardial infarction (MI), non-MI acute coronary syndrome (ACS), stroke, heart failure, or CV death. Secondary outcomes included individual components of the primary composite outcome, any death, and the composite of primary endpoint or any death. We only included these clinical endpoints to our subanalysis, not evaluating renal outcomes in this subpopulation. An adverse event in the study was adjudicated to be serious if it (i) was fatal or life-threatening, (ii) led to significant or persistent disability or any congenital anomaly/birth defect, (iii) caused/prolonged hospitalization, or (iv) was judged by the investigator to pose a significant hazard or harm to the patient, requiring preventive medical or surgical intervention. Hypotension, bradycardia, electrolyte abnormalities, injurious falls, syncope, acute kidney injury or acute renal failure, or any unexpected event that the investigator believed it to be trial-related were categorized as SAEs if they met abovementioned decision-making criteria. Other adverse events not labelled as serious were not included to this subanalysis.The baseline characteristics that were used for predicting clinical outcomes and SAEs included ten variables: age, SBP, diastolic BP, race, smoking status, daily aspirin use, body mass index (BMI), total cholesterol (TC)/high-density lipoprotein cholesterol (HDL-C) ratio, hyperglycemia, and CKD. Among categorical variables, race was self-reported as black or nonblack. Smoking status was grouped as nonsmokers, former smokers, and current smokers. BMI groups were divided into three as under-/normoweight (<25 kg/m2), overweight (25 to <30 kg/m2), and obese (≥30 kg/m2). The cut-off point for TC/HDL-C ratio was chosen as 3.5 where the values above this were reported to be indicative of CV risk among women [17]. The patients who had a blood glucose level of ≥100 mg/dL at baseline were categorized as hyperglycemic [18]. CKD was defined as having an eGFR of 20–60 mL/min./1.73 m2.Study data were analyzed using SPSS version 25.0 software package. Descriptive baseline and outcome data were expressed as the mean (± standard deviation) or percentage of the population. Independent t-test were used for comparing parametric variables, and either chi-square or Fisher’s exact test, where appropriate, for comparing categorical variables between standard- and intensive-treatment groups. The abovementioned baseline characteristics were entered into the Cox proportional hazards regression with a backward selection to determine independent predictors of defined outcomes. The Cox regression was performed for the total population and for each of the standard and intensive-treatment groups separately. Only those characteristics in the last steps that were regarded as statistically significant predictor of the given outcome in the standard- or intensive-treatment groups, or in overall were mentioned in the tables for concise demonstrative purposes. Hazard ratio (HR) was calculated with a 95% confidence interval (CI). An overall 5% type-I error level was used to infer statistical significance.We identified 1247 women who were below 65 years old representing 13.3% of overall SPRINT cohort, where the median duration of follow-up was 3.25 years. The baseline characteristics of this subgroup randomized to either standard-treatment (n = 595) or intensive-treatment (n = 652) were similar except slightly higher percentage of women with increased TC/HDL-C ratio in the latter (Table 1). The primary outcome event occurred in 21 (3.5%) and 18 patients (2.8%) in standard-treatment and intensive-treatment groups, respectively. On the other hand, a total of 341 SAEs were detected; 27.6% in the standard and 27.1% in the intensive strategy. The groups did not differ in terms of clinical or safety outcomes or their individual components. The primary outcome event occurred significantly more in current smokers vs. nonsmokers . While no such effect was found in the standard-treatment group, this effect was also significantly shown in the intensive-treatment group consistent with those of the overall women population. The subjects who were on aspirin at randomization in the intensive-treatment group were significantly more likely to develop the primary outcome  and MI  compared with those who were not using aspirin. This association was not detected in the standard-treatment group or in overall population (Table 2).Among other individual components of the primary outcome, non-MI ACS was found significantly more in women with hyperglycemia than those with normoglycemia . Heart failure occurred significantly more in the presence of CKD in overall  and in the standard-treatment group , whereas this effect disappeared in the intensive-treatment group. On the other hand, those patients who were older at randomization developed less heart failure in the intensive-treatment group , an effect which was not shown in the standard-arm or overall. No predictors of risk were detected in terms of stroke or CV death (Table 2).Death from any cause was found to occur significantly more in current smokers vs. nonsmokers  and in women with vs. without CKD . While similar effects were also consistently observed in the standard-treatment group, no such significant associations were detected in the intensive-arm (Table 2).Primary outcome event or death occurred significantly more in former  or current  smokers vs. nonsmokers. The risk was also significantly higher in patients with vs. without CKD . The subjects who were under aspirin in the intensive-treatment group were more likely to develop the primary outcome or death compared with patients who were not using aspirin at baseline , (Table 2). The risk of overall SAEs was significantly higher in those with black vs. nonblack origin , in current smokers vs. nonsmokers , and those with vs. without CKD . While these risks were also observed in the standard-treatment group, no such association was found in the intensive-treatment group. In addition, the likelihood of SAE significantly increased with the age , and the effect was also observed to disappear in the intensive-treatment group (Table 3).In terms of treatment-related SAEs, a consistently significant effect was shown across groups for the presence of CKD towards more events. Black vs. nonblack women and current smokers vs. nonsmokers developed significantly more related SAEs overall , an effect not observed in the standard- or intensive-treatment group (Table 3).The risk of serious hypotension was elevated in patients with vs. without CKD , which was also observed in the standard-treatment group. No parameter was found to predict a serious bradycardia event in either treatment groups or overall (Table 3).While serious syncope events developed more in current smokers vs. nonsmokers  overall; the risk was also significantly higher in obese vs. normo/underweight women in the intensive-treatment arm . On the other hand, injurious falls occurred significantly more in patients with vs. without CKD only in the intensive-treatment group . Serious electrolyte abnormalities developed less among overweight or obese vs. normo/underweight women, which was especially marked in the intensive-treatment group , (Table 3). The risk of acute kidney injury or renal failure SAE was significantly increased among black population , in former  or current smokers , and in women with CKD ; which were also shown in the intensive-treatment arm compared to those in the standard-treatment arm. In addition, the risk was also significantly higher with elevated basal SBP only in the intensive-treatment group , (Table 3). This study is expected to contribute to the treatment approach of middle-aged hypertensive women which seems to represent a comparably gray zone. The subanalysis of the SPRINT in <65-year-old female population has featured several baseline characteristics for the follow-up of hypertension, managed with either standard or intensive approach: smoking, aspirin use, CKD, hyperglycemia, and the age for the risk of clinical outcomes and SAEs; black race and BMI only for SAEs. These key characteristics seem to be associated with several outcomes or events distinctively, which may depend on selection of the standard- or intensive-treatment strategy. A well-established risk factor for CV disease [10], smoking was reported to affect women more adversely than men . Furthermore, a study showed the largest gap between smoking women and men compared to their never-smoking counterparts in terms of increased MI risk occurred between ages of 55–64 years [20]. Consistently, our subgroup analysis in middle-aged women also showed current smoking to be associated with higher risk for the primary outcome, any death, and several SAEs . Indeed, though less pronounced, the risk was also partially relevant for former smokers vs. nonsmokers, supporting the reduced CV disease risk upon smoking cessation [21]. On the other hand, association of active smoking at baseline with increased CV risk was only significant in the intensive-treatment group, suggesting a failure of the strategies targeting SBP goal <120 mmHg among active smoker vs. nonsmoker middle-aged hypertensive women. In fact, a recent analysis of SPRINT and ACCORD data reported current smokers to benefit less from the intensive treatment [22]. In overall study population, aspirin use at randomization was not associated with the primary outcome. Recently, three consecutive studies have reported that aspirin treatment had only limited and costly preventive effect, if any, in patients with increased CV risks [23–25]. Among these studies, ARRIVE study involved patients comparable to our population, reporting no significant benefit of aspirin in nondiabetic, >60-year-old cases with no interaction by age or sex [23]. Therefore, it may be suggested that aspirin treatment in <65-year-old women, a comparably lower risk group indeed [26], would not be expected to have a substantial preventive effect. On the other hand, the significantly higher risk of the primary outcome and MI among aspirin users in the intensive-treatment arm could have clinical importance. The presence of such an aspirin-based risk gap merely in this group implies that the decision towards intensive treatment in middle-aged hypertensive women should be reevaluated in case that they receive aspirin for a particular indication. Though we did not have data about the purpose of aspirin therapy -primary or secondary-, its concomitance with intensive BP-lowering strategy appears to worsen the outcomes, while it merits further research to elucidate underlying mechanisms or contributing factors.Not just an independent risk factor for CV disease, CKD further seems to compel benefit/risk ratio of BP-lowering therapy in terms of target goals [27]. We observed a positive association of CKD to the increased risk of heart failure near 6.5-fold, and to lesser but significantly raised risks of total mortality, composite endpoints, and several SAEs in this middle-aged hypertensive women population. While this pattern was mainly preserved in the standard-treatment group, CKD in the intensive-treatment group was not associated with increased risk of clinical or safety outcomes, except treatment-related and acute kidney injury/failure SAEs. The difference in favor of intensive strategy is consistent with meta-analyses and CKD subanalysis of the SPRINT , confirming the benefits of such approach also in middle-aged hypertensive women with CKD. Furthermore, a recent subanalysis reported that most of such acute kidney injury events are mild in nature and completely resolved [30], which may help to relieve such potential concerns. It might be suggested that intensive approach could be encouraged in this patient subgroup accompanying CKD.The incidence of hypertension and its associated mortality is higher in blacks vs. whites [6]. Indeed, African American postmenopausal women in Women’s Health Initiative study were reported to have lowest rate of ideal CV health [31]. While black race was associated with greater CV benefit of intensive treatment in the SPRINT overall [22], it did not predict clinical outcomes in our subanalysis of middle-aged women. By contrast, being a <65-year-old hypertensive black women was associated with increased risks of any, related, and acute kidney injury/failure SAEs in our study. Especially the latter may be attributed to the underlying etiology of racial disparities in hypertension, involving reduced ability to excrete sodium loading and different renin-angiotensin-aldosterone system in blacks [32]. In addition, >50-year-old black women were reported to be far more beyond their recommended threshold level of sodium intake than were their white counterparts despite similar amount of consumption [33]. Whether this may contribute to increased SAEs among black women warrants further research.Age was associated with a lower risk of heart failure in the intensive-treatment group. On the contrary, a study on young adult heart failure patients reported similar rates of underlying hypertension and comparable use of antihypertensive drugs between 50–59- and 60–69-years’ age groups, suggesting no age-related association [34]. This is likely to indicate the benefit of selecting intensive antihypertensive strategy to prevent heart failure in middle-aged women as their age increases. It is further enhanced by the absence of increased risk of any SAEs in the intensive-treatment group, unlike the standard-treatment group. While older age is expected to be correlated with higher SAE rates , no association of age to the treatment-related SAEs may help to attenuate potential age-based SAE concerns, if any, in <65-year-old hypertensive women population.Despite being a well-known risk factor for hypertension [37], obesity did not predict clinical outcomes in <65-year-old hypertensive women in this study. By contrast, it appeared to be negatively associated with serious electrolyte abnormalities and positively associated with syncope particularly in the intensive-treatment group. The latter could be partly attributed to the potent reversal of increased sympathetic nervous system activity, an important mechanism of hypertension in obese patients [38]. Whatever the reason, it may be advised for physicians managing obese hypertensive women to pay more attention in accurate BP measurement when considering intensive strategy. This is based on the fact that erroneous measurements due to mismatched cuff size in obese people, still a common practice, lead to overestimated readings and consequently unnecessary treatment [39]. This, in turn, may translate into overshooting of <120 mmHg target, aggravating already raised syncope risk. On the other hand, decreased risk of serious electrolyte abnormalities could be related with expanded intravascular volume among obese hypertensive patients [40] and elevated plasma solute concentrations in obese people [41]. This may imply a higher tolerability to clinically relevant electrolyte imbalances by antihypertensive medication. Considering the CV disease burden caused by obesity, it may be suggested that its relative protective role against electrolyte abnormalities not be overemphasized.The positive association of baseline hyperglycemia with non-MI ACS regardless of antihypertensive strategy should be evaluated in caution. While a recent study reported higher CV or coronary mortality in ever prediabetic patients than normoglycemic patients [42], the lack of such impact of hyperglycemia on other clinical outcomes, i.e. being confined to unstable angina mostly, seem to call the finding in question. In addition, any deterioration in glycemic status during the study might have a confounding effect.More patients with high TC/HDL-C ratio existed in the intensive-arm at baseline, yet this did not reflect as difference in clinical outcomes. Potentially influenced by several factors such as dietary habits, physical exercise, or lipid-lowering drugs [43], TC/HDL-C ratio does not appear as a good predictor to determine either standard or intensive therapy in middle-aged hypertensive women.Polypharmacy is well-recognized risk factor for adverse effects [44]. This, at first sight, imply increased risk of adverse effects in the intensive group. However, SAE rates did not differ between treatment arms, suggesting that it is advisable not to overvalue the role of SAEs in selecting antihypertensive strategy in favor of or against to intensive therapy.Our subanalysis has several limitations. First, in this middle-aged women population, a 3.25-year median follow-up could be regarded as a comparably shorter period to disclose the differences between treatment arms in terms of clinical outcomes. This might also reflect into the predictability of examined variables, as evident by broad CIs in several outcomes where fewer events occurred. Second, rather than being based on individual and single measurements, BP assessments were made with respect to the intended SBP target of the SPRINT, <140 or <120 mmHg. In addition, study design did not force participating clinicians in favor of any antihypertensive class or dose as long as they were guideline-based. Therefore, individual BP recordings and antihypertensive medications and their doses might influence the impact of some risk factors at individual basis. However, it could be suggested that this effect is unlikely to be that much pronounced as the study design applied for both of the treatment arms. Third, there was no baseline data about the use of hormone replacement therapy (HRT) in this subgroup. Though evidence showed that HRT resulted only small BP changes ; there is possibility that HRT could interact with particular antihypertensive medications that might influence future outcomes. On the other hand, menopausal transition was reported not to substantially act on CV outcomes [8]. In fact, the risk was suggested to depend on patients’ lifestyles rather than their hormone profile [47]. Finally, it is important to note that, the examined variables were recorded at the beginning of the study as baseline characteristics and the possible changes during the follow-up period were not evaluated.In conclusion, smoking, aspirin use, CKD, hyperglycemia, black race, age, and BMI seemed as important baseline characteristics in the follow-up of <65-year-old hypertensive women, also depending on the therapeutic strategy. Consistent with total CV risk assessment concept, the success of antihypertensive treatment in daily practice –not confined to only BP values- is enhanced by on-point and rational consideration of suitable baseline parameters of patient subsets. This is especially relevant for populations underrepresented in trials, where subgroup analyses like the current one, aim to shed light on. In this context, such subanalyses could help to customize identify and measure the impact of associated baseline factors in particular patient populations. Therefore, abovementioned baseline parameters should be considered for a rational antihypertensive management, including treatment and follow-up, that contributes to prevent CV outcomes and avoid SAEs in this middle-aged female population with increased CV risk. With a higher life expectancy in the real-world setting, middle-aged hypertensive women might have better outcomes if intensity of BP-lowering strategy is built on predictive factors at baseline. This manuscript was prepared using SPRINT_POP Research Materials obtained from the National Heart, Lung, and Blood Institute (NHLBI) Biologic Specimen and Data Repository Information Coordinating Center and does not necessarily reflect the opinions or views of the SPRINT_POP or the NHLBI.This subanalysis did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The subanalysis was performed after signing data-sharing protocol with SPRINT investigators based on the study approval from local institutional ethics committee .


Coronavirus disease 2019 (COVID-19) has afflicted tens of millions of people, fostering and unprecedent effort in vaccine development and distribution. Healthcare workers (HCW) play a key role in vaccine promotion and patient guidance, and it is likely that hesitancy among this population will have a major impact on the adoption of a successful immunization policy. To investigate HCW attitudes towards anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) vaccination, we developed an anonymous online cross-sectional survey. 1723 Italian HCW responded. Overall, 1155 (67%) intended to be vaccinated, while 443 (26%) were not sure and 125 (7%) declared refusal. In multivariate analysis, factors associated with hesitancy were using Facebook as the main information source and being a non-physician HCW, while predictors of acceptance included younger age, being in close contact with high-risk groups and having received flu vaccination during the 2019–2020 season. Reasons for hesitancy included lack of trust in vaccine safety (85%) and receiving little (78%) or conflicting (69%) information about vaccines. According to our results, adequate investment in vaccine education for healthcare personnel appears to be urgently needed, prioritizing non-physicians and information quality spread through social media. We hope that our data could help governments and policy-makers to target communication in the ongoing COVID-19 vaccination campaign. In January 2020, the World Health Organization (WHO) announced the outbreak of a respiratory syndrome caused by a novel coronavirus in the province of Wuhan, China. Since then, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) has spread to 189 countries, causing nearly 45 million confirmed cases and more than 1 million deaths . So far,Still much remains to be learnt about SARS-CoV2 immunity and its duration , but it Since their introduction in the late 18th century , few pubIn order to advise strategies for vaccination coverage improvement, it is necessary to describe the determinants of vaccine hesitancy in this particular population. From this viewpoint, we conducted the first Italian survey on knowledge, attitudes and practices among healthcare personnel.A cross-sectional, multicenter survey was conducted from 1 October to 1 November 2020, by administering an anonymous online questionnaire to Italian healthcare workers regardless of the category, setting or region in which they were working.Ethical approval was granted by the Research Ethics Committee of the Fondazione Policlinico A. Gemelli before study initiation  Informed consent was requested on the introductory web page prior to survey enrollment.Development of the questionnaire was informed by a literature review, and it was composed by 26 questions with multiple answers or 5-point Likert-style scale, divided into five sections: (i) demographics and occupation-related information; (ii) attitudes and practice toward anti- SARS-CoV-2 and Flu vaccination; (iii) perceptions on reasons of vaccine hesitancy of the general population; (iv) attitude towards vaccines recommendations; and (v) vaccines relevance’ perception Moreover, an additional section was provided for those declaring that they did not want to be vaccinated, exploring the reasons of personal vaccine hesitancy.The questionnaire was developed electronically on SurveyMonkey  and distributed over a 4-week period via mailing list and social media.A descriptive analysis was performed to define the distribution of demographic and occupation-related characteristics of the sample. Vaccine hesitancy was identified as the dependent variable. Vaccine “hesitants” included both those who would not get vaccinated (vaccine resistant/high level of hesitancy) and those being uncertain about their vaccination decision (low levels of hesitancy). Different typologies of postgraduate medical specialties were classified into clinical, non-clinical and surgical areas as for the Italian Ministry of University and Research classification (DM 68/2015).p-value < 0.10 by univariate analysis were included.Independent t test was used to compare groups for continuous variables, whilst a chi-squared test (with the Fisher’s correction if less than five cases were present in a cell) was applied for categorical variables. A logistic regression model was implemented as follows. COVID-19 vaccine hesitancy was considered as a dependent variable and each one of the available factors at the baseline evaluation as independent variables . In the multivariate analysis factors with a p-value < 0.05. Statistical analyses were performed with GraphPad Prism 8.0 .Multicollinearity among covariates was assessed through the variance inflation factor, taking a value of 2 as cut-off to exclude a covariate. However, no variable was excluded according to this pre-specified criterion. Odds ratios (ORs) as adjusted odds ratios (adj-ORs) with 95% confidence intervals (CIs) were used to measure the strength of the association between factors at the baseline (exposure) and COVID-19 vaccine hesitancy (outcome). All statistical tests were two-tailed and statistical significance was assumed for a n = 437, 41%) and 205 (12%) were from a primary care setting, either General Practitioners  or General Practitioner trainees , whilst 544 (32%) were medical school graduates with no further specific educational path. Regarding specialists and medical residents, 356 (44%) were from clinical fields, 154 (19%) from non-clinical sectors and 86 (11%) from surgical sectors  reported to have less than 10 years of length of service in the Health sector. All Italian regions were represented.A total of 1841 healthcare workers accessed the survey and 1723 (93.5%) completed all sections and were considered for the analysis. The mean age of respondents was of 35.5 (SD 11.8) years and 920 (53%) were female. Among all healthcare workers participating in the survey, 378 (22%) were health professionals (including nurses and technicians), 337 (20%) were specialized Medical Doctors, 258 (15%) were medical residents ( sectors . Most pan = 87) reported to have had a previous infection from SARS-Cov2 or to be currently infected, while 36% (n = 636) knew at least one family member or close contact who caught the infection. When asking participants to grade their health status from 0 (very bad) to 10 (excellent) the mean value among all the sample was 9 (SD 1.3), however 275 (16%) participants reported to perceive themselves to be at a higher risk of adverse outcomes due to COVID-19. Sixty-eight percent of respondents (n = 1173) reported to have vulnerable persons between their close contacts or cohabitants, in particular people over 65 , people with disability/chronic conditions  or immunocompromised or in treatment with immunosuppressors  , expert opinions , webinars and scientific meetings , media , Facebook groups  and journal websites . Twenty-four per cent (n = 405) of participants declared to be doubtful about their intention towards recommending of anti-SARS-CoV-2 vaccine to their patients, while 6% (n = 99) reported that they would not recommend vaccination to their patients.Five per cent of participants  . As to in = 810) of respondents reported to have been vaccinated against flu during the last flu season (2019–2020) while, when investigating their attitude during the current season (2020–2021), 79% (n = 1364) of participants declared their willingness to get flu vaccination  HCW reported they wanted to get vaccinated as soon as the vaccine would be available, while 26% (443) declared that they still did not know , and 125p-value < 0.0001) were documented in regards to age group, occupational profile, length of service, having persons at risk among close contacts or cohabitants, information sources on SARS-CoV 2 vaccination, intention towards patients’ COVID-19 vaccine recommendation, flu vaccine undertaken during last season and intention to be vaccinated during the current flu season. Other factors such as sex, geographic or medical area of work, previous infection among family members and close contacts did not have any significant association with vaccine hesitancy.When comparing COVID-19 vaccine hesitants and not , differep < 0.001). On the contrary, being a younger healthcare worker (<30 y) , being in close contact with an high-risk group  and having undertaken seasonal flu vaccine during the 2019–2020 season  resulted to be “protective” towards COVID-19 vaccine hesitancy  and using Facebook as main information source about anti-SARS-CoV-2 vaccination  remained significantly associated with an increased risk of vaccine hesitancy , and vaccine development , implementing an economic incentive  and making vaccination mandatory  (data not shown). To the question “In your opinion, which of the following initiatives would help to increase the number of health workers vaccinating against SARS-CoV-2?”, the answers, in descending order, were increasing information quality about the vaccine  and 45% (n = 767) of participants, while the role of SARS-CoV-2 vaccination by 48% (n = 820) and 35% (n = 609)  .n = 1155) reported to be willful to accept COVID-19 vaccination. Twenty-six percent of the participants indicated uncertainty and 125 HCW (7%) declared that they would refuse to be vaccinated. This finding is particularly striking, in our view, considering that the survey was administered at the verge of the Italian “second wave” [In this large survey conducted among 1723 Italian healthcare workers, only 67%  or conflicting (69%) information about the vaccines. At the same time, the vast majority of respondents reported mistrust on current containment measures.Incertitude related to the rapid development process of COVID-19 vaccines was the second most common reason for responding “No” or “Not sure” to intent to be vaccinated, given the high rate of hesitancy attributed to low trust in pharmaceutical companies and control authorities . These concerns seem to be specific for the new COVID-19 vaccines and do not apply to vaccination in general, as suggested by the impressively high rate of HCW who reported to have received vaccination against influenza for the year 2020–2021: 79% against 47% for the year 2019–2020. In addition, in the present study, respondents who received vaccination against flu for the 2019–2020 season were significantly more likely to report willingness to get vaccinated against SARS-CoV 2.Surprisingly, only 16% of respondents reported to feel at higher risk of bad outcomes due to COVID-19, but more than two thirds declared to have, among their cohabitants or close contacts, people belonging to high-risk categories  and, importantly, this factor resulted significantly associated with willingness to get vaccinated.At the time this paper is written, vaccines are expected to confer substantial benefit in reducing the overall burden and socio-economical stress related to COVID-19 , currentInterestingly, the rate of overall hesitancy towards anti-SARS-CoV2 vaccination reported in our study was similar to the one described in two multinational surveys conducted among the general population ,27. ThisOur study has several limitations. First, respondent selection was not randomized, thus not allowing our results to be generalized to the whole Italian HCW population due to the possibility of a selection bias. In particular, non-physicians HCW and people above 50 years old were likely underrepresented. Another obvious limitation of this study is that the rate of willingness to be vaccinated, and the underlying reasons sustaining hesitancy do not apply to the general population, as it falls outside the study aim. Furthermore, reporting one’s intent to be vaccinated might not necessarily mean that this individual will get vaccinated, as vaccine decision are multi-layered and considerably change over time . In factResults from anti-SARS-CoV2 vaccines phase III trials have outlined  that newGiven the need to achieve and keep the world population above herd immunity threshold for COVID-19, a continuous effort is going to be required from public health authorities to preserve trust and erode vaccine hesitancy. In this fight, healthcare workers will always play a major role, as they will keep being both a high-risk population and the first source to which patients will seek advice. We hope that our data could help governments, public health professionals and policy-makers to target educational interventions and communication in the upcoming COVID-19 vaccination campaign.


Pancreatic cancer remains one of the deadliest cancers worldwide, and it is typically diagnosed late, with a poor prognosis. Early detection is the most important underlying factor for improving the prognosis of pancreatic cancer patients. One of the most effective strategies for detecting cancers at an early stage is screening of the general population. However, because of the low incidence of pancreatic cancer in the general population, the stratification of subjects who need to undergo further examinations by invasive and expensive modalities is important. Therefore, minimally invasive modalities involving biomarkers and imaging techniques that would facilitate the early detection of pancreatic cancer are highly needed. Multiple types of new blood biomarkers have recently been developed, including unique post-translational modifications of circulating proteins, circulating exosomes, microRNAs, and circulating tumor DNA. We previously reported that circulating apolipoprotein A2 undergoes unique processing in the bloodstream of patients with pancreatic cancer and its precancerous lesions. Additionally, we recently demonstrated a new method for measuring pancreatic proton density in the fat fraction using a fat–water magnetic resonance imaging technique that reflects pancreatic steatosis. In this review, we describe recent developments in potential biomarkers and imaging modalities for the early detection and risk stratification of pancreatic cancer, and we discuss current strategies for implementing screening programs for pancreatic cancer. Pancreatic cancer remains one of the deadliest cancers worldwide. In the United States, the 5-year overall survival rate for all cancers diagnosed from 2009 through 2015 was 67%, whereas that for pancreatic cancer was only 9% . ObviousOne of the most effective strategies for detecting cancers at an early stage is screening of the general population. However, as screening the general population is costly, this approach can be less cost-effective if the incidence of the cancer of interest is very low. Thus, determining the incidence of pancreatic cancer is important for developing an effective strategy for early detection. Data from the Global Cancer Observatory website (GLOBOCAN)  indicateIn the general population, the lifetime risk of developing pancreatic cancer is estimated at approximately 1.6% based on United States data from 2015 to 2017 . HoweverIn August 2019, the US Preventive Services Task Force (USPSTF) updated their 2004 recommendations regarding screening for pancreatic cancer using imaging modalities. In the updated statement, the USPSTF did not recommend screening for pancreatic cancer in the general population using imaging methods . In addiThe relative risk for developing pancreatic cancer in patients with known hereditary syndromes associated with an increased risk of developing pancreatic cancer is reportedly 132-fold greater than that of the general population . Other tBRCA1/2, PALB2, ATM, CDKN2A, APC, MLH1, MSH2, MSH6, PMS2, PRSS1, and STK11 [BRCA1/2-mutant tumors are predicted to exhibit increased therapeutic sensitivity to platinum-containing therapeutic agents and inhibitors of poly-(ADP-ribose)-polymerase (PARP) [Investigations of genomic variation or mutations (germ-line mutations) have recently become more important and common in the clinical setting. The development of pancreatic cancer reportedly involves germ-line mutations in multiple genes, such as nd STK11 ,16. The nd STK11 . Genetice (PARP) .Multiple other risk factors related to lifestyle and underlying disease have also been reported. For example, smoking ,20, heavThe early detection and treatment of cancer has been made possible by understanding the precancerous lesions associated with each cancer type ,32,33. WPanIN is a non-invasive macroscopic lesion that occurs in the small pancreatic epithelium. Three types of PanIN were originally described (PanIN-1 to PanIN-3) in 2001 based on the degree of cellular and structural abnormalities . In 2015Although the natural history of PanINs cannot be monitored in living individuals, analyses using autopsy and surgically resected samples have provided several characteristics of the lesions. The number and severity of PanINs are reportedly associated with aging ,38 and pIPMN is an epithelial pancreatic neoplasm that produces mucin and forms cystic lesions. IPMN represents a broad group of pathologies. The grading system for IPMNs was updated in 2015 , such thIPMNs can arise from both the main and branch pancreatic ducts. Due to the increasing use of cross-sectional imaging in recent years, IPMNs are frequently detected incidentally . As mainAlthough both PanIN and IPMN are non-invasive epithelial neoplasms arising in the pancreatic duct, only IPMNs exhibit a morphologic lesion that can be detected by non-invasive imaging. Therefore, the presence of IPMNs is one of the few clues enabling the early detection of pancreatic cancer.As most patients with early stage pancreatic cancer are asymptomatic, establishing a general screening system is the only way to enable early detection. To this end, multiple blood biomarkers are being developed. However, to date, no biomarkers have been approved for the early diagnosis of pancreatic cancer. Therefore, blood tests that enable the detection of resectable pancreatic cancer and/or its precursor in individuals within the asymptomatic population with or without hereditary factors are urgently needed.The glycan carbohydrate antigen 19-9 (CA19-9) is one of the most important and widely used blood biomarkers in the diagnosis of pancreatic cancer. CA19-9 was first described in 1979 as a tumor antigen recognized by the monoclonal antibody NS19-9 . ElevateSeveral limitations preclude the use of CA19-9 as an early detection marker for pancreatic cancer. For example, CA19-9 levels can be elevated in several benign diseases involving not only the pancreas and biliary tract  but alsog overnight at 4 °C, followed by several additional steps [Exosomes are a type of extracellular vesicle (EV) that contains cytoplasmic components, including nucleic acids and soluble proteins. Several kinds of EVs have been described, and they are categorized based on size and biogenesis mechanism . Exosomeal steps . To overal steps . AlthougMicroRNAs (miRNAs) are functional nucleic acids encoded in the genome that undergo a multi-step process that ultimately forms a small RNA of 20 to 25 bases in length . miRNAs Analyses of various tissue samples revealed that several miRNAs, such as miR-21, miR-155, and mi-R 196, are upregulated in pancreatic cancer lesions compared to normal or re-cancerous lesions ,77,78,79n = 15) and healthy subjects (n = 15) and analyzed plasma exosome miRNA expression using qRT-PCR. They found that plasma exosome miR-196a and miR-1246 levels were significantly elevated in pancreatic cancer patients as compared to healthy subjects. The area under the curve (AUC) was 0.81  for miR-196a and 0.73  for miR-1246. Although the fold changes in relative expression levels relative to the control subjects were small (<2 in miR-196a and <3 in miR-1246), this study demonstrated the possibility of using miRNAs as early detection markers for pancreatic cancer.Xu et al. examined whether exosome miRNA could be used as an indicator of localized (stage I and stage IIA) pancreatic cancer . They coCell-free DNA (cfDNA) is composed of non-encapsulated DNA fragments circulating in the bloodstream. These fragments are released into the bloodstream from dying cells and usually removed by macrophages. The average length of a cfDNA is approximately 170 bases, with a half-life of approximately 2 h .The strategy for using cfDNA as an early cancer detection marker is the detection of cancer cell-derived cfDNA  among the total cfDNA. However, this approach has several technical difficulties. Although it was reported that cancer patients have 4 to 40 times higher levels of cfDNA than steady-state healthy individuals due to the overproduction of cancerous cells , increasKRAS gene [KRAS mutation [KRAS mutations are the earliest genetic alteration driving pancreatic neoplasia [KRAS is usually selected as the target gene in screening for pancreatic cancer ctDNA.In using ctDNA as a detection biomarker, a target gene for analysis should be identified. Ideally, it is desirable to target a gene that is mutated in a majority of pancreatic cancer cases. The term “pancreatic cancer” indicates a malignant tumor that occurs in the pancreas. The majority (85–90%) of these cancers are PDAC, and other subtypes are relatively rare ,92. It iRAS gene . Multiplmutation ,95. Moreeoplasia ,97,98. TKRAS mutations in plasma cfDNA as a marker for pancreatic cancer in a large case-control series [KRAS mutations in cfDNA was 10.3%, 17.5%, and 33.3% for samples from patients with local, regional, and systemic stages, respectively. Additionally, cfDNA KRAS mutations were also detected in 3.7% of samples from healthy controls and 4.3% of samples from chronic pancreatitis patients. Cohen et al. also assessed KRAS mutations in cfDNA in 221 patients with resectable PDAC and 182 control patients without known cancer [KRAS mutations in cfDNA was only 30% in the PDAC patients [KRAS mutations were detected in one sample from the control group (0.6%) [Le Calvez-Kelm et al. investigated the utility of detecting l series . They ann cancer . To imprn cancer . Howeverpatients . In addip (0.6%) .KRAS mutations in cfDNA exhibits limited sensitivity as an early detection marker for pancreatic cancer. However, recently, analyzing a patient’s cancer genome has become a common clinical test [KRAS.At this time, even with advanced sequencing technology, the identification of cal test ,103. AccAs discussed above, analyzing mutations in one gene using cfDNA reportedly has insufficient sensitivity to detect early stage pancreatic cancer. This sensitivity could be improved by increasing the number of analyzed genes; however, such analyses are costly. To implement the analysis of cfDNA as a marker for detecting early stage cancers, Choen et al. developed a blood test algorithm known as CancerSEEK, which is capable of detecting eight common types of cancer, including pancreatic cancer . From anAnother group reported the multiparametric analysis of blood samples to develop a novel biomarker signature of early stage PDAC . Using aCirculating serum/plasma proteins and peptides are known to undergo unique post-translational modifications specific to the molecular pathology of each disease. We recently identified a unique post-translational modification of circulating apolipoproein A2 (apoA2) homodimers that can be used to efficiently detect patients with PDAC who can undergo resectable surgery and also risk-stratify PDAC patients. We reported that apoA2-isoforms exhibit a unique processing pattern in pancreatic diseases, including PDAC ,107,108.Circulating apoA2 forms a homodimer, and it consists of 5 isoforms with different C-terminal amino acid sequences: -ATQ/ATQ, -ATQ/AT, -A2-AT/AT, -A2-AT/A, and -A2-A/A. Based on molecular weight, we named the isoforms apoA2-ATQ/ATQ, apoA2-ATQ/AT, apoA2-AT/AT, apoA2-AT/A, and apoA2-A/A. In the bloodstream of healthy subjects, 3 basic isoforms are typically detected in well-balanced proportion: apoA2-ATQ/ATQ, apoA2-ATQ/AT, and apoA2-AT/AT. In contrast, blood samples from patients with PDAC typically contain two aberrantly processed apoA2 isoforms of higher or lower molecular weight, with unique apoA2-isoform processing patterns observed. We defined these as a hypo-processing pattern, in which a heavy isoform (apoA2-ATQ/ATQ) is predominantly recognized, and a hyper-processing pattern, in which light isoforms  are predominantly recognized ,108. In To simplify the measurement of blood concentrations of the intermediate apoA2-ATQ/AT isoform, we developed an enzyme-linked immunosorbent assay (ELISA) in 2015 . A studyThe two aberrant processing patterns of apoA2 isoforms are also observed in individuals at high risk for developing PDAC, and analyses of these isoforms enables an efficient detection of IPMN, which is considered a precancerous lesion, as well as other pancreatic conditions  that predispose patients to pancreatic cancer ,109. TheImaging examinations are currently necessary for the diagnosis of pancreatic cancer. The imaging modalities commonly used to diagnose pancreatic cancer include transabdominal ultrasound, CT, MRI, and endoscopic techniques such as EUS and endoscopic retrograde cholangiopancreatography (ERCP). However, the endoscopic techniques are not suitable for large-scale screening because the examinations are relatively invasive and time consuming. As stated in Transabdominal ultrasound (US) is an imaging modality that allows non-invasive observation of abdominal organs. The reported sensitivity and specificity of transabdominal US for the detection of pancreatic cancer range from 75% to 89% and 90% to 99%, respectively ,114. RecCT is a less invasive imaging modality, and multiple studies have reported that CT is useful for mass screening to detect early stage lung cancer ,117. WitMRI is often used for the diagnosis of pancreatic cancer . One of The reported sensitivity of MRI for the detection of PDAC is 93% , as calculated from pooled data from 11 studies that included 349 PDAC patients, whereas the specificity and accuracy were 89%  and 90% , respectively . HoweverThe primary goals of imaging for the detection of early pancreatic cancer are the detection of solid masses, indirect signs of cancer, and precursor lesions . In addiBased on the above background, we developed a novel method for determining the pancreatic PDFF that calculates average fat fraction values based on all the voxels included in the three-dimensional pancreas B. Our meThe advantages and disadvantages of each biomarker and early detection method introduced in this review are summarized in -omics approaches to radiology are commonly called radiomics [Not only the combination of multiple blood tests but also a combination of blood tests and radiologic images has been studied. For example, an approach that combines blood testing of miRNA or cfDNA with radiographic features has been reported . Such soadiomics , and wheHowever, even if these combination analyses demonstrate high performance, their use in screening the general population may not be feasible when considering their cost-effectiveness. Thus, we need to switch our strategy to the development of a sequential combination of tests. The pool of individuals at high risk within the general population can be enriched using non-invasive biomarkers such as liquid biopsy, with subsequent screening of high-risk individuals using imaging techniques. Imaging modalities should be selected based on comparisons of cost and invasiveness versus effectiveness. If the performance of a blood test used as the first screening technique is quite good, we could proceed directly to more invasive examinations. However, if the performance is poor, less invasive cross-sectional imaging modalities, such as CT or MRI, may be better choices as the first imaging modality. Detailed high-resolution imaging surveys of subjects with high-risk diseases such as IPMN and pancreatitis could enable the detection of pancreatic cancer at a sufficiently early stage to permit curative resection. There is an urgent need to develop screening techniques combining biomarkers and imaging modalities that are non-invasive or minimally invasive to enable the early detection of pancreatic cancer.


The aim of this study was to investigate the incidence and clinical presentation of SARS-CoV-2 infections in a Systemic Lupus Erythematosus (SLE) cohort; to assess correlations with disease characteristics and rheumatic therapy; and to evaluate the occurrence of treatment discontinuation and its impact on disease activity.SLE patients monitored by a single Italian centre were interviewed between February and July 2020. Patients were considered to be positive for SARS-CoV-2 infections in case of 1) positive nasopharyngeal swab; 2) positive serology associated with COVID19 suggesting symptoms. The following data were also recorded: clinical symptoms, adoption of social distancing measures, disease activity and treatment discontinuation.332 patients were enrolled in the study. Six patients (1.8%) tested positive for SARS-CoV-2 infection, with the incidence being significantly higher in the subgroup of patients treated with biological Disease-Modifying Anti-Rheumatic Drugs (p = 0.005), while no difference was observed for other therapies, age at enrollment, disease duration, type of cumulative organ involvement or adoption of social isolation. The course of the disease was mild. Thirty-six patients (11.1%) discontinued at least part of their therapy during this time period, and 27 (8.1%) cases of disease flare were recorded. Correlation between flare and discontinuation of therapy was statistically significant (p<0.001). No significant increase of rate of flare in a subgroup of the same patients during 2020 was observed.Treatment discontinuation seems to be an important cause of disease flare. Our findings suggest that abrupt drug withdrawal should be avoided or evaluated with caution on the basis of individual infection risk and comorbidities. Severe acute respiratory coronavirus 2 (SARS-CoV-2) and related coronavirus disease-2019 (COVID19) represent a serious issue to public health, while data concerning SARS-CoV-2 infections in patients affected by Systemic Lupus Erythematosus (SLE) remain scarce .Patients with SLE represent an interesting study group due to being frequently exposed both to factors that may increase the risk of viral infections and related complications, and to drugs that may improve the outcome. Indeed, alterations in the immune response and immunosuppressive drugs make patients susceptible to viral infections , while gThe aims of this study were: firstly, to investigate the incidence and clinical presentation of SARS-CoV-2 infections in a SLE cohort monitored by a single Italian centre; secondly, to assess correlations with disease characteristics and anti-rheumatic therapies; thirdly, to evaluate the occurrence of treatment discontinuation and its impact on disease activity; finally, to compare the flare rate of the cohort in the pandemic season with that of the equivalent period in 2019.Based on previous reports, our hypothesis is that COVID-19 incidence and course is similar in SLE patients compared to other chronic diseases while personal and societal choices regarding the health emergency could have a significant impact on disease outcomes at least in the short term.This cross-sectional study involved adult patients with SLE satisfying the 2019 European League Against Rheumatism/American College of Rheumatology classification criteria , regularPatients were interviewed during telemedicine visits between February and July 2020 concerning any possible symptoms in the same time period suggesting SARS-CoV-2 infections, results of nasopharyngeal swab and serological test if available, close contacts with confirmed cases, adoption of social distancing measures, concomitant medications and adherence to them, and cases of disease flare. In the case of confirmed infections, we collected data regarding clinical course, need for hospitalisation and requirement for therapy or ventilatory support.We considered cases with positive nasopharyngeal swab or positive serology associated with COVID19 symptoms to be positive for SARS-CoV-2 infections.Disease activity was assessed according to Safety of Estrogens in Lupus Erythematosus: National Assessment-SLE Disease Activity Index (SELENA-SLEDAI)  as well Our sample can be considered representative of a larger population since we included consecutive patients scheduled for visits in our centre.This study was reviewed and approved by local ethical review board “Comitato Etico Area Vasta Nord Ovest”. Informed consent was obtained from patients orally during telemedicine visits, and it was registered on medical records.Continuous variables were described as median and 25–75 interquartile range (IQR) or as mean and standard deviation (SD), as appropriate. Categorical variables are reported as proportions. Cross-tabulated data were analysed using Chi-squared test and student’s T-test. Multivariate analyses were performed using the logistic regression model. P values <0.05 were considered to be statistically significant. All the statistical analyses were performed using STATA V.13 software.Three hundred and thirty-two patients (303 women) with SLE regularly monitored at our centre were enrolled in this study.The median age at enrollment was 47 years (IQR 37.3–56), and the median duration of the disease was 17 years (IQR 9–25). Cumulative organ involvement, concomitant medications, symptoms suggesting SARS-CoV-2 infections, cases of quarantine and contacts with confirmed COVID 19 cases of enrolled patients are reported in Forty-five (13.5%) patients were screened for SARS-CoV-2 antibodies, and two of them  were confirmed to be positive. Testing for SARS-CoV-2 infection using nasopharyngeal swab was performed in 36 patients (10.9%) and 5 of them  were positive.Six patients (1.8%) were positive for SARS-CoV-2 infection and their characteristics and course of infections with related therapy are detailed in Three of these patients (50.0%) required hospitalisation, but none were admitted to an Intensive Care Unit (ICU). Patients 5 was asymptomatic, but her hospitalization was not COVID-19-related.Two patients were treated with low-flow oxygen, and one of them also with antibiotics and medium-high dose of GCs. All patients continued therapy with HCQ and low dose of GCs, both of which began before the pandemic period. One of the positive patients presented articular flare. No other cases of disease flare were recorded in the other five patients, despite the temporary discontinuation of immunosuppressant or biological drugs, and no deaths were recorded.We compared characteristics between patients with and without COVID-19 and no differences were observed with regard to age at enrolment, disease duration or type of cumulative organ involvement.On the other hand, bDMARDs treatment was more frequently assumed by patients with COVID-19 with respect to patients without COVID-19 , while no differences were observed for other therapies nor in cases of combined therapies with more DMARDs or GCs and DMARDs. The association between bDMARDs and SARS-CoV-2 didn’t lose significance at multivariate analysis after correction for disease severity .In the whole cohort, 36 patients (11.0%) discontinued at least one drug for SLE as a result of their physician’s advice or personal choice.During the study period, 27 (8.1%) cases of disease flare were recorded, all of which were mild/moderate. We found a significant correlation between flare and discontinuation of therapies (p<0.001).In the same period (February-July) of 2019, we assessed 147 patients of the 332 enrolled for this study at the clinic. By comparing the same patients in the two time-frames, we can confirm there is no significant difference in term of incidence of flare (11.6% in 2019 vs 8.8% in 2020).Data on social isolation were available only for 193 patients. Social isolation required patients to stay at home at all times, avoiding all face-to-face contact outside home. Within this group, 100 were isolated, and flare occurrence was not significantly different versus not isolated patients (7.1% vs 7.9%). COVID-19 infection occurred in 1 isolated and 5 not isolated patients, and the differences did not reach the threshold of statistical significance (p = 0.08).The results of statistical analysis of characteristics between patients whit COVID-19 and patients without COVID-19, including all p-values for both significant and non-significant results, are reported in In our cohort, the incidence of SARS-CoV-2 infection in SLE patients over a six-month period was 1.8%, and the disease course was mild, with no admittance to ICU.Although a direct comparison is difficult, this was slightly higher than reported in the general Italian or Tuscan population in the same period , 11 Click here for additional data file.


Pro-inflammatory markers play a significant role in the disease severity of patients with COVID-19. Thus, anti-inflammatory therapies are attractive agents for potentially combating the uncontrolled inflammatory cascade in these patients. We designed a trial testing tocilizumab versus standard of care intending to improve the outcomes by inhibiting interleukin-6, an important inflammatory mediator in COVID-19. This open-label multicentre randomized controlled trial will compare clinical outcomes of tocilizumab plus standard of care versus standard of care alone in patients with moderate to severe COVID-19. Two of the following four criteria are required for protocol enrolment: D-dimer > 1,000ng/mL; C reactive protein > 5mg/dL, ferritin > 300mg/dL, and lactate dehydrogenase > upper limit of normal. The primary objective will be to compare the clinical status on day 15, as measured by a 7-point ordinal scale applied in COVID-19 trials worldwide. The primary endpoint will be assessed by an ordinal logistic regression assuming proportional odds ratios adjusted for stratification variables (age and sex). The TOCIBRAS protocol was approved by local and central  ethical committees in Brazil following current national and international guidelines/directives. Each participating center had the study protocol approved by their institutional review boards before initiating protocol enrolment. The data derived from this trial will be published regardless of the results. If proven active, this strategy could alleviate the consequences of the inflammatory response in COVID-19 patients and improve their clinical outcomes. The new virus was first named 2019-nCoV, and little was known about its pathogenesis, transmissibility, and lethality rates.,2 Cases started to spread within China and in 2020 beyond its borders to all continents (except Antarctica). In February 2020, the World Health Organization (WHO) adopted the official name SARS-CoV-2 (instead of 2019-nCoV), and the syndrome associated with the new coronavirus infection was named COVID-19, and declared a pandemic in mid-March. By June 2020, almost 9 million cases and 470 thousand deaths have been reported worldwide. In Brazil, the first reported cases were in late February, and by June, the country accumulates more than one million cases and 50,000 deaths.At the end of 2019, a new coronavirus outbreak in Wuhan (China) made headlines as cases of severe pneumonia and deaths began to emerge.,6 The rapid rise in cytokines reminiscent of that seen in chimeric antigen receptor therapy (CAR-T) is proposed to be an essential element in the rapid clinical deterioration.-9 The COVID-19 SIRS is usually observed in the second week of infection, where a slew of pro-inflammatory signals are triggered, leading to respiratory compromise, and multiorgan failure. After SARS-CoV-2 initially binds to the angiotensin-converting enzyme 2 (ACE2) receptor, several cell types are infected thereafter from the respiratory, gastrointestinal, neurologic, endothelial, and reticuloendothelial systems, giving rise to a wide clinical presentation ranging from oligosymptomatic to multiorgan failure and death.-15 Clinical and laboratory risk factors associated with worse outcomes are being recognized as the pandemic unfolds globally.-18The development of pneumonia progressing to a systemic inflammatory response syndrome (SIRS) and multiorgan failure indicates that pro-inflammatory cytokines are a significant contributor to morbidity and mortality in SARS-CoV-2 infection. Other systems, such as the coagulation cascade, are activated by the crescent inflammatory response, leading to thrombotic and hemorrhagic events both in the lung and systemically.,20 This multisystem havoc triggered by SARS-CoV-2 is now recognized to be a critical element to its pathogenesis; this rationale has guided the proposal of therapeutic strategies that could possibly block its more lethal consequences. For example, an old drug, etoposide, and several more targeted approaches emerged in response to the macrophage activation, including anti-cytokine, anti-chemokine, anti-complement therapies, and Janus kinase (JAK) and Bruton tyrosine kinase (BTK) inhibitors.,21 It is likely that, if proven active, the benefit of these therapies will be incremental, and a combination strategy with antivirals, anticoagulants, or possibly convalescent plasma, will provide a better therapeutic approach.-24Macrophage infection and activation, initially in the lungs and then systemic, emerges as an essential source of pro-inflammatory cytokines such as interferon gamma (IFN-γ), interleukin (IL)-6, IL-12, tumoral necrosis factor (TNF), IL-1RA, and C-X-C motif chemokine 10 (CXCL10) among others.,26 Preliminary data have shown that tocilizumab and other IL-6 blockers may have a role in severe SIRS cases, but its role in COVID-19 patients has not yet been confirmed, and its use should be considered experimetal.,28 Given its mode of action, it is plausible that tocilizumab is active in patients with already established SIRS (that could worsen), as opposed to the early use during the infection cycle with no demonstrable inflammatory activity. Thus, to test this hypothesis, we devised a trial where tocilizumab will be tested to avert the more lethal consequences of COVID-19-related SIRS. To investigate the effects of blocking an ongoing inflammatory response, we designed a randomized controlled trial comparing the efficacy of tocilizumab plus standard of care to standard of care alone in patients with moderate to severe SARS-CoV-2 infection who require supplemental oxygen and have increased systemic inflammation markers levels. The requirement of having increased levels of systemic inflammatory markers, in our hypothesis, will better select those more likely to benefit from this strategy.Tocilizumab is an IL-6 inhibitor approved for rheumatoid arthritis, giant cell arteritis, and cytokine release syndrome during CAR-T.The TOCIBRAS  was developed according to SPIRIT guidelines  and regiBP - A Beneficência Portuguesa de São Paulo, HCor-Hospital do Coração, Hospital Israelita Albert Einstein, Hospital Sírio-Libanes, Hospital Alemão Oswaldo Cruz, Hospital Moinhos de Vento, Brazilian Clinical Research Institute (BCRI), and the Brazilian Research in Intensive Care Network (BRICNet)  .Confirmed diagnosis of SARS-CoV-2 infection.Chest computed tomography (or chest X-ray) consistent with COVID-19. COVID-19-related symptoms for more than three days.18 years of age or older.2) > 93% OR need for mechanical ventilation less than 24 hours before the randomization.Need for oxygen supplementation to maintain oxygen saturation  > 5mg/dL.Ferritin > 300mg/dL.Lactate dehydrogenase (LDH) > upper limit of normal.Need for mechanical ventilation for 24 hours or more before the randomizationHypersensitivity to tocilizumabPatients without therapeutic perspective or in palliative careActive non-controlled infections (other than COVID-19)Neutrophil count < 0.5 x 109/LPlatelet count < 50 x 109/LLiver disease, cirrhosis or elevated aspartate aminotransferase (AST) or alanine aminotransferase (ALT) above 5 times the upper limit of normal.Renal disease with estimate glomerular filtration below 30mL/min/1.72 m2 .Breastfeeding women.Pregnancy.Other clinical conditions that contraindicate tocilizumab, according to the attending physician.To assess the effect of tocilizumab plus standard of care compared to standard of care alone on the clinical status on day 15, as measured by a 7-point ordinal scale, in adults hospitalized patients with moderate to severe COVID-19. The 7-point ordinal scale applied in this study is as follows: Patient not hospitalized, with no limitation in activities.Patient not hospitalized, with limitation in activities.Patient in the hospital, without supplemental oxygen.Patient in the hospital, with supplemental oxygen.Patient in the hospital on non-invasive positive pressure ventilation (NIPPV) or high flow nasal cannula.Patient on mechanical ventilation.Death.To assess the treatment effect with tocilizumab plus standard of care compared to standard of care, in adults hospitalized with COVID-19, on the following outcomes:All-cause mortality from randomization until day 28.Hospital mortality.Degree of organ dysfunction assessed with the Sequential Organ Failure Assessment (SOFA) score on day 8 and day 15 after randomization.Clinical status on days 8 and 29 after randomization, using the 7-level ordinal scale. Ventilator-free days within 29 days.Time until oxygen support independence within 29 days.Length of hospital stay.Incidence of secondary infections. Thromboembolic events .Incidence of adverse events (AE). To assess the inflammatory markers and cytokines’ association with the clinical outcomes.To assess the kinetics of hemostatic parameters, inflammatory markers, cytokines, peripheral blood flow cytometry, complete blood count, renal and liver function tests. To assess the viral clearance of SARS-CoV2 on D8.The following exploratory tests will be conducted and correlated with clinical outcomes:- Biomarker measurements: D-dimer, CRP, LDH, ferritin, IL-6, TNFα, IL2 receptor (CD25), and IL-10. - Peripheral blood cytometry for T , B  and NK  lymphocytes, monocytes , plasmacytoid and myeloid dendritic cells, eosinophils, basophils, and neutrophils. - Coagulation studies PT/PTT, fibrinogen, vWF, ristocetin cofactor, and factor 8.Details of the methodology for all these tests is provided in The concomitant use of hydroxychloroquine, azithromycin, corticosteroids, or other therapies are allowed in this trial as part of the standard of care if local institutional guidelines/protocols include these agents as part of the standard management for COVID-19 patients. Antibiotics are permitted at any time during the study at the discretion of the treating physician. The standard of care treatment for COVID-19 is not yet defined thus investigators can apply what is considered their standard approach for these patients per local policies. After providing written informed consent, the eligible patients will be randomized to receive either tocilizumab plus standard of care (n = 75) or standard of care alone (n = 75). In the experimental arm, tocilizumab will be given as a single intravenous infusion at 8 mg/kg dose. The maximum dose is 800 mg. To allow for homogenous dose rounding between centers, the following bodyweight-based scale will be applied:- < 50kg - 8mg/kg/dose.- From 50 to 56kg - 400mg/dose. - From 57 to 68kg - 500mg/dose. - From 69 to 81kg - 600mg/dose. - From 82 to 93kg - 700mg/dose. - ≥ 94kg to 800mg/dose (max dose).No dose adjustment for renal or hepatic impairment will be used. Infusion-related reactions will be treated with antihistamines and corticosteroids as needed and reported as AE. All infusions will be administered in hospitalized patients in a regular ward or intensive care unit. The participants will be randomly assigned to either the experimental (tocilizumab) or the control (standard of care) group with a 1:1 allocation considering blocks  with random variation and using age (< 60 and ≥ 60 years) and gender as strata, according to a computer-generated randomization schedule. The random sequence will be generated by a statistician not involved with patient’s care using a Software R 3.6.3 algorithm.,31Allocation concealment is ensured by a web-accessed system (REDCap) which only displays the assigned treatment after the participant is properly registered into the trial system and all eligibility criteria are met.Patients, investigators, and caregivers will not be blinded to the assigned treatment. Clinical outcome assessors and statisticians performing the analysis are not involved with the patient care teams and are independent of the treating sites.BP - A Beneficência Portuguesa de São Paulo.,31REDCap is a secure, web-based software platform designed to support data capture for research studies, providing an intuitive interface for validated data capture; audit trails for tracking data manipulation and export procedures; automated export procedures for seamless data downloads to common statistical packages and procedures for data integration and interoperability with external sources.Study data will be collected and managed using Research Electronic Data Capture (REDCap) electronic data capture tools hosted at  This system’s functionalities include patient registration, concealment of randomization, data entry, data cleaning, data export for statistical analysis, and event adjudication. Clinically relevant laboratory assessments will be conducted in real-time, while those included in the exploratory list will be analysed in a later time, from frozen serum or plasma. Data from the primary and secondary endpoints will be included in REDCap in the appropriate time points. The independent Data Monitoring Committee (DMC) will have full access to the study data .Data will be entered by the local study personnel who have a unique center designated access, which is not transferable. A limited number of staff members are provided access to REDCap for study-related interactions.The data will be kept on the institutional server, under the recommended REDCap Consortium security requirements.The follow-up data for this trial are collected until day 29. A one-year follow-up study of the hospitalized patients enrolled in all Coalition trials will keep monitoring those patients enrolled in this study accepting to participate. Patients who die within this period will be censored at the time of death, and those still hospitalized will be followed on the study until discharge before enrolling in the long-term study. Contacts by telephone and other means will be used to ensure the highest possible retention rate during follow-up. We anticipate a high retention rate up to D29, considering that most of the patients will still be recovering from their recent viral pneumonia. Adherence will be monitored by reconciling protocol specified data collection and the data entry into REDCap.Considering an ordinal seven stages outcome with probabilities of 30%, 20%, 8%, 8%, 4%, 15%, 15%, respectively for stages 1 to 7, under the model of proportional odds ratios for the accumulated probabilities for the outcome levels, a sample of 75 cases per arm (150 cases) has an 80% power to detect an odds ratio of 0.44, with a 5% significance level.The main analysis will follow the intention-to-treat principle. The primary endpoint will be assessed by ordinal logistic regression assuming proportional odds ratios adjusted for the stratification variables (age and gender). Logistic regression models will assess binary cumulated outcomes. If odds proportionality does not hold in the final analysis, the primary outcome will be changed to a binomial endpoint collapsing categories 1 to 5 and 6 to 7 . Secondary outcomes will be evaluated by generalized linear regressions using appropriate distributions. All models will be adjusted for age and the results will be presented with their 95% confidence intervals for the effect measures. Subgroup analyses will be presented in forest plots evaluated with interaction terms of group and the following variables: age (< or > 60 years), sex, types of comorbidities , and inflammatory markers increased at the time of the study entry. Analyses will be performed with R software.An interim analysis will be performed when 50% of the planned accrual is reached (n = 75). The interim analysis will be performed by an independent DMC who will analyze efficacy and safety data, recruitment rate, adherence to the protocol, data quality, and follow-up losses. These data will be provided by the study coordinating site to the committee via a report. The Lan DeMets method and O’Brien Fleming thresholds will be applied as pre-defined criteria for study interruption. If the committee recommends continued accrual, the study will go onto completion (n = 150).Adverse events are defined as any untoward medical occurrence, including an exacerbation of a pre-existing condition, in a subject during a clinical trial. The event does not necessarily have to have a causal relationship with the treatment.A serious AE is defined as any AE that results in death, offers immediate risk to life, results in persistent or significant disability, requires or prolongs the patient’s hospitalization, or if it is a major medical event that based on proper medical judgment could threaten the patient’s life or could require medical or surgical intervention to prevent one of the other results listed above. Adverse events classified as serious will be notified within 24 hours to the coordinating site.The AEs will be graded according to the most recent version of the Common Terminology Criteria for Adverse Events (CTCAE). Monthly monitoring will evaluate the integrity of the screening, inclusion/criteria, and data reporting from the sites for adequacy and consistency. Every 15 days, a statistical methodology will be applied to look at inconsistency and data errors. Given the biologic characteristics of tocilizumab, the following AEs will be of specific interest and will be captured: - Secondary infections.- Anemia.- Liver function test abnormalities.- Diverticulitis. - Herpes zoster.- Headache.- Hemorrhage.- Thromboembolic events.- Serious infusion-related toxicities.Comissão Nacional de Ética em Pesquisa - Conep) is the entity in Brazil regulating ethical standards for clinical research. TOCIBRAS was developed and will be conducted under the normative set forth by Conep, which adopts international ethical standards in clinical research. Each participating center had the study approved at their institutional review boards before initiating protocol enrolment. A first amendment was submitted to both central and local ethical committees, dealing with issues that were not clear in the original version of the protocol. This amendment did not change the primary or secondary endpoints, the study design, statistics, safety/efficacy monitoring, Informed Consent, exclusion criteria, or interim analysis. The inclusion criteria were not unchanged, except for the limit for the time of intubation up to 24 hours before the inclusion, as the benefit of an anti-inflammatory strategy, including tocilizumab, is more likely to be seen before a fully established SIRS.The TOCIBRAS study was approved by local and central  ethical committees following current national and international guidelines/directives. The National Committee in Research and Ethics  before it becomes fully established with multiorgan compromise. This approach differs, for example, from antiviral strategies where the more significant benefit is anticipated in the earlier stages of the infection (first week). Therefore the study was designed to detect a difference of tocilizumab plus standard of care versus standard of care in this more select ‘window’ in the disease course in moderate to severe COVID-19. As with most COVID-19 studies, assumptions had to be made in regards to anticipated treatment differences given the scarcity and inconsistency in the best available data available at the time of protocol development in regards to outcomes of patients with COVID-19. Nevertheless, we included a robust design and several secondary clinical and laboratory exploratory endpoints, which will allow us to detect if there is an activity of tocilizumab in this patient population. Our work will complement all the anti-inflammatory and other approaches being developed to mitigate the consequences of COVID-19.- The randomized controlled study design permits a more definitive conclusion regarding the activity of tocilizumab in COVID-19.- The well-defined patient population with a higher propensity to develop inflammatory complications of COVID-19 may define the subset of patients in which a potent anti-inflammatory approach is warranted.- The 7-point ordinal scale is a robust endpoint for COVID-19 trials.- The study is not blinded which presents a weakness of the study design.- The total n is relatively small for a controlled study with more limited statistical power.Click here for additional data file.