Blood pressure (BP) lowering reduces the risk of recurrent stroke after intracerebral haemorrhage (ICH). However, implementation of BP lowering in clinical practice in the UK is unknown.We identified adults with first-ever incident ICH to quantify the proportion who survived >14 days after hospital discharge and were prescribed BP-lowering medication in a prospective, population-based, inception cohort study in the Lothian region of Scotland during June 2010–May 2012 and January–December 2019. After the first cohort, we analysed reasons for avoiding BP-lowering medication in a sample from the Lothian region of the Scottish Stroke Care Audit during January 2017–November 2017, which informed a quality improvement intervention that was implemented in the second cohort.After efforts to improve monitoring and lowering of BP amongst ICH survivors, there was an increase in the proportion of patients prescribed BP-lowering medication at hospital discharge between the first and second population-based cohorts . Compared with patients not prescribed BP-lowering medication at hospital discharge, patients prescribed BP-lowering medication presented with higher systolic BP , and were more likely to have pre-morbid hypertension  and atrial fibrillation .In this population-based study, the proportion of patients with ICH who were prescribed BP-lowering medication at hospital discharge increased after a quality improvement intervention. Our findings informed a quality improvement intervention that aimed to increase the use of BP monitoring and BP-lowering medication in Lothian. We compared the proportion of ICH survivors who were prescribed BP-lowering medication at hospital discharge in a population-based cohort study before and after the quality improvement intervention to identify a temporal change in practice, and assessed patient characteristics associated with the prescription of BP-lowering medication to identify areas for further improvement.http://www.isdscotland.org), the Scottish Stroke Care Audit (http://www.strokeaudit.scot.nhs.uk), and records at the Office of the Procurator Fiscal.8We identified patients with ICH  in The Lothian Audit of the Treatment of Cerebral Haemorrhage (LATCH) from 1 June 2010 to 31 May 2012, and 1 January 2019 to 31 December 2019. LATCH ascertained cases from the Lothian Health board region of Scotland  in residents aged ≥16 years diagnosed with first-ever or recurrent ICH  through multiple routes: prospective hot pursuit sources through a trust-wide network of physicians, neurologists, neurosurgeons, radiologists, pathologists, specialist nurses and audit personnel, daily neuroradiology meetings and review of all computed tomographic (CT) head scans; and retrospective searches of electronic patient notes, International Classification of Diseases 10th Revision (ICD-10) coded hospital discharge records  database to identify patients with ICH in Lothian from 2 January 2017 to 9 November 2017, and examined their hospital records to identify reasons why BP-lowering medication was not prescribed at hospital discharge.9We defined ICH as a symptomatic event , referable to a discrete parenchymal bleed . We included patients with incident first-ever ICH presumed due to cerebral small vessel disease who survived >14 days after hospital discharge. We reviewed patients’ medical records and investigations to exclude cases of recurrent ICH, isolated extra-axial intracranial haemorrhage, and ICH definitely attributable to a macrovascular cause, tumour, trauma, and haemorrhagic transformation of ischaemic stroke. Standard clinical practice in NHS Lothian involves targeted use of CT angiography informed by clinical predictors of the risk of underlying macrovascular pathology.blood pressure-lowering therapy should be prescribed by hospital discharge if systolic BP is >130 mm Hg, unless there is a contra-indication to the use of these drugs .’ Therefore, the primary outcome was the prescription of BP-lowering medication at hospital discharge, irrespective of prior BP-lowering medication use or prior hypertension diagnosis.The LATCH audit standard relating to long-term BP lowering after ICH is as follows: ‘10 by implementing a quality improvement intervention on 1 November 2018 . The intervention involved the introduction of Lothian-wide guidance on the treatment of ICH , and the opportunity for ICH survivors to use a home BP tele-monitoring service (Florence tele-monitoring [https://www.getflorence.co.uk/]) or participate in one of two ongoing research studies of BP monitoring and BP lowering after ICH .The 2017 audit indicated that there was a persisting unexplained deficit in proportion of ICH survivors that received a prescription for BP-lowering medication at hospital discharge, so we sought to improve the availability and use of BP monitoring and BP-lowering medication where it was indicated by clinical guidelines10To promote the quality improvement intervention, we organised a series of education sessions about ICH-ACT and the various BP monitoring and lowering activities for key stakeholders in secondary care . We worked closely with clinical colleagues to maintain engagement with the intervention: this included weekly departmental meetings attended by stroke clinicians, and monthly email reminders to LATCH collaborators . We anticipated that these activities would improve the uptake of guideline-recommended BP management by raising awareness of this important intervention amongst patients and their hospital-based healthcare practitioners, and by providing several options for their long-term BP management.11 In view of prescribing patterns in the first population-based cohort, we assessed reasons for the avoidance of BP-lowering medication in the audit and the second population cohort (these data were not available for the first population-based cohort). To assess participation in several BP-monitoring activities available from 2018 onwards, we collected information about the long-term BP monitoring plan from the electronic discharge summary in the second population cohort.We retrospectively collected prospectively recorded data on BP-lowering medication use after ICH from paper and electronic patient records associated with the corresponding hospital admission including initiation of, or changes to, BP-lowering medication during admission and BP-lowering prescription on discharge. In the population-based cohorts, we extracted additional data from patients’ medical records: medical history; use of BP-lowering, antiplatelet and anticoagulant medication at ICH diagnosis; clinical data on admission ; and haematoma characteristics on baseline CT brain scan (anonymised scans were prospectively collected and held on a secure server). We rated ICH location on baseline CT brain scans according to the CHARTS rating system.To identify a temporal change in practice, we compared the proportion of ICH survivors who were prescribed BP-lowering medication at hospital discharge before (first population-based cohort) and after (second population-based cohort) the quality improvement intervention using the chi-squared test. We assessed the associated change in the proportion of ICH survivors who were prescribed BP-lowering at hospital discharge in a multivariable logistic regression model adjusted for clinically important baseline confounders, which included age (≥80 years vs. <80 years), Glasgow coma scale (GCS) score (≤12 vs. >12), ICH location , presence of intraventricular haemorrhage, presence of any cardiovascular comorbidity, and prior use of anticoagulation. We also compared the two population-based cohorts by their patient characteristics, stratified by prescription of BP-lowering medication at hospital discharge (yes vs. no), using the Kruskal-Wallis test for continuous variables, and the chi-squared test for categorical variables. The significance level was set at P = 0.05.In the first population-based cohort , there were 268 cases of incident first-ever ICH of whom 130 (49%) survived >14 days after hospital discharge . Among 1The subsequent audit that informed the quality improvement intervention  revealed that of 61 patients with ICH who survived >14 days after hospital discharge, 33 (54%) were prescribed BP-lowering medication, 19 (31%) were not prescribed BP-lowering medication and had a reason for avoiding them and 9 (15%) were not prescribed BP-lowering medication and did not have a clear reason for avoiding them .Following the introduction of the quality improvement intervention, the second population-based cohort  revealed that of 136 cases of incident first-ever ICH, 89 (65%) survived >14 days after hospital discharge. A comparison of the baseline characteristics of the first and second population-based cohorts is shown in Supplementary Table 1: compared to the first population-based cohort, anticoagulant use was more common, and subarachnoid extension less common in the second population-based cohort. Among 89 patients with ICH who survived >14 after from hospital discharge, 68 (76%) patients were prescribed BP-lowering medication, 15 (17%) patients were not prescribed BP-lowering medication and had a reason for avoiding them, and 6 (7%) patients were not prescribed BP-lowering medication and did not have a clear reason for avoiding them . CompareThe distribution of reasons for avoiding BP-lowering medication was similar in the audit and second population-based cohort: among patients with a reason for avoiding BP-lowering, there were 11/19 (58%) and 9/15 (60%) with systolic BP levels <130 mm Hg; 5/19 (26%) and 3/15 (20%) with frailty/poor function; and 3/19 (16%) and 3/15 (20%) with a contraindication to BP-lowering medication in the audit and second population-based cohort, respectively.Compared with patients not prescribed BP-lowering medication at hospital discharge, patients prescribed them presented with higher systolic BP , and were more likely to have pre-morbid hypertension  and atrial fibrillation , and be prescribed BP-lowering medication (none vs. one vs. many: P < 0.001 and P < 0.001) and anticoagulant medication (none vs. one: P = 0.003 and P = 0.041) at symptom onset. Overall, there were 48/130 (37%) and 19/89 (21%) prescribed antiplatelet medication, and 13/130 (10%) and 18/89 (20%) prescribed anticoagulant medication at the time of ICH onset in the first and second population-based cohorts, respectively.In two epochs of this prospective, population-based, longitudinal cohort study and an interim audit, between half and three quarters of ICH survivors were prescribed BP-lowering medication at hospital discharge; around a third of patients who did not receive BP-lowering medication did not have a clear reason for avoiding them. In 2019 (the second population-based cohort), there was a significant increase in the proportion of patients who received BP-lowering medication to 76% from 62% in the first population-based cohort; 24% were prescribed 3 or more agents, and the use of calcium channel blockers overtook renin-angiotensin system blockers. Premorbid hypertension and atrial fibrillation were more common among patients prescribed BP-lowering treatment at hospital discharge compared with patients who were not; age, sex and the proportion of patients with deep/infratentorial haematoma location were similar.12 and updates to the UK National Clinical Guidelines for Stroke in 20127 and European Stroke Organisation guidelines in 2014,10 prescription of BP-lowering medication for the secondary prevention of recurrent stroke after ICH could be further improved. Yet, while little change occurred between 2010–2012 (first population-based cohort) and 2017 (audit), we found an increase in the proportion of ICH patients that received BP-lowering at hospital discharge in 2019 (second population-based cohort), which was associated with the introduction of a quality improvement intervention involving Lothian-wide guidance on ICH management and opportunities to recruit patients to two randomised controlled trials (TRIDENT and PROHIBIT-ICH) and a home BP tele-monitoring initiative (Florence) from 2018 onwards. These findings are encouraging, and may reflect increasing awareness of the benefits of BP-lowering after ICH among clinicians due to embedded quality improvement.Despite evidence of benefit emerging from the PROGRESS trial nearly two decades ago,13 61% in a large US study4; and when considered alongside all-cause strokes, varying but substantial under-prescribing is reported in Canada14 and the UK.15 To understand patient factors that influence prescribing habits, we stratified patients by population-based study epoch and BP-lowering medication prescription at hospital discharge. We found pre-morbid hypertension and atrial fibrillation were more common among patients who received BP lowering in first and second population-based cohorts, and hyperlipidaemia was more common in patients who received BP lowering in the second population-based cohort; differences in prior myocardial infarction, transient ischaemic attack, and diabetes mellitus did not meet statistical significance. These findings are consistent with those from an Australian registry where detailed comorbidity data were collected in 1,498 ICH patients,13 and when considered together, probably reflect the continued use of pre-morbid BP-lowering medication in patients with a diagnosis of hypertension or other cardiovascular comorbidity, and β-blockers prescribed for atrial fibrillation. The PROGRESS trial results showed that benefits from BP-lowering treatment are likely to exist irrespective of baseline BP and other cardiovascular comorbidities.12 Therefore, it is important to reach all patients without a contraindication to BP-lowering medication.Our findings are similar to those from other observational studies of prescribing practice after ICH: 74% of patients were prescribed BP-lowering medication after ICH in a recent, large Australian study,16 where BP lowering to a systolic BP target <140 mm Hg is recommended during the first 7 days due the potential for modest benefits on function and health-related quality of life,17 followed by continued treatment to achieve systolic BP levels <130 mm Hg in the long term. However, haematoma location, and presence of extension into intraventricular, subarachnoid and subdural spaces did not significantly differ according to discharge BP lowering. These findings are encouraging in view of evidence to suggest benefits from long-term BP lowering exist in both lobar and non-lobar ICH,18 although our small sample size may have precluded the detection of any true differences.By the same criteria, we assessed differences in admission BP and haematoma characteristics on diagnostic CT scan: compared with patients who were not prescribed BP-lowering medication at hospital discharge, patients prescribed them had significantly higher BP on admission to hospital. We also identified that a systolic BP level <130 mm Hg was a common reason for avoiding BP-lowering medication. This indicates some degree of concordance with clinical guidelines,20; and more frequent use of anticoagulants in patients with atrial fibrillation, where reductions in the risk of ischaemic stroke are greater than in patients taking antiplatelets.21 However, our small sample and multiple comparisons increase the likelihood of chance findings.A key methodological strength of this study is its population-based design: to our knowledge, all incident ICH cases in the Lothian population were captured through multiple sources of ascertainment. Patient characteristics associated with the prescription of BP-lowering medication at hospital discharge were internally consistent in two population-based cohorts separated by nearly a decade. These similarities suggest that changes in the proportion of patients who were prescribed BP-lowering medication may have resulted from the introduction of quality improvement measures rather than changes in patient characteristics. Other temporal changes in prescribing appeared to reflect emerging evidence, including: more frequent use of calcium channel blockers at hospital discharge where data suggest greater efficacy for stroke prevention22 randomised evidence from the TRIDENT randomised controlled trial is awaited to determine the effects of this strategy on recurrent stroke and vascular events in ICH survivors.We acknowledge there are limitations to our study. First, the increase in the proportion of patients with ICH who were prescribed BP-lowering at hospital discharge associated with the quality improvement intervention is prone to confounding from unmeasured factors, including temporal changes in practice related to early intensive BP lowering. Second, in the absence of long-term follow up data from patients identified by our population-based study in 2019, we were unable to assess temporal changes in the level of concordance with treatment in the community, nor the proportion achieving optimal systolic BP control in the longer term. In view of our finding that multi-agent treatment regimens are common among ICH survivors, and recent evidence to suggest a fixed low-dose polypill in addition to standard care is safe, tolerable and lowers BP more than standard care alone in middle-aged adults with hypertension,In summary, the prescription of BP-lowering medication at hospital discharge for the prevention of recurrent stroke after ICH over the last decade could be improved upon. A quality improvement intervention that involved Lothian-wide guidance on the management of ICH, and the opportunity for ICH survivors to participate in several BP monitoring and BP-lowering activities, was associated with an increase in the proportion of patients who received BP-lowering medication at hospital discharge. Further improvements may be possible if BP lowering is considered in patients without a previous diagnosis of hypertension or other cardiovascular comorbidities.Click here for additional data file.Supplemental material, sj-pdf-1-eso-10.1177_2396987320975724 for Prescription of blood pressure lowering treatment after intracerebral haemorrhage: Prospective, population-based cohort study by Karl Bonello, Amy PK Nelson, Tom J Moullaali, Rustam Al-Shahi Salman and for the Lothian Audit of the Treatment of Cerebral Haemorrhage Collaborators in European Stroke Journal


Using the Council of State and Territorial Epidemiologists (CSTE) classification guidelines, we characterized coronavirus disease 2019 (COVID-19)–associated confirmed and probable deaths in Puerto Rico during March–July 2020. We also estimated the total number of possible deaths due to COVID-19 in Puerto Rico during the same period.We described data on COVID-19–associated mortality, in which the lower bound was the sum of confirmed and probable COVID-19 deaths and the upper bound was excess mortality, estimated as the difference between observed deaths and average expected deaths. We obtained data from the Puerto Rico Department of Health COVID-19 Mortality Surveillance System, the Centers for Disease Control and Prevention’s National Electronic Disease Surveillance System Base System, and the National Center for Health Statistics.During March–July 2020, 225 COVID-19–associated deaths were identified in Puerto Rico (119 confirmed deaths and 106 probable deaths). The median age of decedents was 73 ; 60 (26.7%) deaths occurred in the Metropolitana region, and 140 (62.2%) deaths occurred among men. Of the 225 decedents, 180 (83.6%) had been hospitalized and 93 (41.3%) had required mechanical ventilation. Influenza and pneumonia (48.0%), sepsis (28.9%), and respiratory failure (27.1%) were the most common conditions contributing to COVID-19 deaths based on death certificates. Based on excess mortality calculations, as many as 638 COVID-19–associated deaths could have occurred during the study period, up to 413 more COVID-19–associated deaths than originally reported.Including probable deaths per the CSTE guidelines and monitoring all-cause excess mortality can lead to a better estimation of COVID-19–associated deaths and serve as a model to enhance mortality surveillance in other US jurisdictions. The Puerto Rico Department of Health (PRDoH) reported its first case of the novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on March 9, 2020, and the first COVID-19–associated death on March 17, 2020. As of July 31, 2020, the PRDoH had reported 17 872 confirmed COVID-19 cases .Puerto Rico is home to an estimated 3.6 million people. Since April 5, 2020, the Council of State and Territorial Epidemiologists (CSTE) has recommended that all COVID-19 deaths be reported to local public health authorities. Based on the CSTE guidelines, a confirmed COVID-19 death case is defined as meeting confirmatory laboratory evidence (positive RT-PCR test result); a probable COVID-19 death case is defined as meeting 1 of the following criteria: (1) meets clinical criteria and epidemiological evidence without a positive RT-PCR test result, (2) meets presumptive laboratory evidence (serology test) and either clinical criteria or epidemiological evidence, or (3) meets vital records criteria (“a death certificate that lists COVID-19 disease or SARS-CoV-2 as a cause of death or significant condition contributing to death”) without a positive RT-PCR test result.Estimating and characterizing COVID-19–associated deaths in the United States during the current pandemic has been challenging because of limited testing, variable testing sensitivity, misclassification of COVID-19–associated deaths, lack of completeness of COVID-19 case reports and surveillance forms, and reporting delays to local surveillance systems. The objectives of our study were to characterize COVID-19–associated deaths in Puerto Rico during March–July 2020 (using CSTE guidelines) and estimate the range of COVID-19–associated mortality that may have been underestimated.In Puerto Rico, death from COVID-19 is a reportable event to the PRDoH Mortality Surveillance System (MSS). Accurate classification of COVID-19–associated deaths (confirmed and probable) might vary by reporting source and in some cases requires review by a multidisciplinary team at PRDoH. However, counting only confirmed and probable COVID-19–associated deaths might underestimate the number of deaths attributed to the pandemic. Deaths are not counted by MSS when they are not directly associated with SARS-CoV-2 infections, such as deaths that occur outside a health care setting or deaths that are misclassified by the attending health care provider, which may contribute to an underestimation of deaths associated with COVID-19. Estimating excess deaths can help explain the severity or burden of pandemics and public health emergencies. from Puerto Rico’s laboratory reporting system and the National Electronic Disease Surveillance System Base System. The National Electronic Disease Surveillance System Base System is an integrated information system developed by the Centers for Disease Control and Prevention (CDC) to help local, state, and territorial public health departments, such as PRDoH, manage notifiable disease data and send reports to CDC. Puerto Rico’s regional epidemiologists receive death information from all hospitals and collect and verify additional COVID-19 death data, including pending laboratory test results, requested by the central PRDoH MSS team. Regardless of where the COVID-19 death occurs , death certificates are completed by physicians and sent to vital statistics. The PRDoH MSS central team links its data with data on COVID-19 deaths from vital statistics using the International Classification of Diseases, Tenth Revision code U07.1, COVID-19. The PRDoH MSS central team reconciles classification (confirmed or probable) from all reporting sources and reports the daily number of deaths (per date of death) due to COVID-19 into an electronic centralized system at the PRDoH. Deaths that were undefined or under investigation were reviewed by a multidisciplinary team when COVID-19 testing (confirmed and probable) and clinical or pathology information became available. Because public health surveillance data collected by PRDoH and analyzed in this article are not considered research, no institutional review board review was required.PRDoH MSS is a passive surveillance system that was enhanced for the COVID-19 response by including additional data sources to verify information on deaths attributed to COVID-19. PRDoH MSS includes information on deaths from regional epidemiologists, vital statistics from physicians, and COVID-19 laboratory results (confirmed [RT-PCR molecular test for SARS-CoV-2 RNA] and probable [SARS-CoV-2 serology test]) We calculated excess mortality using the Farrington surveillance algorithm applied to death data for Puerto Rico from 2013 through the present. Negative values, in which the observed count fell below the NCHS threshold, were set to zero and were not depicted. Details about NCHS methods and excess death data associated with COVID-19 can be found elsewhere. We calculated a range of deaths potentially associated with COVID-19. The lower bound of the range was the sum of confirmed and probable COVID-19 deaths reported by PRDoH during March 17–July 31, 2020 . The upper bound of the range of COVID-19–associated deaths was the sum of excess deaths in Puerto Rico reported by NCHS during March 2–August 2, 2020 . We calculated 95% CIs for the sum of excess deaths using the standard error of excess deaths for the study period. This range, the difference between the lower bound and the upper bound, represents an estimate of missed deaths that likely were associated with the COVID-19 pandemic. We also depicted the excess estimates for all cases of death excluding COVID-19 deaths to visualize the gap between excess mortality and COVID-19 deaths during the study period. We did not analyze data on underlying medical conditions, symptomology, or course of clinical illness and care received because this information was missing for 171 (76%) cases.Excess mortality is defined as the difference between observed deaths and average expected deaths in the same period as reported by CDC’s National Center for Health Statistics (NCHS).As of July 31, 2020, PRDoH had officially reported 225 COVID-19 deaths (119 confirmed cases and 106 probable cases). Of the 225 COVID-19 deaths, 140 (62.2%) decedents were male . The medDuring March–July 2020, a total of 863 deaths were reported to NCHS and PRDoH; of these deaths, 638  were found to be in excess of the expected baseline. Included in the 638 deaths were the reported 119 confirmed COVID-19 cases and 106 probable COVID-19 cases, leaving as many as 413 possible additional COVID-19–associated deaths that were assigned another cause of death . Considering excess COVID-19–associated mortality might improve our understanding of how many deaths are attributed to the pandemic. Although these methods are being used in the surveillance of COVID-19 mortality, the results of our analysis suggest that many COVID-19–associated deaths in Puerto Rico during the pandemic were missed—approximately 2 times higher than originally reported—during the study period; as many as 413 COVID-19–associated deaths might have been uncounted. Previous national studies support that deaths from COVID-19 are likely underreported by local surveillance systems. Increased COVID-19 testing and completeness of death reporting under local surveillance could enhance the understanding of the number of deaths caused by this evolving disease and pandemic. We suspect that reported deaths associated with COVID-19 could increase substantially as testing and local surveillance systems improve case ascertainment and classification.Since the early stages of the COVID-19 pandemic, monitoring and estimating COVID-19–associated deaths in the United States has been challenging. Public health officials have raised concerns about estimating the “true” death toll resulting from COVID-19. The inclusion of probable cases is particularly important because, at the time of the study, Puerto Rico had the highest number of probable COVID-19 cases in the country. This new pathogen has placed new and unique demands on local public health surveillance systems and infrastructures everywhere. Therefore, complementing data with NCHS data on excess deaths  is a more sensitive measure than only counting COVID-19 confirmed and probable deaths.Our study results also reaffirm the importance of using CSTE COVID-19 death case definitions in reporting deaths. Of the 225 COVID-19 cases in Puerto Rico, almost half were probable cases. The CSTE case definition was also used in New York City COVID-19 mortality surveillance, with 5048 probable cases added to 13 831 laboratory-confirmed COVID-19 deaths. We hypothesized that people in Puerto Rico might have delayed or avoided medical care because of concerns about exposure to COVID-19. Delay or avoidance might have exacerbated uncontrolled chronic conditions or circulating infections, as shown by the conditions that contributed to death indicated on death certificates. Local public health officials should emphasize the importance of attending regular medical appointments to enhance the public’s awareness and action on medical care. Public health actions could be strengthened by tailoring public messaging to populations that may require special assistance to attend their medical appointments.Based on our reported data, deaths due to COVID-19 in Puerto Rico are particularly affecting older people and people living in the most populated regions. Data show that most people die in health care settings . Similar to national data, early local pandemic data show that the number of hospital visits was lower than usual, perhaps because of strict public health mitigation measures related to COVID-19. Second, the fragmented surveillance systems for the COVID-19 response in Puerto Rico likely underestimated COVID-19 death counts. However, the underestimation of deaths could be a common pattern during the current COVID-19 response in many jurisdictions in the United States and elsewhere. Third, excess mortality trend data do not align with the PRDoH epidemiological curve depicted for COVID-19 deaths, as previously reported in COVID-19 analyses. This misalignment may have resulted from the lag between the occurrence of the death and the completion and submission of the death certificate by local public health officials to NCHS. Per NCHS, such data lags can range from 1 to 8 weeks. Therefore, use of local data to depict mortality trends might buffer possible differences in national reporting delays. Fourth, conditions contributing to death based on death certificates might be incomplete or under investigation. Therefore, our results should be interpreted with caution, as more detailed analyses are needed. Finally, overattributing deaths directly to COVID-19 during the current pandemic could underestimate data on mortality from other causes. Therefore, a more detailed analysis of deaths, considering delay or access to care, might be warranted.This study had several limitations. First, both incomplete and missing data on COVID-19 deaths in the PRDoH MSS database limited our ability to better understand contributing mortality factors or course of illness among the population; national data have had similar limitations . all-cause excess mortality proved to be a useful public health tool to monitor the current COVID-19 pandemic. Only counting laboratory-confirmed or probable COVID-19–associated deaths likely underestimates the true number of deaths associated with the pandemic. To better understand underlying medical conditions that contribute to deaths during the pandemic, more complete investigations, including the completion of CDC’s standardized case-report form, with medical record abstraction and including additional data sources, could be enhanced. Given the variability of reporting sources and laboratory COVID-19 test results for deaths, PRDoH MSS could consider expanding its passive surveillance efforts to an active or hybrid surveillance to increase death case ascertainment and completeness. Ascertainment of deaths during a pandemic could also take into consideration public health restrictions  and how they might have affected a person’s ability to attend regular medical appointments for chronic or new conditions. Public health officials could consider enhancing the public’s awareness of the importance of attending regularly scheduled medical appointments to avoid exacerbation of chronic or acute illness. Enhanced mortality surveillance can provide vital information to help monitor the severity and progression of the COVID-19 pandemic by estimating deaths directly and indirectly associated with COVID-19. Health department monitoring of all causes of excess death during the COVID-19 pandemic could help guide public health decision making, messaging, and interventions to help prevent additional deaths.Puerto Rico, as with many other US jurisdictions, has been affected by the COVID-19 pandemic. Similar to a previous postdisaster study conducted on the island,


Myasthenia gravis (MG) with antibodies to the muscle-specific receptor tyrosine kinase (MuSK) is a distinct sub-group of MG, affecting 5–8% of all MG patients. MuSK, a receptor tyrosine kinase, is expressed at the neuromuscular junctions (NMJs) from the earliest stages of synaptogenesis and plays a crucial role in the development and maintenance of the NMJ. MuSK-MG patients are more severely affected and more refractory to treatments currently used for MG. Most patients require long-term immunosuppression, stressing the need for improved treatments. Ideally, preferred treatments should specifically delete the antigen-specific autoimmune response, without affecting the entire immune system. Mucosal tolerance, induced by oral or nasal administration of an auto-antigen through the mucosal system, resulting in an antigen-specific immunological systemic hyporesponsiveness, might be considered as a treatment of choice for MuSK-MG. In the present study we have characterized several immunological parameters of murine MuSK-EAMG and have employed induction of oral tolerance in mouse MuSK-EAMG, by feeding with a recombinant MuSK protein one week before disease induction. Such a treatment has been shown to attenuate MuSK-EAMG. Both induction and progression of disease were ameliorated following oral treatment with the recombinant MuSK fragment, as indicated by lower clinical scores and lower anti-MuSK antibody titers. Myasthenia gravis (MG) is an autoimmune disease characterized by skeletal muscle weakness as a result of an immunological attack at the neuromuscular junction (NMJ). In 5–8% of MG patients the autoantibodies present in the sera are against the muscle-specific receptor tyrosine kinase (MuSK) classifying these patients as a distinct MG sub-group called MuSK-MG . MuSK isThe use of currently immunosuppressive therapies for MuSK-MG patients is challenging as patients are prone to develop severe facial weakness and bulbar symptoms, including dysphagia, dysarthria and respiratory crisis with some atrophy of facial muscles making the treatment less effective , 6. ThymTo date, general immunosupression is the mainstay treatment for autoimmune diseases. The main challenge for immunologists is to develop novel treatments that will manipulate specifically or correct the abnormal immune response leaving the overall immune response intact.Specific systemic tolerance to an antigen can be achieved by exposing mucosal surfaces to a particular antigen , and it We have shown in our previous studies that mucosal  administration of torpedo AChR prior to disease induction resulted in EAMG suppression and was accompanied by inhibition of the humoral response as well as cellular responses to AChR , 12. MorShigemoto et al. have developed a MuSK-EAMG model  in whichIn this study we have induced the MUSK-EAMG experimental model disease in mice and characterized some of its immunological properties in order to apply it as a model for therapeutic experiments. Specifically, we report on our successful efforts to induce mucosal tolerance to the MuSK antigen by oral application of the recombinant extracellular domain of the MuSK protein.Female FVB/N mice aged 6-8 weeks were obtained from the Animal Breeding Center of The Weizmann Institute of Science, Rehovot, Israel and were maintained at the Institute's animal facilities. All the experiments in this study were performed according to the institutional guidelines for animal care.pCEP-PU vector containing the His-tagged extracellular domain of recombinant rat MuSK  was kinOn day 0, adult female mice were anesthetized (Ketamine: 111 mg/kg and Xylazine: 22 mg/kg) and immunized subcutaneously, each with a total volume of 200 μl of recombinant MuSK (20 or 40 μg/mouse), emulsified in complete Freund's adjuvant (CFA) from Sigma-Aldrich , as follows: 20 μl in each hind foot pads, 40 μl at the base of the tail and 20 μl in each of 6 well-separated sites on the back. On day 14 post-injection, all mice were boosted by 6 well-separated sites on the back with 20–40 μg MuSK emulsified with incomplete Freund's adjuvant (IFA) from Sigma-Aldrich . Control mice were immunized by CFA and IFA only.Mice were observed, weighed and scored blindly on alternate days, for the clinical severity of disease as follows: 0 - Healthy mouse; 1 - Body weight loss; 2 - Body weight loss, weakness, prominent cervicothoracic hump; 3 - Body weight loss, weakness, prominent cervicothoracic hump, tremor and ungroomed fur; 4 - Dead.The method of blinding employed in our experiments was as follows: FVB/N mice were randomly divided into experimental groups: MuSK and CFA. One operator was assigned to the experimental treatments (MuSK immunization) while a second person who assessed the mice  remained blinded to the experimental groups until the end of the experiment.All experimental groups consisted of 10 mice each, unless otherwise specified and all experiments were repeated 2–3 times.Sera of treated mice were collected by retro-orbital bleeding 6 weeks following disease induction. The levels of anti-MuSK antibodies were determined by standard ELISA as follows: Microtiter plates were coated with recombinant MuSK protein , and reacted with 100 μl of the tested mouse serum, at a dilution of 1:1000 for total IgG. Rabbit anti-Mouse alkaline phosphatase antibody  was added followed by alkaline phosphatase-conjugated streptavidin. Antibody levels were evaluated by measuring the optical density at 405 nm.Flow cytometry analysis was performed on splenocytes of MuSK-immunized mouse and of control mouse. Spleen cells were suspended in FACS wash buffer  and incubated for 60 min at 4°C in the dark with antibodies to the tested cell surface molecules. Cells were washed and analyzed on a FACScan flow cytometer. The following antibodies were used for flow cytometry: FITC-conjugated anti-mouse CD4 (L3T4) and APC-conjugated anti-mouse CD25 (PC61.5). For intracellular staining, cells were fixed and permeabilized using the fixation/permeabilization kit from e-Bioscience  followed by staining with PE-conjugated anti-FoxP3 antibody (e-Bioscience).Total RNA was extracted from mice splenocytes and muscles at the end of the experiment (8 weeks following immunization with MuSK). Extraction was performed using the high pure RNA Isolation Kit  according to the manufacturer's instructions. The concentration of total RNA was measured by NanoDrop ND-1000 Spectrophotometer . Complementary DNA was prepared and quantitative real-time reverse transcription (PCR) was performed using the LightCycler system (Roche) according to the manufacturer's instructions. Primer sequences  are given in Musk-specific oral tolerance was attempted by feeding of FVB/N mice with recombinant MuSK, essentially as described by us for the induction of oral tolerance of AChR-EAMG by a recombinant AChR fragment . FVB/N mDisease evaluation was performed as mentioned above: FVB/N mice were randomly divided into experimental groups: MuSK and OVA oral administration. One operator was assigned to the experimental treatments (feeding by gavage and MuSK immunization) while a second person who assessed the mice  remained blinded to the experimental groups until the end of the experiment.t-test. p values < 0.05 were considered as significant.The results are presented as mean ± SEM. Two-way ANOVA test was used to compare disease assessment vs. control and treatment vs. control groups all along the experiment. Differences in mean values were compared between treatment and control groups by the Student's MuSK-EAMG, an experimental model of MuSK-MG, has been established in our lab in FVB/N female mice, according to Mori et al. , which oAnti-MuSK IgG antibodies were analyzed by ELISA and were detected in all MuSK-immunized mice, whereas control CFA-immunized mice had no detectable antibodies to MuSK . InteresIn order to test whether the induction of MuSK-EAMG results in muscle damage, the mRNA expression of several genes was examined in samples derived from masseter muscles from sick (MuSK-immunized) and control mice.The initiation of protein degradation involved among others the lysosomal endopeptidase enzyme Cathepsin l. We have observed that the level of cathepsin 1 mRNA expression is significantly increased in MuSK-immunized mice, as depicted in +CD25+Foxp3+ Treg cells in MuSK-EAMG mice, their splenocytes were stained for CD4, CD25 and Foxp3, by specific antibodies. As shown in + cells among CD4+CD25+ cells, in the spleens of MuSK-EAMG mice  cells. Dysfunction and/or altered Treg cell numbers can result in the development of autoimmune diseases . Animal Induction of oral tolerance involves many mechanisms that modulate the immune response against auto antigens. Oral tolerance has an impact on the numbers and function of Treg cells, on the secretion of pro and anti-inflammatory cytokines and on Th-1/Th-2 effector cells . This obHere we demonstrate for the first time an attempt to modulate MuSK myasthenia gravis by feeding mice with low doses of recombinant MuSK. The immune response acts differently to the dose of the antigen administered. High doses of antigens leads to anergy or apoptosis of antigen specific immune cells while administration of low doses of antigen leads to induction of antigen specific Tregs –32.Administration of recombinant MuSK protein resulted in a significant therapeutic effect in treated mice, accompanied by a corresponding effect in weight loss. Additionally, the titer of total MuSK specific IgG in the serum at the end of the experiment was lower in MuSK fed mice, indicating amelioration of the disease. Moreover, expression level of Foxp3 and TGFβ were elevated in MuSK fed-mice suggesting that oral administration of MuSK modulated Tregs.Taken together, our results demonstrated oral tolerance efficacy in MuSK-EAMG model. This therapeutic potential should be further explored and considered as a novel approach for MuSK-MG treatment and hopefully be effective and safe for the benefit of MuSK-MG patients.All datasets generated for this study are included in the article/supplementary material.The animal study was reviewed and approved by the Weizmann Institutional Animal Care and Use Committee.DR, SF, and MS designed and guided the research. DR and RA performed the animal experiments and analyzed most of the experiments. DR and SF wrote the manuscript. All authors reviewed and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Heparin administration in COVID-19 patients is recommended by expert consensus, although evidence about dosage, duration and efficacy are limited. We aim to investigate the association between different dosages of low molecular weight heparin (LMWH) and mortality among COVID-19 hospitalized patients.Retrospective study of 450 laboratory-confirmed COVID-19 patients admitted to Sant’Orsola Bologna Hospital from March 01 to April 10, 2020. Clinical, laboratory and treatment data were collected and analyzed. The in-hospital mortality between COVID-19 patients treated with standard prophylactic LMWH dosage vs. intermediate LMWH dosage was compared. Out of 450 patients, 361 received standard deep vein thrombosis (DVT) prophylaxis enoxaparin treatment (40-60mg daily) and 89 patients received intermediate enoxaparin dosage (40–60 mg twice daily) for 7 days. No significant differences in the main demographic characteristics and laboratory testings at admission were observed in the two heparin regimen subgroups, except for older age and prevalence of hypertension in the group treated with “standard” prophylaxis LMWH dosage. The intermediate LMWH administration was associated with a lower in-hospital all-cause mortality compared to the “standard” prophylactic LMWH dosage . This difference remained significant after adjustment with the propensity score for variables that differed significantly between the dosage groups .Intermediate LMWH dosage seems to be associated with lower incidence of mortality compared to standard DVT prophylaxys in hospitalized COVID-19 patients. Our study paves the way to further pathophysiological investigations and controlled studies of anticoagulation therapy in Covid-19 disease. Recently, a novel coronavirus (2019-nCoV), officially known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), caused a severe pandemic infection worldwide with considerable morbidity and mortality. The name for the disease resulting from SARS-CoV-2 infection has also been identified with the acronym COVID -19 . This is associated with an increase of systemic inflammation, cytokine storm and diffuse endothelial injury, resulting in the most severe cases in multi-organ failure with consequently poor outcomes. Fortunately, the clinical spectrum appears to be wide, including asymptomatic infection, mild upper respiratory tract illness and severe viral pneumonia with occurrence of respiratory failure . DespiteTo address such unsolved issues, we analyzed data from a single referral center to investigate the potential association between different dosages of LMWH enoxaparin administration and mortality among hospitalized COVID-19 patients.Consecutive patients admitted to Sant’Orsola Bologna University Hospital with laboratory-confirmed COVID-19 were included in this retrospective cohort study, which was conducted from March 01, 2020, to April 10, 2020. The diagnosis of COVID-19 was established according to World Health Organization interim guidance and confirmed by RNA detection of the SARS-CoV-2 in the microbiology laboratory of the hospital (Outcomes between COVID-19 patients treated with standard prophylactic LMWH enoxaparin dosage (subcutaneous enoxaparin 40–60 mg daily) or intermediate LMWH enoxaparin dosage (subcutaneous enoxaparin 40–60 mg twice daily) for 7 days were compared  and/or hydroxychloroquine , when appropriated . ClinicaOutcome measurements included all-cause-death, severe respiratory failure requiring admission to the intensive care unit (ICU) and/or orotracheal intubation and/or renal failure requiring continuous venovenous hemofiltration (CVVH) and/or extracorporeal membrane oxygenation (ECMO), acute kidney injury (AKI), acute myocardial infarction, atrial fibrillation and heart failure.2/FiO2 ≤ 300 mm Hg. We did not include respiratory rate ≥30 breaths/min according to the Diagnosis and Treatment Plan of COVID‐19 suggested by National Health Commission of China due to the considerable inter-observed variability.Severe COVID‐19 was defined as meeting arterial oxygen saturation ≤93% at rest or PaONon evidence-based treatments were administered according to DL 18, art.17/3/2020 of the National Health Service for the Emerging Infectious Disease. The need for informed consent was waived accordingly.Continuous and categorical variables were presented as median (IQR) and n (%), respectively. We used the Mann-Whitney U test, χ. test, or Fisher’s exact test to compare the two groups of heparin dosage, as appropriate. To compare the all-cause in-hospital mortality between the two groups of heparin dosage, logistic regression analysis with propensity score adjustment was used, to control for the imbalance in the group characteristics. The propensity score, i.e. the conditional probability of being treated with the intermediate LMWH dosage given the set of variables that differed significantly between the dosage groups, was estimated using a multiple logistic regression model.2/FIO2 value; p = 0.003) was observe in the group of standard prophylaxis LMWH, however the severity of COVID 19 was similar between the two groups. Patients treated by the intermediate LMWH dosage received hydroxychloroquine and i.v. Tocilizumab more frequently . Despite a similar baseline profile, no differences in ICU admission, orotracheal intubation, renal failure requiring continuous venovenous hemofiltration and extracorporeal membrane oxygenation between the groups were observed. Interestingly, the intermediate LMWH dosage was associated with a lower in-hospital all-cause mortality compared to the standard prophylaxis LMWH dosage . This difference remained statically significant after adjustment with the propensity score for potential confounder variables that differed significantly between the dosage groups .We evaluated clinical records of 510 consecutive adult patients with confirmed COVID-19 diagnosis referred to our Hospital. Twenty-one treated with warfarin, 13 patients with new oral anticoagulation and 26 patients taking anticoagulant enoxaparin dosage (100 mg twice daily) were excluded. A total of 450 hospitalized patients with COVID-19 infection were included in the final analysis  in the standard prophylaxis LMWH group and 2  in the intermediate LMWH dosage group occurred . Moreover, hemoglobin values remained stable after one week of therapy in the two groups analyzed .Our data suggest that the standard prophylaxis LMWH dosage for DVT may be less effective than the intermediate LMWH dosage. In fact, in-hospital mortality rate is significant lower in our cohort of COVID-19 patients treated with intermediate LMWH dosage and this difference remained significant after adjustment with the propensity score for variables that differed significantly between the dosage groups.Previous report suggested that standard prophylaxis LMWH dosage is associated with an improve outcomes in COVID-19 patients. Tang et al. demonstrated that LMWH treatment was associated with better prognosis in severe COVID‐19 patients meeting “Sepsis-Induced Coagulopathy” (SIC) criteria or with markedly elevated D‐dimer . HoweverRecent guidance document recommends the use of standard-dose regimens with LMWH in the management of venous thromboembolism (VTE) in hospitalized COVID-19 patients, primarly in severe COVID-19 patients, although data on efficacy and safety of this approach are limited . So far,In the present article, we evaluated the different LMWH regim dose (“standard” versus intermediate LMWH enoxaparin dosage) in all hospidalized patients without stritifyng the severity of COVID-19 infection. The main novelty of our work is that for the first time we demonstrated that intermediate LMWH enoxaparin dosage improved the prognosis both in severe and non-severe COVID-19 patients. However, we do not examine the exact mechanism of this evidence. The beneficial effect of intermediate LMWH regimen observed in our study could be correlated to the prevention of or pulmonary microthrombosis and/or disseminated intravascular coagulation, which are considered as possible pathophysiological mechanisms leading to a worse prognosis in COVID-19 patients .Although in this study we did not assess the thromboembolic complication, literature data demonstrated that massive pulmonary thrombosis/embolism was the cause of death until one third of the case . HoweverOur findings of significantly less in-hospital mortality rate in patients treated with intermediate LMWH dosage supported the hypothesis that LMWH accomplish simultaneous antithrombotic, anti-inflammatory and antiviral activity in COVID-19 infection. These effects seem to be more effective in intermediate LMWH dosage.In our study, a significantly higher number of patients in the intermediate LMWH dosage were treated with both hydroxychloroquine and tocilizumab. Tocilizumab, which is the IL-6 receptor antagonist, has been approved for the treatment COVID-19 patients. Although, the administration and dosage of tocilizumab for COVID-19 are still being explored and the risk of serious infection  must be investigate. In this patients the association of LMWH might help to reduce inflammatory burden and consequently the dosage of tocilizumab. Regarding the role of hydroxychlorochine, some observational studies have recently demostrated that administration of hydroxychloroquine did not reduce the risk of intubation or death in COVID-19 patients and its benefits remain very weak and conflicting . So far,However, there are substantial limitations of our observation owing to its retrospective and non-randomized nature, namely the absence of a formal hypothesis testing and some unbalanced features of the study subgoups.In conclusion, although the optimal thromboprophylactic regimen is not known, our study opens new insights in heparin therapy for patients hospitalized with COVID-19. Large clinical trials are needed to define: a) a more precise dose-range of LMWH to balance the anti-thrombotic action and potentially severe bleeding complications; b) the more appropriate disease stage in which LMWH administration should be started; c) the clinical fingerprint of patients most likely to benefit from LMWH therapy.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.PP, LB, ECD, and MG contributed conception and design of the study. FD, ST, RP, MBa, and GT organized the database. LB and MBi performed the statistical analysis. PP wrote the first draft of the manuscript. PP, LB, and MG wrote sections of the manuscript. BC, PV, CP, MBi, and NG revised the article. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Internet-based interventions are a promising strategy for promoting healthy lifestyle behaviors. These have a tremendous potential for delivering electronic health interventions in scalable and cost-effective ways. There is strong evidence that the use of these programs can lead to weight loss and can lower patients’ average blood pressure (BP) levels. So far, few studies have investigated the effects of internet-based programs on patients who are obese with hypertension (HTN).The aim of this study is to investigate the short- and long-term efficacy, in terms of body composition and BP parameters, of a self-administered internet-based intervention involving different modules and learning techniques aimed at promoting lifestyle changes  in patients who are obese with HTN.A randomized wait-list controlled trial design was used. We recruited 105 adults with HTN who were overweight or obese and randomly assigned them to either a 3-month internet-based intervention group (n=55) or the wait-list control group (n=50). We assessed BMI (primary outcome), body fat mass (BFM), systolic (S)BP and diastolic (D)BP, blood glucose and insulin levels, physical activity levels, and functional capacity for aerobic exercise at Time 0 (preintervention) and Time 1 (postintervention). All the patients in the wait-list control group subsequently received the intervention, and a secondary within-group analysis, which also included these participants, was conducted at Time 2 (12-month follow-up).P=.005), BFM , DBP , and blood glucose .A 2-way mixed analysis of covariance showed a significant decrease in BMI, BFM, and blood glucose at 3 months in the internet-based intervention group; the effect size for the BMI and BFM parameters was moderate to large, and there was also a borderline significant trend for DBP and insulin. These results were either maintained or improved upon at Time 2 and showed significant changes for BMI (mean difference −0.4, 95% CI −0.1 to −0.6; Implementation of our self-administered internet-based intervention, which involved different learning techniques aimed to promote lifestyle changes, resulted in positive short- and long-term health benefits in patients who are obese with HTN.ClinicalTrials.gov NCT03396302; https://clinicaltrials.gov/ct2/show/NCT03396302 Cardiovascular disease is the leading cause of morbidity and mortality in developed countries and continues to be a major public health issue that accounts for over 4 million deaths per year in Europe . ObesityPromoting healthy behaviors such as physical activity and healthy eating through lifestyle counselling is recommended as the first-line therapy for the treatment of these patients and may be an effective tool for treating obesity and preventing obesity-related health burdens ,8,9. HowInternet-based interventions (IBIs) are a promising strategy for promoting healthy lifestyle behaviors and have not been entirely explored; these have a tremendous potential for delivering electronic health  interventions in scalable and cost-effective ways. IBIs can provide immediate, easy to access, relatively inexpensive, and individually tailored support for self-management and the promotion of behavioral change to large segments of the population. Moreover, there is strong evidence that the use of these programs can lead to weight loss -21 and cThis study aimed to examine the short- and long-term efficacy in terms of body composition and BP parameters of a self-administered IBI involving different modules and learning techniques, aimed at promoting lifestyle changes  in patients who are obese with HTN.Compared with the wait-list control (WLC) group receiving standard medical care, we hypothesized that the IBI group will have significantly better improvements for body composition, BP, blood glucose and insulin levels, physical activity levels, and functional capacity for aerobic exercise from baseline to postintervention.This prospective, single-center, wait-list controlled trial  with balanced randomization (1:1) was approved by the Hospital of Sagunto Human Ethics Committee and followed the ethical guidelines set out in the Declaration of Helsinki.2 and <30 kg/m2) or had type 1 obesity (BMI>29.9 kg/m2 and <35 kg/m2). HTN was defined as systolic (S)BP≥140 mmHg or diastolic (D)BP≥90 mmHg, or current use of antihypertensive medication. All patients in our study were on antihypertensive treatment. The exclusion criteria included a diagnosis of diabetes, previous ischemic heart disease, cerebrovascular disease, or a severe psychiatric disorder; taking more than three antihypertensive drugs; physical impairments precluding participation in physical activity; receiving any treatment for weight loss elsewhere; or having no internet access.Eligible participants were all adults between 18 and 65 years of age with HTN and who were overweight , from January 2018 to March 2019. The study included 105 participants. Before the start of the trial, Researcher 1, who was not involved in the recruitment or inclusion of the participants, generated a random sequence (based on simple randomization) using a computerized random number generator; this was concealed from all other study investigators throughout the entire study period. Upon enrollment in the study and after completing the primary and secondary outcome measures (baseline), the participants were randomly assigned either to the 3-month IBI group (n=55) or the WLC group (n=50). As shown in the participant flowchart in After the Time 1 assessment, WLC participants received the intervention, which was offered to comply with the instructions of the hospital’s human ethics committee, and the patients were subsequently assessed at a 12-month follow-up (Time 2) . Thus, aThe intervention program we used is called “Vivir mejor” (translated from Spanish as “Live better”) ,30. For During the first 3 months of the study and prior to starting the intervention, the WLC group received standard medical care that focused on reducing their cardiovascular risk factors . StandarThis IBI was delivered using a web-based platform called Wix. Wix is a self-hosted website builder and content management system with more than 90 million users . This cl2) was calculated using an electronic balance scale .BMI and secondary outcomes  were measured using validated and reliable tests. BMI . SBP and DBP were measured with a validated, semi-automatic, digital tensiometer (Colin Press-Mate BP-8800) according to the European Society of Hypertension and European Society of Cardiology guidelines, as described elsewhere ,37. All At Time 0, the patients’ habitual levels of physical activity were objectively monitored for 7 consecutive days using an accelerometer (Actigraph GT3X) . The parA submaximal exercise test, the 6-minute walk test (6MWT), was used to assess the participants’ functional capacity for aerobic exercise. This test evaluates the maximum distance that they could cover along a 30-m long corridor during a 6-minute period. Participants were instructed to walk along the walkway as fast as possible and to stop when needed. The assessor walked alongside the participants to ensure their safety and provided them with standardized verbal encouragement at 1, 3, and 5 minutes, and the total distance covered (in meters) was recorded. Participants were instructed to avoid smoking for 48 hours, caffeine for 12 hours, and strenuous exercise for 24 hours prior to their assessment.We directly recorded participants’ participation and activity in the program (to assess their engagement) and determined adherence to the intervention by tracking the number of program activities they completed. We also registered the percentage of participants who completed all the activities in the program .To detect a reduction in BMI of 1 (SD 1.7), which agrees with the data of a previous study , with a P<.05 for all our analyses. The data in this study are presented as mean (SD).This study used both a between-subjects controlled  and a within-subjects uncontrolled  design; therefore, it involved two different statistical approaches. The between-group comparison (IBI vs WLC) assessed the effect of the IBI on outcomes at 3 months; the within-subject uncontrolled analyses assessed the effect of the IBI over time at the individual level (using pooled data from both groups). This approach was selected because all the WLC participants also subsequently received the IBI to comply with the instructions of the hospital’s human ethics committee. The statistical analysis was performed according to intention-to-treat. Statistical analyses were performed using SPSS version 19.0 for Windows, and the statistical significance was set at 2 and interpreted following the Cohen guidelines [2=0.01), moderate effect sizes , and large effect sizes .Two-way mixed analysis of covariance (ANCOVA) tests were used to compare the study effects on the BMI, BFM, SBP, DBP, blood glucose and insulin levels, physical activity levels, and functional capacity for aerobic exercise, using time (baseline vs 3 months postintervention) as the within-group factor and group (IBI vs WLC) as the between-group factor. The analysis was adjusted for sex, age, and use of antihypertensive drugs. Effect sizes were estimated using partial etaidelines  for smalt tests on related samples to compare the baseline vs 12-month follow-up values for the anthropometric variables , SBP, DBP, and blood glucose; and (2) Wilcoxon tests to compare the preintervention vs 12-month follow-up values for insulin.After testing the normality (using the Shapiro-Wilk test) of the pooled data from both groups (N=105) at the 12-month follow-up, the following statistical tests were carried out as follows: (1) We did not record the patients’ levels of physical activity or their functional capacities for aerobic exercise at 12 months because of the implied complexity of asking participants to record these measurements for 1 year.Program engagement was analyzed by calculating the percentage of participants who completed the entire program.We screened 510 participants in this randomized controlled trial. A total of 405 consecutive subjects were not allocated for randomization, because they declined to participate (n=99) or did not meet the inclusion criteria (n=306): diabetes (106), severe psychiatric disorder (5), taking more than 3 antihypertensive drugs (78), no internet access (87), or other (30). The general characteristics of the study population are shown in P=.05) for DBP and insulin , BFM , DBP , and blood glucose levels  The results of the Wilcoxon tests did not show any changes in insulin values. Regarding engagement in the program, 73.3% (77 out of 105 participants) completed the entire program, comprising of nine modules.The results at the 12-month follow-up  showed sLive better that aims to promote lifestyle changes in patients who are obese with HTN. Our results showed a significant decrease in the BMI, BFM, and blood glucose levels at 3 months in the IBI group, with a moderate to large effect size for BMI and BFM, and also highlighted a trend toward significance for DBP and insulin levels. Moreover, these improvements in BMI, BFM, and BP were sustained and reached statistical significance for DBP at the 12-month follow-up. When evaluating specific IBIs, several attributes may be associated with increased e-counselling efficacy. These include the duration [The present study evaluated the efficacy of a 3-month completely self-administered IBI called duration ,39, the duration ,40, the duration , whetherduration ,41.2) after 3 months that was sustained after 12 months. Moreover, the mean decrease in BFM (by 1.1 kg) achieved after 3 months had doubled 12 months later (to 2.4 kg). Although this parameter is less frequently used because of its lower feasibility, BFM may be a better marker of cardiovascular risk compared to BMI, which has been criticized because the latter does not always reflect true body fat content [For any intervention to have a significant and sustained effect, a minimum follow-up time of 6 months is required . Indeed, content ,43. Inde content  and in iConsistent with our results, a Cochrane meta-analysis concluded that interactive computer-based interventions, compared to no intervention or minimal interventions , are an effective tool for enhancing weight loss and weight maintenance . In addiConcerning BP values, we found a nonsignificant decrease in SBP and DBP after 3 months, which was sustained and even reached statistical significance after 12 months in the case of DBP (−1.8 mmHg). These results are slightly lower than those reported in the Internet Lifestyle Counselling meta-analysis (−3.8 mmHg for SBP and −2.1 mmHg for DBP) by Liu et al  and are Two recent meta-analyses that evaluated the BP lowering  and weigAll the behavioral change techniques mentioned by Liu et al  and KhayLive better intervention focused on modifying both of these target behaviors, because it has been shown that tackling them simultaneously is more effective at promoting weight loss than targeting either alone [Exercise and diet are two major target behaviors for modification by internet-based e-counselling interventions designed to prevent or treat cardiovascular disease risk factors such as obesity and HTN. Numerous studies suggest that automated, self-guided, internet-based lifestyle counselling (e-counselling) programs can evoke meaningful improvements in daily physical activity ,58 and der alone .2=0.213) in the IBI group. Nevertheless, caution must be taken in interpreting these results, as this variable also increased with a moderate effect size in the WLC group . It is worth considering that a learning effect over a 2-month period has been documented with repeated administration of the 6MWT [After 3 months, our results showed improvements in the patients’ functional capacities for aerobic exercise with a large effect size in both sexes  and was similar to the levels reported in related eHealth interventions ,65. IncrThe internet is an efficient way to prevent and treat chronic conditions by promoting healthy lifestyles because it can reach more individuals  and can provide more intensive contact at potentially lower costs than conventional face-to-face programs -74; it cHowever, despite the encouraging results of this study and other studies on internet-based programs, the goal of web-based interventions is not to replace in-person care but rather to maximize care. Of note, internet interventions also have potential disadvantages such as their inability to recognize comorbidities that would not have become apparent without the patient implementing lifestyle changes. Furthermore, internet use depends on age, income, education level, and digital skills, and there may be participation bias and lower response rates because of technical problems or different levels of computer experience among participants . In addiThe main limitation of this study was its lack of a control group for the analysis carried out at 12 months. Although the positive results at 3 months remained at the 12-month follow-up visit, the follow-up analyses were based on uncontrolled data and, thus, should be interpreted with caution. Controls eliminate possible alternate explanations of experimental results, especially those of confounding variables and experimental bias, which allows investigators to control for threats to validity.Furthermore, the participants we enrolled had demonstrated an initial level of motivation to engage in an eHealth program. Therefore, our findings may only be generalizable to individuals with internet access who are similarly interested in such eHealth interventions . In addiIn summary, we explored the effects of implementing a 3 month, self-administered IBI involving different learning techniques and aimed at promoting lifestyle changes  in patients who are obese with HTN. Overall, the participant engagement was high, and we observed moderate to large effect sizes in relation to BMI and BFM reductions. In addition, there was a favorable trend towards a relation to BP, which reached statistical significance at the 12-month follow-up. Taken together, these findings suggest that our tailored approach for delivering a lifestyle change intervention to patients who are obese with HTN provides positive health benefits. Simple strategies that can easily be incorporated into daily living in a scalable and cost-effective way can empower patients by educating them about health, thus, increasing their confidence and promoting self-management. Future research should investigate the acceptability and cost-effectiveness of self-administered internet-based interventions in populations with different sociodemographic status.


Prevotella 9, Lachnospiraceae UCG-001 and Bacteroides, associated with a higher weight loss after 12-month of follow-up, whereas in the cross-sectional analysis, Prevotella 2 and Bacteroides were enriched in the lowest tertile of baseline BMI. Our findings suggest that fecal microbiota plays an important role in the control of body weight, supporting specific genera as potential target in personalized nutrition for obesity management. A more in-depth taxonomic identification method and the need of metabolic information encourages to further investigation.Gut microbiota is essential for the development of obesity and related comorbidities. However, studies describing the association between specific bacteria and obesity or weight loss reported discordant results. The present observational study, conducted within the frame of the PREDIMED-Plus clinical trial, aims to assess the association between fecal microbiota, body composition and weight loss, in response to a 12-month lifestyle intervention in a subsample of 372 individuals (age 55–75) with overweight/obesity and metabolic syndrome. Participants were stratified by tertiles of baseline body mass index (BMI) and changes in body weight after 12-month intervention. General assessments, anthropometry and biochemical measurements, and stool samples were collected. 16S amplicon sequencing was performed on bacterial DNA extracted from stool samples and microbiota analyzed. Differential abundance analysis showed an enrichment of  Overweight and obesity are considered a worldwide public health problem which has rapidly increased up to reach global epidemic proportions . ObesityBacteroidetes are commonly less abundant in people with obesity, with this abundance increasing along with weight-loss [Firmicutes phylum as some of their genera as Lactobacillus and Clostridium have been associated to metabolic dysregulations related to obesity [Phascolarctobacterium, Dialister, Prevotella-to-Bacteroidetes ratio) were associated with a higher or lower weight loss [In the last few years, gut microbiota has been highlighted as an important factor related to obesity and its associated comorbidities . Causal ght-loss , whereas obesity , suggest obesity . In a we obesity . In contght loss ,12.Accordingly, the aim of the present study is to identify, in a large sample size, specific genera associated with baseline body mass index (BMI) and changes in body weight in response to a lifestyle intervention, in an elderly population with overweight/obesity and metabolic syndrome.2), aged between 55 and 75 years old and who at least met 3 criteria for metabolic syndrome [http://www.predimedplus.com, accessed 18 November 2020, and was registered at the International Standard Randomized Controlled Trial . This trial was approved by the institutional review board of all participating institutions, and participants provided written informed consent.This study was conducted within the frame of the PREDIMED-Plus clinical trial, that aims to evaluate the long-term effect of an intensive weight-loss lifestyle intervention on cardiovascular disease and mortality in a population with overweight and obesity  recruited in the PREDIMED-Plus centers of Reus and Málaga in Spain, randomly selected, matched by sex, age, and BMI, and with stool samples available at baseline and after 12-month of intervention.A cross-sectional analysis was conducted stratifying the sample by tertiles of baseline BMI. In addition, a longitudinal analysis was conducted stratifying the sample by tertiles of changes in body weight after 12-month intervention.Information about disease prevalence, lifestyle and medication use was collected. At baseline and 12-month timepoint, waist circumference was measured midway between the the lowest rib and the iliac crest using an anthropometric tape, body weight was measured using high-quality electronic calibrated scales, height was measured using a wall-mounted stadiometer. Systolic and diastolic blood pressure was measured 3 times using a validated semiautomatic oscillometer  and the mean value recorded.Blood samples were collected at both timepoints after an overnight fast. Plasma fasting glucose, total cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides concentrations were measured using standard enzymatic methods, low-density lipoprotein (LDL) cholesterol concentrations were calculated with the Friedewald formula whenever triglycerides were less than 300 mg/dL, and glycated hemoglobin was measured by a chromatographic method.Baseline and 12-month timepoint stool samples were collected and kept frozen till the delivery to the laboratory. In case of antibiotic treatment or fiber supplements, samples were collected 15 days after treatment completion. Stool samples were then separated into 250 mg aliquots stored at −80 °C, until analysis.Microbial DNA was extracted using the QIAmp PowerFecal DNA kit  following the manufacturer’s instructions. In the first step of the extraction, an additional lysing of 5 min using FastPrep-24™ 5G Homogenizer  was conducted. DNA concentration and purity were assessed with the Qubit 2.0 Fluorometer-dsDNA Broad Range Assay Kit .Targeted sequencing libraries were created with the 16S Metagenomics kit , using a pool of primers to amplify multiple hypervariable regions  of the 16S rRNA gene, in combination with Ion Plus Fragment Library Kit , to ligate barcoded adapters. Synthesized libraries were pooled and templated on the automated Ion Chef system  followed by a 400 bp sequencing on the Ion S5 . Sequenced reads were generated in BAM (Binary Alignment Map) format and then converted in FASTQ format using the File Explorer plugin of the Torrent Suite Server software , interfaced with the Ion S5.A customized Python script  was usedP value < 0.05 was deemed statistically significant.Baseline characteristics of participant were described as means and standard deviations or median and interquartile range (as appropriate) for quantitative variables, and numbers and percentages for categorical variables. Population was stratified by tertiles of baseline BMI and by tertiles of changes in body weight after 12-month intervention irrespective of the intervention group of the trial. Differences across tertiles were evaluated through one-way analysis of variance (ANOVA) or Kruskal–Wallis test for numerical variables, as appropriate, and with Pearson’s chi-square test for categorical variables. Student’s t-test or Mann–Whitney U test were used to calculate differences between tertiles for numerical variables, Pearson’s chi-square test was used for categorical variables. Statistical analysis was carried out using IBM SPSS Statistics version 23 . All statistical tests were 2-sided and ASV counts and taxonomic information generated with QIIME 2, were imported into R (version 3.6.2) and processed with the package Phyloseq, version 1.30.0 . ASVs coChao1, Shannon and Simpson indexes were calculated and pairwise comparison using Wilcoxon rank sum test performed to evaluate differences in microbial diversity among tertiles of baseline BMI. Bray-Curtis, Jaccard, Weighted and Unweighted Unifrac distance matrices were calculated and permutational multivariate analysis of variance (PERMANOVA) performed using the adonis function , to test differences in groups compositions, whereas permutation test for homogeneity of multivariate dispersions was performed to test variability among groups.Firmicutes-to-Bacteroidetes (F/B) ratio was computed based on the relative abundance between the phylum Firmicutes and Bacteroidetes, the log-normalized Prevotella-to-Bacteroides ratio (P/B) was computed based on the relative abundance between the genus Prevotella and Bacteroides. One-way ANOVA was used to test if F/B and P/B ratios were statistically significant different between tertiles of baseline BMI and tertiles of changes in body weight after 12-month intervention.The log-normalized P value < 0.05) were identified between tertiles of baseline BMI and tertiles of changes in body weight after 12-month intervention, using Wald’s test in the DESeq2 package, version 1.26.0 [Differential abundant significant ASVs , this study has some limitations that deserve comments. First, in our study we did not evaluate short-term changes in body weight and therefore, we cannot determine resilience of the gut microbiota; second, the design of our study did not allow it to establish causality; and finally, as this study was conducted in elderly Spanish individuals with obesity and metabolic syndrome, it cannot be extrapolated to other populations.We identified specific fecal microbiota signatures at genus level potentially related to changes in body weight in response to lifestyle intervention in an elderly population with overweight and obesity. These finding offer a promising novel perspective to support clinicians to tailor personalized interventions for obesity treatment, in which successful strategies can be predicted according to the microbiota composition. In any case, the validity of these microbial signatures has to be reproduced in other populations, taking into account the gut microbiota at species level. Furthermore, metabolic data are necessary to integrate these results and identify potential pathways involved, encouraging the need for further investigation in this field.


Background: Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) is a rare T-cell neoplasm, accounting for approximately 3% of adult non-Hodgkin lymphomas. Although NPM1 is the most common fusion partner with ALK, many others have been described, necessitating break-apart FISH studies for confirmation of the diagnosis. TNF receptor-associated factor 1 (TRAF1) is a rare ALK partner that is thought to confer a worse prognosis in patients. We describe the utility of next-generation sequencing (NGS) RNA analysis in detection of this uncommon ALK partner.Case Description: A 42-year-old male with cervical lymphadenopathy presented for excisional biopsy. Following a tissue diagnosis of ALCL, ALK+, RNA from the biopsy was extracted from Formalin-fixed paraffin-embedded (FFPE) tissue and prepared for Anchored Multiplex PCR using the Archer® FusionPlex® v2 assay, which employs unidirectional gene-specific primers using NGS to detect novel or unknown gene partners.Results: Histologic evaluation of the excised lymph node showed atypical cells, including “horseshoe/kidney”-shaped nuclei. Neoplastic cells were immunoreactive against CD30, ALK , CD2, CD4, granzyme B, and TIA-1. A diagnosis of ALCL, ALK+ was made. The pattern of ALK immunostaining suggested a non-NPM1-associated ALK translocation pattern, prompting further investigation. NGS fusion analysis showed a translocation involving exon 7 of TRAF1 and exon 20 of ALK.Conclusion: ALK positivity suggests an overall favorable prognosis of ALCL as compared to ALK-negative cases. However, in the rare published cases of TRAF1-ALK, an aggressive clinical course has been observed, which may reflect the aggressive propensity of this particular fusion, as these cases appear to be refractory to standard chemotherapy and also to the first generation ALK inhibitors. This study highlights the advantage of using NGS in RNA-based fusion assays to detect rare translocations, which can be of some clinical importance in detecting rare but aggressive fusion partners of ALK. As these technologies become more available, there is potential to identify such changes and effectively stratify the prognosis of ALCL patients. Although only a few cases have been reported, some have exhibited more aggressive clinical features. We highlight in this report another TRAF1-ALK-associated ALK+ ALCL and describe the clinical course of this case. This case also highlights the utility of RNA-based next-generation sequencing (NGS) analysis to detect novel and rare translocations involving genes such as ALK.Although the majority of ALK+ ALCL exhibit the NPM1-ALK reciprocal translocation, many variant translocations have been identified. Morphologically and immunohistochemically, there is no significant difference between the NPM1-associated ALK+ ALCL and those with the variant translocations . With reA 42-year-old Caucasian male presented history of progressive fatigue, chills, night sweats, and unwanted weight loss. He was found to have left cervical lymphadenopathy. Magnetic resonance imaging (MRI) revealed widespread adenopathy, innumerable small sclerotic lesions throughout the bones and one small lucent lesion in the right side of the pelvis. He underwent excisional biopsy of the cervical lymph node.Histologic evaluation of the excised lymph node showed effaced architecture with many atypical cells, including those with “horseshoe/kidney”-shaped nuclei. The neoplastic cells were immuno-reactive against CD30, ALK , CD2, CD4, granzyme B, and TIA-1 . Many ba2 area of the slide. After drying, this area was removed from the slide and transferred to a sterile tube. The product was treated with proteinase K, ethanol, and RNA extraction buffer. The RNA concentration of the final product was determined by the Qubit fluorometric quantification system (Life Technologies). The target-enriched library was prepared with the Archer FusionPlex Lung/Thyroid Panel  as per manufacturer's instructions (Next-generation sequencing (NGS) fusion analysis utilizing RNA from the sample was performed as described previously . Brieflyructions . This kiructions . SequencFollowing confirmation of the diagnosis with NGS translocation analysis , a bone TRG gene rearrangement studies were performed on the bone marrow from the aspirate sample. DNA extraction was performed using Qiagen PureGene kits as described previously  as previously described  positive lymphomas , juxtaposes the chromosomal region 5q35, which includes the NPM1 gene, with 2p23 which includes the gene encoding for ALK, a receptor tyrosine kinase. This leads to increased expression of ALK. The translocation results in a chimeric gene that encodes an 80-kD fusion protein. This protein combines the N-terminal portion of NPM1 to the entire cytoplasmic domain of the receptor tyrosine kinase ALK  translocation, the particular cytoplasmic, nuclear, and nucleolar staining can be explained by the formation of dimers between wild-type NPM and the fusion NPM-ALK protein. When the NPM1-ALK fusion protein forms homodimers, the dimerization process mimics ligand binding. This leads to autophosphorylation of the tyrosine kinase domain of ALK and constitutive activation, resulting in the oncogenic properties seen in anaplastic lymphomas  translocation, at least 11 variant translocations involving the ALK gene at p23 have been recognized. In all these translocations, the ALK gene is placed under the control of the promoter of genes that are constitutively expressed in lymphoid cells leading to elevated ALK gene expression. The most common variant translocation is t, in which the TPM3 gene, located on chromosome 1, is fused to the ALK catalytic domain  translocation is found in about 15–20% of ALK+ anaplastic lymphoma cases.However, not all ALK-positive lymphomas of carry the c domain . Howevert, involves the TFG gene. It produces two different fusion transcripts, of 85 and 97 kDa. These are referred to as TFG-ALKshort and TFG-ALKlong (ATIC gene (formerly known as pur-H), which codes for a protein involved in purine biosynthesis  other than NPM1-ALK. They found that although there are some morphologic differences between non-NPM1-ALK fusion cases from the classical ALK+ ALCL with NPM1-ALK1 fusion, the clinical features are largely equivalent. These cases mostly occurred in children or young adults who presented with advanced disease associated with extranodal involvement and B symptoms . Additionally, the overall prognosis of the variant ALK-positive lymphomas was similar to the classical NPM1-ALK cases with regard to relatively superior survival rates. The variant fusion cases were also significantly different in terms of prognosis from ALK-negative ALCLs . Other shan NPM1  (Table 1TRAF1) gene. Feldman et al. described a case of a 41-year-old male who suffered from an almost 30-year history of relapses of his lymphoma. He achieved remission only after high-dose chemotherapy and autologous stem cell transplantation (Another non–NPM fusion partner which has been described recently is the tumor necrosis factor receptor (TNFR)-1 associated factor  receptors. Once they link with TNFRs, they lead to activation of downstream kinase pathways. Binding of TNF to its receptor leads to recruitment of various TRAF proteins to result in activation of transcription factors such as nuclear factor κB (NF-κB) and activator protein-1 (AP-1) –30. To dIn this regard, Abate et al. have studied the properties and functions of the TRAF1-ALK fusion protein. The N-terminal of the TRAF1 region and its interaction with ALK leads to alterations in NF-kB function, contributing to tumorigenesis and maintenance of the neoplastic phenotype . The chit translocation involving NPM1-ALK. One methodology that has been developed in cases when the identification of the partner gene is desired is rapid amplification of cDNA ends (RACE)  . This telocation .TRAF1-ALK fusion. However, the utility of NGS is the discovery of novel fusions which may, in the future, be associated with adverse prognosis.The advent of next-generation sequencing and other deep sequencing platforms have allowed the rapid discovery of unique and novel translocations in cancer. However, this technology has not been utilized widely in the detection of translocations in ALK+ ALCLs. Here, we employed next-generation sequencing in an RNA-based fusion assay to detect this rare translocation partner. The advantage of the Archer assay is the ability to detect novel translocation partners for ALK in the case of ALCLs and lung carcinomas as well as other translocations in many other tumors. As NGS is employed increasingly in clinical and research settings, one may expect further studies investigating the biologic and clinical significance of these genetic lesions. Based on a cost prospective, it may be prohibitive to employ such testing on all ALK+ cases. It may also be possible to design a dual-fusion FISH probe to detect the specific TRAF1-ALK, an aggressive clinical course has been observed, which may reflect the aggressive propensity of this particular fusion, as these cases appear to be refractory to standard chemotherapy and also to first generation ALK inhibitors. In our case, aggressive chemotherapy and bone marrow transplantation likely aided in remission of disease. The current modalities to detect ALK translocation are IHC and FISH, but neither would identify these rare ALK partners. This study highlights the utility of using NGS in RNA-based fusion assays to detect rare translocations, which can be of clinical importance in detecting rare but aggressive fusion partners of ALK, e.g., TRAF. As these technologies become more available, there is potential to identify such changes and effectively stratify the prognosis of ALCL patients.ALK positivity suggests an overall favorable prognosis of ALCL as compared to ALK-negative cases. However, in the rare published cases of www.ncbi.nlm.nih.gov/nuccore/MT163661).The original contributions presented in the study are publicly available. This data can be found here: the NCBI GeneBank (MA and IA contributed to the design of the study, review of relevant clinical and pathologic data, and development of the manuscript. LS provided technical review of the data and editing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.