Acute exacerbation (AE) in idiopathic pulmonary fibrosis (IPF) is unfortunate a deadly event with a very high mortality rate. Its occurrence is highly unpredictable, though few baseline risk factors have been identified. The revised definition of AE is more precise with clarity on defined parameters. However, no clear guidelines exist on treatment, with most therapies showing inconsistent benefits. Both the approved anti-fibrotic (pirfenidone and nintedanib) have shown equal efficacy in reducing the decline in lung functions, with few studies suggesting a drop in AE. We report a case of a patient with IPF with mildly impaired lung functions who was initiated on pirfenidone with dose titrated on a weekly basis but developed AE-IPF on day 10 of starting pirfenidone and after four days of doubling the dose from 600 mg/day to 1,200 mg/day. This raised the suspicion of whether pirfenidone played any role in this unfortunate event. With no response to conventional therapy of steroids and non-invasive ventilation for AE-IPF, initialization of nintedanib led to recovery with discharge of the patient in two weeks of hospitalization. This case highlights inadequacy in knowledge about the effects of these anti-fibrotics in IPF and recommends close monitoring in the future. Acute exacerbation (AE) of idiopathic pulmonary fibrosis (IPF) is defined as a clinically significant deterioration in respiratory symptoms characterized by the presence of new widespread alveolar abnormalities . The repWe present a case of an 84-year-old man, a reformed smoker from the last 12 years (bidi smoking index of 2000) with a history of hypertension and type 2 diabetes mellitus on regular treatment since the last 20 years. He presented to us with complaints of progressive breathlessness for the last three months , cough, and scant expectoration associated with a weight loss of around 6 kg. The patient was seen by his physician who noticed basal crackles and obtained a chest X-ray, which was reported as increased markings with bibasilar linear opacities, shaggy cardiac borders, and both diaphragms borders. The patient was further seen in our Interstitial Lung Disease Clinic and underwent high-resolution computed tomography (HRCT) of the chest with a complete evaluation of lung functions and autoimmune profile. There was no history of any known exposures. HRCT of the chest revealed inter-lobular, intra-lobular, and sub-pleural interstitial thickening involving the basal and peripheral regions, along with traction bronchiectasis, honeycombing, and mediastinal lymphadenopathy (largest node being 10 mm in short axis) , normal liver and renal function tests (LFT and KFT), and normal serum LDH (lactate dehydrogenase) levels (154U/L), with a negative autoimmune profile. His lung functions showed moderate restriction  and significant de-saturation (21%) on the six-minute walk test (6MWT) with a distance covered of 224 meters (58% predicted).2 of 85% at room air. There was no history of fever, and chest X-ray showed worsening with bilateral middle and lower zone non-homogeneous opacities , a confident diagnosis of IPF (GAP I) was made. Although he had mild functional impairment, but in view of symptoms and to reduce AE, he was started on pirfenidone at 200 mg thrice a day, increasing to 400 mg thrice a day from day 7, and was advised review at two weeks with LFTs for further escalating the dose. He received standard supportive care with influenza and pneumococcal vaccinations and proton pump inhibitors. However, on day 10 of starting pirfenidone, he presented to the emergency department with an increase in breathlessness (mMRC grade IV) and baseline SpOs Figure . Routines Figure -2d.Echocardiography revealed a left ventricular ejection fraction (LVEF) of 55%, no left ventricular (LV) regional motion abnormality, right ventricle (RV) normal in size with adequate systolic function, and mild concentric LV hypertrophy. Sputum examination showed no acid-fast bacilli (AFB) and no growth on pyogenic cultures. Multiplex PCR (polymerase chain reaction) of sputum for viral and other infections was also negative. Pro-calcitonin (<0.05 ng/mL) and NT-proBNP  levels 387pg/mL) were normal. His Krebs von den Lungen (KL-6) level was 3,867U/mL  [7pg/mL we2 of 92% at room air.The patient was diagnosed as having AE-IPF and was managed conservatively with parenteral methylprednisolone at 40 mg every six hours and parenteral amoxicillin-clavulanic acid, with non-invasive ventilation (NIV) support, oxygen, and other supportive treatment. However, the patient did not respond and continued to worsen. Family members declined consent for invasive mechanical ventilation (IMV). After discussion with family members, nintedanib 150 mg twice daily was added to his treatment regimen. The patient was gradually weaned from NIV over the next week and steroids were tapered off. The patient was discharged on day 16 on domiciliary oxygen and tapering steroids. The patient was followed in the outpatient department at two weeks, which showed marked improvement in GGOs on CT scan Figure  and, theAE-IPF is a major cause of morbidity and mortality in patients with IPF of unknown etiology and is reported as unpredictable acute worsening, which is not due to heart failure or fluid overload. It carries 40-60% hospital mortality with 90% mortality in six months following discharge . Since iOur case developed an AE of IPF within 10 days of initiating anti-fibrotic therapy with pirfenidone. AE in IPF is unpredictable, with a recent retrospective study reporting one- and three-year incidences rate of AE of 14.2% and 20.7%, respectively . Hence, If we consider that AE-IPF, in this case, was idiopathic, we can look for baseline risk factors reported to predict future development of AE in IPF, such as lower FVC, never smoker status, and female sex. Another recent study showed baseline cardiovascular diseases, pulmonary hypertension, higher GAP stage (≥II), higher serum LDH level (≥180 U/L), and higher neutrophil (≥1.77 %) and eosinophil ≥3.21 %) percentages in BAL .21 % per, or treaSince alveolar epithelial cell injury is the predominant factor in the pathogenesis of AE-IPF, we used the KL-6 marker, which is a sensitive biomarker for type II alveolar epithelial proliferation and/or injury  and predOur case highlights AE of IPF while being initiated on pirfenidone and treated with nintedanib. AE-IPF episode is estimated to occur in 5%-10% of patients per year , with veAlthough it is debatable if AE in our patient occurred by chance when initiated on pirfenidone within 10 days, but as our patient had none of the known risk factors for AEs at baseline, possible implication of drug cannot be denied completely. Also, once AE-IPF sets in, there is no way to distinguish the cause behind it. We did not come across any such occurrence of AE-IPF with pirfenidone in safety studies in clinical trials as well as real-world experience. Also, prior use of pirfenidone has been shown to reduce postoperative AE in IPF cases undergoing thoracotomy for lung cancer . But thePirfenidone, a pyridone derivative, suppresses transforming growth factor (TGF)-beta induced myofibroblast differentiation and fibrogenic activity of human lung fibroblasts, though the exact mechanism of action in IPF is still unknown. The possible paradoxical effect of rapid dose escalation, thereby inducing worsening of our case, needs to be explored further. This would require in-depth analysis, as in the LOTUSS trial, a longer titration schedule (four weeks versus two weeks) was associated with better tolerability against a 14-day titration routinely recommended .Clinical improvement in response to nintedanib being given during AE has been reported earlier also . Our casThe effect of pirfenidone in inducing AE-free period in IPF cases had conflicting results from various studies. The first trial was terminated early as there were AEs only in the placebo arm, but these results were not duplicated in the second trial. Safety data on anti-fibrotics are still being collected and submitted to regulators, and it would be worthwhile to relook from this perspective too as any patient of IPF developing AE would be considered as a failure of treatment rather than because of it. Hence, the impact of IPF therapies on risks and outcomes of AE in IPF needs further elucidation.


Aim: Long-term studies addressing the outcomes of single immediate implantation and provisionalization at the maxillary esthetic zone are needed. The current study aimed to assess such outcomes along a follow-up period of up to 18 years. Materials and methods: The current study is a continuation follow-up of our previously published up to 6-year follow-up study, dated between the years 2002–2008, performed in a private clinical practice in Tel-Aviv, Israel. A total of 15 patients (23 implants) who had been treated for single-tooth replacement at the maxillary esthetic zone since 2002, underwent clinical and radiographic follow-up evaluations. Primary outcomes included mean Marginal Bone Levels (MBL), with Bleeding on Probing (BOP), implant success rate, prosthetic and esthetic complications evaluated as secondary outcomes. Results: The implant success rate was at 100%. Bone remodeling processes were observed over the follow-up period, with 0.9 mm mean marginal bone loss observed during the first 6 years of observation, followed by −0.13 ± 0.06 mm mean loss after 6 to 18 years. The last finding suggests bone deposition, as reported by other studies . At the final radiographic evaluation, a mean MBL of 1.35 mm ± 0.16 was demonstrated. No differences with respect to implant type or site were found. A generalized absence of BOP and esthetic complications occurred in two cases as a result of continuous adjacent teeth eruption versus obvious implant ankylosis. Conclusions: Adhering to careful clinical protocols and 3D bone to implant considerations while immediately placing an anterior implant, this treatment approach offers both stable and esthetically acceptable results for the replacement of missing teeth at the maxillary esthetic zone. Immediate implant insertion and provisionalization at the maxillary esthetic zone is considered to be a viable treatment option for those with missing teeth in that area ,3,4,5,6.There might be several disadvantages of immediate loading such as micromotion and implant instability ,30, whicMarginal bone levels (MBL) after immediate implant insertion and provisionalization seem to be equally successful to those levels after early and conventional implant loading, as described by recent systematic reviews and meta-analyses ,26. It mAs most existing data regarding immediate implant placement and provisionalization deal with short-term follow-ups, as described by several systematic reviews and meta-analyses, for example, , there iTherefore, the aim of the current retrospective study is to evaluate the long-term follow-up period of up to 18 years of immediately placed and provisionalized implants in fresh-extraction sites at the maxillary esthetic zone, in terms of marginal bone levels, bleeding on probing, implant success rates, prosthetic and esthetic complications.The current study is a continuation follow-up of our previously published up to 6-year follow-up study, dated between the years 2002–2008, performed in a private clinical practice in Tel-Aviv, Israel [In this retrospective study, 15 patients  ranging in age from 23 to 62 years, who were treated for single-tooth replacement at the maxillary esthetic zone since 2002, were selected for clinical and radiographical follow up evaluations, with a total of 23 implants. Following a thorough explanation of the treatment alternatives as well as risks, those accepting replacement by immediate loading and provisional crown placement were included. Informed consent was obtained from all patients before the clinical procedure.The current study addresses clinical and radiographical evaluations between the years 2002–2020 of fifteen patients with 23 implants, as one of the patients was considered as ‘drop-out’ as he did not show for follow-ups due to unknown reasons.The oral examination was focused on stable bilateral occlusion, soft tissue condition, buccolingual and mesiodistal width of soft and hard tissues and intermaxillary relationship. Periapical radiographs using the parallel technique, panoramic radiographs and computerized tomograms were also obtained, as necessary.Older than 18 years of age;Tooth loss due to periodontal attachment loss,Non-restorable crowns,Endodontic failures,Root fracture,Dentoalveolar trauma.Inclusion criteria for tooth procedure of extraction and immediate implant placement and provisionalization included:Uncontrolled diabetes;Severe para-functional habits (bruxism or clenching);Infected adjacent teeth;The need for tissue augmentation procedures during surgery.Exclusion criteria for this clinical procedure included:Implant placement and immediate provisionalization were performed by a single experienced clinician (E.M.). All patients were given Amoxicillin 1 gr. 1 h prior to surgery. Gentle elevation of the tooth root was performed to preserve the alveolar housing around the extraction site. Flaps were avoided. Atraumatic extraction was done using a periotome to release the periodontal ligament. Once the tooth was removed, the socket was carefully debrided and irrigated with sterile saline. Tapered titanium implants were placed .Implants with diameters of 3.3 to 5.5 and lengths of 13 to 16 mm were selected based on the size of the tooth socket and mesiodistal diameter of the tooth to be replaced.Implants were placed according to an ideal three-dimensional bone to implant position, in the apico-coronal, anterior-posterior, and mesio-distal dimensions.The platform of the implant was set approximately 1.5–2 mm below the level of the buccal bone. Implant placement respected the minimal 1.5–2 mm space between the adjacent tooth and the implant. Implants were positioned palatally in relation to the natural ‘central’ position where the tooth should have been, and an autogenous bone graft obtained by using a drill was used to fill space discrepancies in the cervical area when gaps were 2 mm or greater.Implant stability was monitored by using a manual torque wrench and the insertion torque was recorded in N/cm. If insertion torque values were 32 N/cm or greater, the implants were included in the study.Following placement, each implant was connected to a prefabricated plastic provisional abutment, as previously described ,31. The Patients were asked to limit their diet to soft food for one month and were routinely examined once a week for 3 weeks and then once a month for 6 months.The primary outcome parameter of this retrospective study was the measurement of MBL. The marginal bone level was recorded at the mesial and distal aspect of each implant after provisionalization (baseline) and during follow-ups up to 18 years. The distance between the implant-abutment interface to the first bone-to-implant contact  was assessed on periapical radiographs obtained using a positioning device and the long-cone parallel technique at baseline and at follow-ups. The implant length served as the reference distance during these measurements. The marginal bone level was calculated at follow-ups by subtracting bone levels at these years from baseline bone levels. Mesial and distal values were averaged in order to obtain a single value per implant.The secondary outcome parameters were the BOP, implant success, and prosthetic complications. BOP was assessed at six locations  around the implant at follow-ups. Each location was scored 0 or 1 . Implant success was defined as the mere presence of the implant irrespective of other clinical parameters. Prosthetic and esthetic complications were recorded as well. All measurements of peri-implant tissue responses were done by a single examiner (E.M). Statistical analyses were conducted utilizing SPSS Statistics version 26 .The mean MBL around the remaining implants, as measured in the last follow-up radiograph was at 1.35 mm ± 0.16. The pre and post-operative radiographs compared with the follow-up radiographs showed a mean marginal bone loss from placement to the 6-year follow-up, from placement to last follow-up, and between the 6-year follow-up to the last follow up time point, as described in In the current retrospective clinical follow-up study of up to 18 years, a 100% survival rate was observed among immediately placed and provisionalized implants in the esthetic zone of the maxilla. This high rate coincides with other long-term follow-up studies using immediate placement and loading protocols at the esthetic zone ,34,35,36The mean marginal bone loss in immediate implants with the non-occluding provisional crown during the first follow-up period of up to 6 years was found at 0.9 mm, as previously described . In the In the initial healing period following immediate provisionalization of implants in the esthetic area, there may be an accelerated bone resorption period, as suggested by Donati et al. , and wasIn the current study, BOP served as the marker for gingival inflammation. Donati et al.  reportedAlthough absent in our current long-term follow-up study, prosthetic complications may arise over long-term follow-up of immediate implantation and provisionalization protocols. Maló et al.  reportedFollowing immediate placement and provisionalization of anterior maxillary single implants, a continuing gingival recession of the facial gingival tissue may also occur, along with possible spontaneous papilla regeneration over time, as suggested by a recent long term follow up (2–8 years) study , and supDual or co-axis subcrestal angle correction implants with variable platform switching (SAC/VPS) have recently been shown to increase peri-implant soft tissue thickness and reduce gingival recession in case of immediate implant placement and provisionalization at the esthetic maxillary zone . It seemTo sum up, for soft and hard tissue preservation, both conventional implant loading protocols and immediate implant placement and provisionalization at the esthetic maxillary zone may yield similar beneficial results, as found by randomized controlled trials, systematic reviews, and meta-analyses ,49,50,51Presently, 3D preoperative planning through cone-beam computed tomography analysis (CBCT) allows for the verification of the maxillary anatomy and qualitative and quantitative bone structures before dental implant placement ,53. The Guided implant surgery allows the transfer of planned rehabilitation projects directly into the surgical field hence it should be considered for adoption especially for these highly technique-sensitive surgical procedures in the esthetic zone of the anterior maxilla.The current study has several limitations, including a relatively small number of patients and implants as part of its design. On the other hand, the benefit of such study design could be the ability to maintain a steady longitudinal long-term follow-up study (up to 18 years follow up) as dropouts are quite common in large scale studies, varying between 10–54.7% dropouts, for example, in ,46,60,61In conclusion, in a selected group of patients with very good oral practices and oral hygiene maintenance, immediate implant insertion and non-occluding provisionalization aid in the preservation of soft and hard tissue architecture at the esthetic maxilla zone. Proper case diagnosis utilizing 3D analysis, clinical and radiographic measures, as well as careful patient selection, are of paramount importance to promote success. Altogether, this treatment approach serves as a viable treatment option for those with missing teeth in this area, with acceptable esthetics, long-term survival rate, and marginal bone loss over a long-term follow-up period (14–18 years).


Background and purpose: Diagnosis of dementia with Lewy bodies (DLB) is highly challenging, primarily due to a lack of valid and reliable diagnostic tools. To date, there is no report of qualitative signature for the diagnosis of DLB. We aimed to develop a blood-based qualitative signature for differentiating DLB patients from healthy controls.Methods: The GSE120584 dataset was downloaded from the public database Gene Expression Omnibus (GEO). We combined multiple methods to select features based on the within-sample relative expression orderings (REOs) of microRNA (miRNA) pairs. Specifically, we first quickly selected miRNA pairs related to DLB by identifying reversal stable miRNA pairs. Then, an optimal miRNA pair subset was extracted by random forest (RF) and support vector machine-recursive feature elimination (SVM-RFE) methods. Furthermore, we applied logistic regression (LR) and SVM to build several prediction models. The model performance was assessed using the receiver operating characteristic curve (ROC) analysis. Lastly, we conducted bioinformatics analyses to explore the molecular mechanisms of the discovered miRNAs.Results: A qualitative signature consisted of 17 miRNA pairs and two clinical factors was identified for discriminating DLB patients from healthy controls. The signature is robust against experimental batch effects and applicable at the individual levels. The accuracies of the-signature-based models on the test set are 82.61 and 79.35%, respectively, indicating that the signature has acceptable discrimination performance. Moreover, bioinformatics analyses revealed that predicted target genes were enriched in 11 Go terms and 2 KEGG pathways. Moreover, five potential hub genes were found for DLB, including SRF, MAPK1, YWHAE, RPS6KA3, and KDM7A.Conclusion: This study provided a blood-based qualitative signature with the potential to be used as an effective tool to improve the accuracy of DLB diagnosis. Dementia with Lewy bodies (DLB) is the second most common cause of neurodegenerative dementia, accounting for up to 15–20% of dementia patients . An accuet al. developed a serum signature with four peptides  for discriminating DLB patients from AD patients and healthy controls  and two clinical factors (age and APOE ε4 genotype) to differentiate DLB patients from healthy controls  , amyloidcontrols . Anothercontrols .Although these reported quantitative signatures for DLB have achieved reasonable discriminatory capability, their application may be limited due to widespread batch effects. Therefore, it is of great significance to identify qualitative signatures that are insensitive to batch effects for DLB diagnosis. Some studies have indicated that REO-based qualitative signatures are robust against batch effects . MoreoveThe flowchart of this study is shown in Homo sapiens (organisms). Then, the inclusion criteria were used as follows: 1) datasets contained DLB patients’ and healthy controls’ miRNA expression profiles; 2) samples were blood samples; and 3) information on age and APOE 4 genotype were provided. Finally, only one dataset GSE120584 was screened out and downloaded. The normalized miRNA expression matrix, platform set, annotation file, and corresponding clinical information were downloaded and parsed via the GEOquery package  Feature generation. Given that the expression values of a miRNA pair  are denoted as Ei and Ej. The REO pattern of the miRNA pair is denoted as 1 (or 0 or −1 ) if Ei > Ej (or Ei = Ej or Ei < Ej). We calculated the values of the REO patterns for all miRNA pairs in each sample. The REO patterns of all miRNA pairs were used as new features for feature selection. 2,547 miRNAs constructed 3242331 miRNA pairs. 2) Feature selection. All feature selection methods were run on the training set. One miRNA pair was defined as a reversed stable miRNA pair when its REO pattern was identical in most control samples and was opposite in most patient samples. We first quickly identified 962 reversed stable miRNA pairs by setting the threshold at 60%. Then, the random forest (RF) was used to select 400 top-ranked important reversed stable miRNA pairs. The RF was implemented by the RandomForestClassifier function of the scikit-learn’s ensemble package. The random state was 16, and all other parameters were kept at default. Lastly, the support vector machine-recursive feature elimination (SVM-RFE)  with strTwo commonly used machine learning methods, logistic regression (LR)  and SVM Sensitivity (SE), specificity (SP), overall prediction accuracy, F1 score, and area under the receiver operating characteristic (ROC) curve (AUC) were calculated. ROC curves of the models were also plotted.p-value < 0.05 was considered significant. The miRWalk 3.0 online database  and the mirDIP online database (http://ophid.utoronto.ca/mirDIP/) were used to predict target genes of these dysregulated miRNAs. TargetScan, miRDB, and miRTarBase datasets were incorporated into the miRwalk framework (https://string-db.org/cgi/input.pl), an online tool for retrieving interacting genes, was applied to construct a protein-protein interaction (PPI) network. The confidence score threshold was set to 0.9. Then, the CytoHubba . The expressions of hsa-miR-92a-2-5p and hsa-miR-6813-5p were downregulated in DLB patients with healthy controls. And the expressions of 7 miRNAs, including hsa-miR-5698, hsa-miR-5001-3p, hsa-miR-5195-3p, hsa-miR-4687-3p, hsa-miR-4793-5p, hsa-miR-1322, and hsa-miR-4756-3p, were upregulated. The remaining 12 miRNAs, including hsa-miR-3148, hsa-miR-335-3p, and hsa-miR-3677-3p, etc., were not significantly altered in DLB patients and healthy controls. More details are given in Based on 17 miRNA pairs and the signature, we built four models by SVM and LR, namely DLB1, DLB2, DLB3, and DLB4, respectively. These established models were firstly evaluated by the stratified-3-fold cross-validation method. The cross-validation results of the four models are shown in Then, an independent test set was used to evaluate the predictability of these four models. Prediction results for the test set are shown in Overall, among these four models, the DLB3, which was developed by SVM based on the signature, outperforms other models in the training set. However, the DLB4 constructed by LR based on the signature provides the best prediction in the independent test set. Comparatively speaking, it is more important to classify external samples outside of the training set correctly. Therefore, in our study, the DLB4 is suggested to discriminate DLB patients from healthy controls. Meanwhile, we noted that the signature-based models perform better than 17-miRNA-pairs-based models in the training and test sets. These results indicate that integrating the clinical factors (age and APOE ε4 genotype) and 17 miRNA pairs improves the prediction performance.Firstly, 328 genes were predicted as target genes of these nine miRNAs by miRWalk and mirDIP . DetaileThe object of this study is to identify a qualitative signature for DLB diagnosis. We conducted an analytical study of serum miRNA profiling and clinical information of 169 DLB patients and 288 healthy controls. The significant findings of the study were: 1) a qualitative signature that consisted of 17 miRNA pairs and two clinical factors was identified for the diagnosis of DLB; 2) Based on the signature, prediction models were established by LR and SVM. Among them, the DLB4 model performed the best, which offered an accuracy of 82.61% for the test set; 3) Five potential hub genes were discovered for DLB.The main differences between our analysis and previous studies are exhibited in two aspects. On the one hand, as far as we know, a few quantitative signatures and no qualitative signatures have been reported for the diagnosis of DLB. This study discovered a blood qualitative signature consisted of 17 gene pairs and two clinical factors based on the REO pattern of the miRNA pair. The signature shows favorable discrimination capability, and it is robust and applicable to individual analysis. This is mainly because many biological and technical noise presented in the raw data is absorbed by the use of discrete classes (REO pattern). Several advantages of REO-based signatures have been demonstrated in numerous previous studies . For exaOn the other hand, we conducted a comprehensive bioinformatics analysis of potential target genes of nine dysregulated miRNAs of 21 miRNAs. Five hub genes were identified, including SRF, MAPK1, YWHAE, RPS6KA3, and KDM7A. Few, almost none of the studies so far have reported an association of them with DLB, but some evidence has implicated that they may play critical roles in other dementia subtypes. For example, SRF/MYOCD are suggested as novel targets for AD . They fuSeveral limitations need to be acknowledged and addressed for this study. Firstly, to use these miRNA pairs as biomarkers, multicenter prospective studies will be required to evaluate the accuracy of DLB diagnosis. Secondly, some clinical characteristics associated with dementia were not analyzed in this study due to insufficient clinical information of these samples, such as hypertension, dyslipidemia, and diabetes. Thirdly, more basic studies will be required to study the possibility of these miRNAs being developed as biomarkers for DLB diagnosis. Lastly, we will focus on discovering signatures for differentiating DLB patients from other dementias in the future.Overall, a blood qualitative signature consisted of 17 miRNAs and two clinical factors was identified to distinguish DLB patients from healthy controls in this study. The signature is highly robust against batch effects, and it is suitable for individual clinical applications. It is expected that the signature discovered in our research can be used as an effective tool to improve the accuracy of the diagnosis of DLB. Moreover, these new hub genes found may be potential targets for the treatment of DLB. More future studies will be required to explore the possibility of these hub genes being developed as targets in DLB.


The SARS-CoV-2 virus has taken more than 2 million lives on a global scale. Over 10 million people were confirmed with COVID-19 infection. The well-known spot of primary infection includes the lungs and the respiratory system. Recently it has been reported that the cardiovascular system and coagulation mechanisms were the second major targets of biological system affected due to the viral replication. The replication mechanism of SARS-CoV-2 involves the angiotensin-converting enzyme 2- (ACE2) surface receptors of endothelial cells belonging to various organs which act as the binding site for the viral spike (S) protein of SARS-CoV-2. The COVID-19 virus has been recently listed as a primary risk factor for the following cardiovascular conditions such as pericarditis, myocarditis, arrhythmias, myocardial injury, cardiac arrest, heart failure and coagulation abnormalities in the patients confirmed with COVID-19 viral infection. Direct and indirect type of tissue damage were the two major categories detected with cardiovascular abnormalities. Direct myocardial cell injury and indirect damage to the myocardial cell due to inflammation were clinically reported. Few drugs were clinically administered to regulate the vital biological mechanism along with symptomatic treatment and supportive therapy. COVID-19 virus has created a global pandemic situation that had never seen by the world when compared to the other major infectious outbreaks. 2,244,713 patients were died due to COVID 19 based on the World health organization (WHO) report dated on 03.02.2021 , high blood pressure, diabetic clinical condition, hyperglycaemia, people diagnosed with congestive cardiac failure (CCF), cancer, coronary heart disease, Atherosclerotic heart disease act as absolute lethal factors in patients confirmed with SARS-CoV-2 .The symptoms for SARS-CoV-2 includes common symptoms associated with common cold and flu, intermittent diarrhea, anosmia, and ageusia , 6. The Chest pain representing the following heart conditions including ischemia, MI detected by the elevated levels of troponin, (CK) creatine kinase, natriuretic peptide (NT-Pro-BNP) were significantly noticed with few primary coronary occlusions. Both direct and indirect mechanisms of tissue damage were documented as a major cause for cardiomyopathy in the dead patient –12. MortBased on the mode of cellular damages caused to various tissues and organs, by viral replication mechanism, it has been categorized into direct and indirect modes resulting in cardiac and pulmonary damages .COVID-19 virus can directly invade the cardiac and pulmonary tissues by using extreme target specificity . The damThe angiotensin-converting enzyme (ACE) are protein units distributed on the surface of endothelial cells which act as a primary target for viral invasion into the cells belonging to the cardiovascular system, gastrointestinal tract, renal system and respiratory system and type 2 pneumocytes . InjuredThe viral S-protein recognizes the extracellular domain of this ACE2 type 1 integral protein-containing peptidase as a targeted binding site. The diagrammatic representation of SARS-CoV-2 was depicted in 2 supply, and destabilizes the coronary plaque and formation of microvascular thrombosis. Persistent CVD (cardiovascular disease) becomes unpredictable and unstable due to an abnormal balance of high metabolic demand induced by a viral infection and decreased cardiac reserve. Acute Myocardial infraction (AMI-type 2) due to the uprise of metabolic demand by cardiomyocytes were documented in patients confirmed with SARS-CoV-2. There is an extreme chance for rupturing of coronary plaque and cardiac failure followed by SI in individuals with existing CAD (coronary artery disease). Abnormal coagulation has been recorded but the biochemical mechanism remains undiscovered. Arrhythmias were widely reported in COVID-19 infected individuals. Lymphopenia was highly associated with cytokine storm. Hypoxia, tachypnoea were few other clinical complications developed due to indirect viral mechanism. Multiple organ dysfunction Syndrome due to hyperimmune activity is a major cause of fatality (Systemic inflammation (SI) is the indirect mode of the viral mechanism causing damages to the cardiomyocytes. It has been reported that there is an elevated level of plasma troponin detected with a linear positive correlation with elevated CRP in blood. Procalcitonin (PCT) was also expressed along with CRP. This indicates that SI is a root cause for the damage of cardiomyocytes. Cytokine storm is also a part of an indirect mechanism leading to the damages of cardiomyocytes. Huang et al.  emphasizfatality .2 supply leading to tissue injury and cell death in the heart  occurs as a consequence of hypercytokinemia and Tumor Necrosis Factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6  and ARBs (Angiotensin receptor blockers) are angiotensin receptors inhibitors and blockers used to inhibit the communicating between the host cell with the virus. In the earlier stage, it was uncertain but based on the recent data it was found to be useful for protecting acute pulmonary injury caused by internalization of SARS-CoV-2 with host cell-based on murine model test results . Anti-ACfficient –9. Adminfficient . The usefficient , 78. Indfficient , 79.Recent reports of cardiovascular complications due to COVID-19 was clinically reported on a wide scale. Infection to SARS-CoV-2 has become a risk factor for cardiac disorders lately. Compared to adults, aged persons were prone to more complications and severity of cardiac conditions due to COVID-19 infection. Symptomatic and Supportive treatment was clinically administered for COVID-19 infected patients. Phase I and II clinical trials are currently in progress in many countries. The vaccine under clinical trials exhibits only short immunogenic effects which lead to administration of booster dose after 72–150 days to increase its efficacy up to 90%. To avoid infection preventive measures including usage of face mask, sanitiser, social distancing has to be followed until the discovery of a vaccine with prolonged immunogenic effects.SR: conceptualization and investigation. RG: resources and validation. SK, PK, and JR: formal analysis. VS: conceptualization and supervision. All authors contributed to the article and approved the submitted version.SK was employed by the company Devansh Lab Werks, Inc and PK was employed by the company Microgen Health Inc. The remaining 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.


Onboard quarantining has been only partially effective to control outbreaks of coronavirus disease on cruise ships. We describe the successful use of the ship as a quarantine facility during the response to the outbreak on the MS Artania, which docked in Western Australia, Australia. The health-led 14-day quarantine regime was based on established principles of outbreak management and experiences of coronavirus disease outbreaks on cruise ships elsewhere. The attack rate in the crew was 3.3% (28/832) before quarantine commencement and 4.8% (21/441) during quarantine on board. No crew members became symptomatic after completion of quarantine. Infection surveillance involved telephone correspondence, face-to-face visits, and testing for severe acute respiratory syndrome coronavirus 2. No serious health issues were reported, no response staff became infected, and only 1 quarantine breach occurred among crew. Onboard quarantine could offer financial and operational advantages in outbreak response and provide reassurance to the shore-based wider community regarding risk for infection. Legionella pneumophila  was identified as the causative organism of an infectious respiratory disease affecting residents of Wuhan, China. Coronavirus disease (COVID-19) rapidly spread around the world and was declared a pandemic on March 12, 2020 , on March 25, 2020, carrying 832 passengers (age range 7–89 years) of 12 nationalities and 503 crew members (age range 23–61 years) of 30 nationalities.The cruise ship MS Artania, which is 230 meters long, 9 decks, and built in 1984, can carry On arrival, the ship’s medical team reported to WA health authorities that 2 passengers had tested positive for SARS-CoV-2 upon their return to Germany after disembarking the ship in Sydney, Australia, on March 14, 2020, and that a further 15 passengers and 10 crew had reported fever, mild respiratory symptoms, or both during March 21–25. Point-of-care test kits for influenza A and B were not available on board. According to standard ship protocols, these persons were immediately isolated in their cabins and released 48 hours after symptoms resolved. On March 25, specimens were collected from 9 persons who remained in isolation; 7 (5 passengers and 2 crew members) tested positive for SARS-CoV-2 by reverse transcription PCR (RT-PCR). On the same day, 2 further persons evacuated for non–COVID-19 medical reasons also tested positive for SARS-CoV-2, and an outbreak was declared.The government of Australia has legislated responsibility for human biosecurity for international maritime arrivals and took the lead role in a multiagency response to the outbreak. It tasked an Australian Medical Assistance Team (AUSMAT) to coordinate the operational aspects of managing the outbreak. The AUSMAT team worked closely with federal agencies involved in biosecurity and border control, the state health department and law enforcement agency, and the local port authority.Vessel command divided the crew remaining on board into 2 groups, determined by the Minimum Safe Manning Certificate of the vessel: essential crew (EC), whose role was to maintain the safety  and vital functions (power supply and remote or direct systems monitoring) of the ship, and nonessential crew (nEC). Before quarantine began, all known SARS-CoV-2–positive case-patients (7 passengers and 2 crew members), along with their cabin-sharing contacts (7 passengers and 2 crew members) disembarked and were transferred to a hospital or hotel, depending on their clinical condition. An additional 2 passengers disembarked for other medical reasons and tested positive for SARS-CoV-2 upon hospital admission. Once they disembarked, no passengers or crew members returned to the ship, even if cleared of SARS-CoV-2 infection. Asymptomatic persons from Europe (791 passengers and 23 nEC) who were medically fit-to-fly (not tested for SARS-CoV-2 infection) repatriated to Germany on March 29, 2020, aboard 4 Condor Flugdienst charter flights.>38.0°C, and a confirmed case required a positive test result by RT-PCR on an oropharyngeal and bilateral deep nasal specimen. After commencement of quarantine, a health questionnaire based on the same guidelines was used for daily screening of nEC by using a cloud-based short message service (SMS) system or fixed telephone lines in cabins. Any health screening failure prompted a face-to-face interview and temperature measurement; otherwise, a face-to-face interview was conducted every 3 days. EC were monitored by daily face-to-face health screening and temperature measurement. The ship’s doctor  provided additional information daily because crew reported symptoms directly. We collated and analyzed data pertaining to demographics, symptomatology, temperature recording, and laboratory results in Excel .We defined a case according to Australia’s public health guidelines for COVID-19  and to respond to vessel emergencies. All nEC were accommodated individually in a separate area of the vessel in either unused or decontaminated cabins vacated by the disembarked passengers. They remained in strict quarantine for 14 days. EC and nEC could disembark, under escort, only if they tested positive for SARS-CoV-2, for other medical reasons, or because of a vessel emergency. The doors of all occupied cabins were marked to identify crew and food drop locations and for emergency evacuation purposes.The dock alongside the vessel and the adjacent terminal building were considered free of SARS-CoV-2 contamination (green zone). The access gangway, stairwells, and corridors to cabins were considered at low risk for contamination (yellow zone) and functioned as buffer zones. Contaminated areas (red zones) consisted of EC work areas and all occupied cabins .Entry to the green zone did not require personal protective equipment (PPE), but surgical masks and gloves were required for entry to the yellow zone. Within the red zone, different levels of PPE were mandated . All extAccess to interpreter services was available, but because the official language on board was English, all crew had a reasonably good command of the language. All could communicate by using their own mobile phones (top-up credit vouchers were provided) and fixed-line telephones in cabins. AUSMAT also received, attended, and assessed health-related calls from crew. Initial contact was by telephone and escalated to a cabin visit or engagement of onshore WA health emergency resources, if required. Medical facilities on board were not used.Efforts to minimize psychological stress and feelings of isolation and improve compliance included encouraging communication by individual 2-way and mass-SMS messaging systems, by using daily health checks as opportunities for high-quality contact time, and by using the public address and closed-circuit television (CCTV) systems to keep crew accurately informed. Other measures included acknowledging special events (birthdays and religious days), daily brain teaser exercises, and unsolicited local community support (handwritten postcards from primary school students).Before quarantine began, refrigerators in each cabin were stocked with several days’ supply of bottled water and long-life food and beverage items. To limit potential fomite spread, kitchen and catering facilities on board were not used. A 15-person external catering company prepared and delivered food for all persons on board, under direct supervision of AUSMAT. No food allergies were declared and meals consisted of culturally appropriate dish options not dissimilar to those normally available on board.Food delivery was conducted through evening-only food-drops, consisting of a cold breakfast and lunch and hot dinner. Food was dropped in front of each cabin, and the bridge informed the relevant nEC by public address to open the door and collect the food package after delivery.Waste bags were prelocated in each cabin, collected from the front of each cabin, and disposed of by the nightly cleaning team. Judicious food packaging resulted in minimal waste.To minimize traffic, 2 sets of bed linens were placed in each occupied cabin; a contingency procedure for special circumstances was available through an external contractor. At the end of the quarantine period, all laundry and linen were collected in plastic bags and heat-cleaned at 60°C by using the washing facilities aboard the vessel.A comprehensive brief detailing the quarantine process, requirements, and restrictions was communicated to the crew. Compliance was continuously monitored by a temporary 16-camera internal CCTV system, supplemented by 5 security guards whose responsibilities included immediately, reporting any breaches of quarantine protocols.Before quarantine began on April 3, a total of 28 of 832 passengers and 30 of 503 crew members experienced symptoms and tested positive for SARS-CoV-2. The earliest symptoms in crew were recorded on March 21 in a motorman who later tested positive for SARS-CoV-2. We identified 2 distinct crew clusters: 5 security guards in whom symptom onset occurred during March 25–April 2, and 9 food service staff (6 wait staff and 3 food preparation staff) in whom symptom onset occurred during March 22–30.During quarantine, 39 nEC disembarked: 21 (4.8%) symptomatic and SARS-CoV-2– positive persons , and 18 close contacts, none of whom tested positive. After clearance testing on day 13 of quarantine, 2 asymptomatic EC tested positive, which resulted in all EC disembarking for a further 14-day onshore quarantine. All close contacts remained negative for SARS-CoV-2. A previously identified EC backup team from nEC subsequently managed the vessel.The attack rate in crew before quarantine was 6.0% (30/503); during quarantine, the rate was 4.8% (21/441) (4.2% [18/427] in nEC and 21.4% [3/14] in EC). We recorded 1 COVID-19–related death in a male crew member 42 years of age. By the end of quarantine, 81 persons (30 passengers and 51 crew members) tested positive for SARS-CoV-2 ; of thosWe performed health screening through 2,934 SMS messages, 3,339 telephone calls, and 1,033 face-to-face visits; we also reviewed 13 medical calls made by crew to the onboard doctor. A total of 245 RT-PCR tests were performed, including those used for clearance testing. No serious mental or physical health issues were reported; the main complaints conveyed by crew were constipation, lack of access to exercise, and lack of fresh air in some cabins.A breach of quarantine was reported on day 5 when 3 nEC shared a kettle between adjacent rooms. Subsequently, 1 nEC became symptomatic and tested positive for SARS-CoV-2. A member of the external catering staff reported headache and fever (38°C) on day 7 of quarantine. She tested negative for SARS-CoV-2 and quarantined at home until symptoms resolved.MS Artania departed Fremantle on April 18, 2020, carrying 403 crew and 8 passengers to return to its home port in Bremerhaven, Germany. During the voyage, crew were repatriated in Jakarta, Indonesia (56 crew members); Manilla, Philippines (236 crew members); and Cairo, Egypt (1 crew member). Because of the possibility of asymptomatic infection and transmission during the 51-day voyage, the vessel command continued to impose AUSMAT recommendations for social distancing and mask-wearing in communal areas. The medical team continued rigorous COVID-19 symptom screening and temperature measurement of all persons. No crew members demonstrated elevated temperature or symptoms of acute respiratory infection before arrival in Germany on June 8, 2020 .Outbreaks of infectious diseases on cruise ships are a known risk, and cruise companies are well-versed in managing outbreaks of various types. However, SARS-CoV-2 poses a new and more severe threat, for which established prevention and response methods are inadequate. Early in the pandemic, several COVID-19 outbreaks on cruise ships drew global attention, and the level of risk and complexities involved in their control led to a shutdown of the cruise industry. We demonstrate that under certain circumstances, a COVID-19 outbreak aboard a cruise ship can be successfully controlled by using the vessel as a quarantine facility, which can have substantial financial, operational, and safety advantages.The outbreak aboard the MS Artania occurred in a setting of low prevalence of the disease in the WA community but intense political and community concern about the risk of importing the virus into the state , gave us a high level of confidence that the WA responders were not themselves a risk vector for infection. In geographic locations where SARS-CoV-2 activity is higher, introduction of the virus on board by infected responders would need to be mitigated by regular symptom and temperature checks, SARS-CoV-2 testing, or both.Solitary quarantine is challenging and potentially detrimental to physical and mental health. We focused on minimizing the length of quarantine through strict adherence to its principles and excellent communication by using a variety of technologies. Daily health checks provided the opportunity to build rapport and support and to reinforce and encourage compliance with quarantine requirements, and efforts were made to acknowledge special events. This approach might have contributed to a lack of reported serious mental or physical issues. The ultimate measure of success of this operation was that no symptoms consistent with SARS-CoV-2 infection were detected in any crew member after the 14-day quarantine period on board ended.The first limitation of our study is that it does not provide a complete description of the outbreak on board the MS Artania. Whereas no passengers or crew had to quarantine or be tested upon arrival in Bremerhaven, some crew who disembarked in other countries had to quarantine or be tested upon arrival home. None of these crew were symptomatic, but we could not obtain further details after their arrival. Our attempts to identify other cases from the cohort of repatriated passengers and crew before the start of quarantine have not been successful. Second, we could not be certain that all asymptomatic nEC were not infected and infectious during quarantine and at its conclusion because we did not test asymptomatic nEC as a condition of release. However, none subsequently experienced symptoms of COVID-19, and ongoing IPC measures for the duration of the voyage back to Germany mitigated this small risk even further. The guidelines for screening and testing for SARS-CoV-2 are constantly evolving. Our screening and testing protocols reflected best practices in Australia at that time, and a similar vessel outbreak would now be managed under a more rigorous testing regime.Although the international cruise industry was effectively halted because of the COVID-19 pandemic, some cruises have restarted and the risk for COVID-19 outbreaks will endure. The severe consequences of such outbreaks to human life and to the viability of the cruise industry necessitate a precautious approach, including the ability to manage outbreaks effectively and efficiently.In conclusion, use of the ship itself as a quarantine facility during an onboard outbreak offers financial and operational advantages, and we have demonstrated its feasibility under certain circumstances. Onboard quarantine should be considered as an option in COVID-19 outbreak response plans for cruise ships.


LEP-CpG7 and CpG15 methylation in patients were significantly higher than in healthy controls (P < 0.05). The LEP-CpG11, CpG33.34.35, CpG36 methylation, and mRNA expression decreased significantly in patients compared with healthy controls (P < 0.05). After controlling gender, age, BMI, dose of antipsychotic and duration of illness, LEP-CpG7 methylation was negatively associated with PANSS positive symptoms subscore . In addition, LEP-mRNA expression was negatively correlated with PANSS total score  and positive subscale . Nevertheless, only the LEP-CpG7 methylation level remained negatively correlated to the PANSS positive subscore following multiple stepwise regression . These results suggest that leptin methylation and mRNA expression might contribute to the pathogenesis of schizophrenia. LEP-CpG7 methylation may be negatively associated with positive symptoms in patients with schizophrenia.Leptin involved in the regulation of dopaminergic neurons of the central nervous system may confirm the hypothesis of neurodevelopment in schizophrenic patients. However, specific genetic mechanisms are undefined. Therefore, we aimed to explore the regulation of DNA methylation of leptin promoters and mRNA expression in patients with schizophrenia. A cross-sectional study enrolled 40 patients and 40 healthy controls from the Beijing Huilongguan Hospital in China. The leptin methylation levels and mRNA expression were examined by highly sensitive mass spectrometry based on the MassARRAY System and real-time quantitative polymerase chain reaction (qPCR). The Positive and Negative Symptoms Scale (PANSS) was applied to estimate the clinical symptoms of patients. The  Schizophrenia is deemed to a chronic and severe psychosocial disease with a lifetime prevalence of ~1% of the world's population . HoweverDNA methylation primarily occurs on cytosine residues in CpG dinucleotides close to gene promoters. The process is catalyzed by DNA methyltransferases, without changing the gene sequence . It has Leptin, a 16 kD protein, a primary product of the Ob gene, is an abundant hormone synthesized and secreted by adipocytes in adipose tissue, consistent with the amount in peripheral blood . AdditioLEP-mRNA expression in peripheral blood are utile and profound means of exploring the mechanisms of action of leptin that underlie complex variation in mental and psychological features of schizophrenia.Recently, the hypothesis of leptin dysregulation has been associated with psychopathology in schizophrenia. A case-control study illustrated that lower leptin levels in peripheral blood was closely related to more severe positive symptoms of schizophrenia . In contBased on the studies described above, the aims of the current study were as follows:To detect dysregulation of leptin CpG site methylation in the promoter region and mRNA expression in peripheral blood leukocytes, by comparing schizophrenia and healthy controls (HC) using the MassARRAY System and RT-PCR.To explore the relationship between leptin methylation, mRNA expression and the psychopathological status of schizophrenia patients as assessed by the Positive and Negative Symptom Scale (PANSS).To evaluate potential clinical and biological indices that could predict the severity of psychiatric symptoms in schizophrenia.All patients were recruited as inpatients of Beijing Huilongguan Hospital, China, and HC were recruited via written and electronic advertisements in the local community from October 2017 to March 2018. This study initially enrolled 49 Chinese Han schizophrenic patients and 42 healthy subjects, who were matched for gender, age, and BMI.All patients with schizophrenia was rigorously diagnosed by the fourth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV). The inclusion criteria for patients were as follows: (1) between 18 and 60 years old; (2) right-handed; (3) on stable antipsychotic treatment for longer than 6 months (without dose change for at least 4 weeks). The daily chlorpromazine equivalent dosages were shown in The inclusion criteria for HC were as follow: (1) between 18 and 60 years old; (2) were right-handed; (3) no history of medicine or neurological or psychiatric disorders. The exclusion criteria for the controls subjects were following: (1) a family history of mental disorders among first-degree relatives; (2) were pregnant or lactation; (3) an Intelligence Quotient (IQ) < 70; (4) healthy controls with the endocrine, fever, immune and metabolic disease were also excluded.Following screening, nine patients were excluded from the analysis due to not detecting the leptin methylation level or mRNA expression (three patients) or rejection (five patients) or exclusion criteria (one patient). They were therefore considered outliers. Forty HC were finally included in the analysis while two healthy people were excluded due to physical conditions. Hence, all analyses were performed on 40 inpatients and 40 controls in the study .The study was approved by the Ethics Committee of Beijing Huilongguan Hospital and was consistent with the Declaration of Helsinki (version 2004). All participants, including schizophrenia and HC signed written informed consent after a detailed explanation of the study contents.2), which was computed by height and weight.The PANSS  was usedIn particular, depressed factor , one of five factor models of PANSS was found to connect with clinical features and biomarkers in recent studies –24. Henc260/280nm < 2, OD260/230nm > 0.6) and gel electrophoresis detection (>20 ng/uL). DNA methylation levels at CpG sites in the promoter region were assessed using mass spectrometric technology, which proved to have high sensitivity, superior performance, high resolution, and easy repeatability on mass spectrometry based on the MassARRAY System  for quantitative real-time sequencing of DNA methylation  following the manufacturer's protocol. The quality of leptin DNA was assessed by OD values (1.5 < ODhylation .http://www.epidesigner.org) for CpG islands methylation prediction. The probe was designed according to the basic property requirement of Taqman probes. The primer sequences for human leptin gene were 5′-aggaagagagATTTAGAGTTGTGTGGGGTTTTGT-3′ (forward) and 5′-cagtaatacgactcactatagggagaaggctCACCTTCCCAAAAAACTAATCCTTA-3′ (reverse). LEP DNA methylation was measured at CpG sites near 222~648 bp in the promoter region of leptin. There was a portion of CpG sites without detection and some neighboring CpG islands to merge analysis. Finally, 12 CpG islands of leptin promoter were found to be involved in the analysis  were adopted. Following the manufacturer's protocol, reverse transcription was induced by reverse transcriptase to cDNA. The primer sequences for leptin mRNA were 5'-AGCTGTGCCCATCCAAAAAG-3′(forward) and 5′-TGGAGGAGACTACTGCGTG-3′(reverse). Due to the relatively constant expression in blood, tissue, and organs, GAPDH  was regarded as the reference gene when comparing leptin mRNA expression. The primer sequences for GAPDH were 5′-CTGCCAACGTGTCAGTGGTG-3′ (forward) and 5′-TCAGTGTAGCCCAGGATGCC-3′ (reverse). Real-time PCR (RT-PCR) was operated with a QuantiTect SYBR Green RT-PCR kit . And in the end, the comparative cycle threshold (2−ΔΔCt) method was used to accurately calculate relative quantitative leptin mRNA expression with GAPDH as a housekeeping gene  was extracted using the QIAamp RNA Blood Mini Kit . The quality of RNA was evaluated by its absorbance at ODThis was the first exploratory study about the relationship between leptin methylation and schizophrenia. There was no reference to calculate sample size in previous studies. So we included 40 cases in each group according to the actual clinical situation and usable expenses, which was it know that this is incomplete for a rigorous clinical study. However, we believe that the sample size will be accurately calculated according to this study in our follow-up studies.T-test between schizophrenia and HC groups, while the Mann-Whitney U-test was used when the distribution was not normal. Partial correlation analysis was adopted to examine the relationship between leptin methylation, mRNA expression and psychopathology of schizophrenia, with the following covariates when appropriate: gender, age, BMI, dose of antipsychotics and duration of illness. Taking the psychopathology scores as dependent variables and gender, age, BMI, dose of antipsychotic, duration of illness, LEP-CpG methylation and LEP-mRNA expression as independent variables, we used multiple linear stepwise regression analysis to explore potential factors influencing psychopathology symptoms. The significant statistical level was set at P-value < 0.05.Statistical analyses were performed using the Statistical Package for the Social Sciences . The demographic data were calculated using descriptive statistics. The categorical data were presented as frequencies and percentages (%). The numerical data were reported as the mean and standard deviation (SD) or the median and quartile. The chi-squared test for categorical variables, such as gender, Was taken into account. All continuous variables with normal distribution were calculated by two independent-sample P < 0.05).All analyses were performed on 40 patients and 40 controls. There were no significant differences in age, gender or BMI between the HC and schizophrenic patients (as shown in P > 0.05). LEP-CpG7  and LEP-CpG15  methylation surrounding the leptin promoter region in schizophrenic inpatients were significantly higher than those in HC. In contrast, markedly lower DNA methylation levels were observed in LEP-CpG11 , LEP-CpG33.34.35  and LEP-CpG36  methylation in patients. In addition, patients with schizophrenia exhibited significant lower LEP-mRNA expression  when compared with HC groups after using the Mann-Whitney U-test (P > 0.05)   (details > 0.05) .LEP-CpG7 methylation and PANSS positive subscore . In addition, LEP-mRNA expression demonstrated a negative correlation with PANSS total score  and positive subscale .After controlling gender, age, BMI, dose of antipsychotic and duration of illness, partial correlation analysis showed a negative correlation between LEP-mRNA expression, the LEP-CpG7 methylation level remained negatively correlated to PANSS positive subscore, with correlation coefficients of −0.485 . Meanwhile, LEP-mRNA expression was not significantly correlated with PANSS total score due to being excluded from the linear regression  polymorphism in the promoter neighborhood of LEP was not associated with significantly greater olanzapine- and clozapine-induced weight gain  for the publication of any potentially identifiable images or data included in this article.JS, YC, DC, and WL wrote and revised the manuscript. JS, KC, and HL analyzed the data. QZ, JY, and WF contributed to data collection and management. DC provided essential suggestions and revised the manuscript. All authors contributed to and have approved the final manuscript.This study was supported by the Capital Health Research and Development of Special (Grant: SF2018-2-2131).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


How does low-value care use vary across health systems?This cohort study measured and reported the use of 41 individual low-value services and a composite measure of 28 services for 556 health systems serving a total of 11 637 763 Medicare beneficiaries across the US. Systems varied widely in the provision of low-value care; those with a smaller proportion of primary care physicians, without a major teaching hospital, serving a larger proportion of non-White patients, headquartered in the South and West, and serving areas with higher health care spending delivered more low-value care.This study suggests that claims-based definitions can be used to measure low-value care within systems, providing granular, actionable feedback to promote health care quality and affordability. Low-value health care remains prevalent in the US despite decades of work to measure and reduce such care. Efforts have been only modestly effective in part because the measurement of low-value care has largely been restricted to the national or regional level, limiting actionability.To measure and report low-value care use across and within individual health systems and identify system characteristics associated with higher use using Medicare administrative data.This retrospective cohort study of health system–attributed Medicare beneficiaries was conducted among 556 health systems in the Agency for Healthcare Research and Quality Compendium of US Health Systems and included system-attributed beneficiaries who were older than 65 years, continuously enrolled in Medicare Parts A and B for at least 12 months in 2016 or 2017, and eligible for specific low-value services. Statistical analysis was conducted from January 26 to July 15, 2021.Use of 41 individual low-value services and a composite measure of the 28 most common services among system-attributed beneficiaries, standardized to distance from the mean value. Measures were based on the Milliman MedInsight Health Waste Calculator and published claims-based definitions.P < .001), no major teaching hospital , larger proportion of non-White patients , headquartered in the South or West , and serving areas with more health care spending .Across 556 health systems serving a total of 11 637 763 beneficiaries, the mean (SD) use of each of the 41 low-value services ranged from 0% (0.01%) to 28% (4%) of eligible beneficiaries. The most common low-value services were preoperative laboratory testing , prostate-specific antigen testing in men older than 70 years , and use of antipsychotic medications in patients with dementia . In multivariable analysis, the health system characteristics associated with higher use of low-value care were smaller proportion of primary care physicians (adjusted composite score, 0.15  for systems with less than the median percentage of primary care physicians vs −0.16  for those with more than the median percentage of primary care physicians; The findings of this large cohort study suggest that system-level measurement and reporting of specific low-value services is feasible, enables cross-system comparisons, and reveals a broad range of low-value care use. This cohort study uses Medicare data to measure and report low-value care use across and within individual health systems and identify system characteristics associated with higher use. We then identified health system characteristics associated with higher use of low-value services.15 This study was approved by the Dartmouth Committee for the Protection of Human Subjects. We received permission from IQVIA to report individual health system performance. To comply with Centers for Medicare & Medicaid Services reporting restrictions, we reported ranges of the number of beneficiaries attributed to each health system and suppressed system-level, low-value measure results with fewer than 11 beneficiaries.16For this cohort study, to generate health system–level low-value care measures, we used 2015-2017 Medicare fee-for-service administrative data from the Beneficiary Summary file, claims and administrative records , the Long Term Care Minimum Data Set, and First Databank. We examined the care of beneficiaries older than 65 years of age who were enrolled for at least 12 months or until death in Medicare Parts A and B in either 2016 or 2017. For select measures, we also used a random 40% sample of fee-for-service prescription (Part D) data. For measures using prescription information for eligibility exclusions, we required Part D enrollment for all months (≥12 months or until death) of Parts A and B enrollment. For measures in which prescription receipt was an outcome, we required at least 1 month of Part D enrollment. We excluded members with any hospice claims in 2016-2017. To determine health system and area-level characteristics, we used the Agency for Healthcare Research and Quality 2016 Compendium of US Health Systems (AHRQ Compendium), IQVIA OneKey, and MedInsight developed by Milliman (formerly Leavitt Partners’ Torch Insight).13We identified health systems from the AHRQ Compendium. These systems are relatively large and include a range of workforce compositions, reimbursement models, and organizational structures. The AHRQ Compendium defines systems as including at least 1 hospital and at least 1 group of physicians providing comprehensive care; the hospital(s) and physician group(s) are linked through common ownership or joint management.17 We restricted our analyses to systems that were not predominantly pediatric (based on name) and with 250 or more attributed beneficiaries.We linked Medicare beneficiary claims data to health systems by attributing each beneficiary to a single health system based on the plurality of primary care services received across 2016 and 2017, a method adapted from previous analyses.18 The calculator flags services as necessary, likely to be wasteful, and wasteful; to be conservative, we included only the wasteful services. Two physician researchers (I.G. and N.E.M.) reviewed measures to identify 36 Milliman low-value care definitions relevant to our study population. We defined and coded 5 additional measures described in previous studies.23 For each measure, we identified the beneficiaries eligible  for the potentially low-value service within each health system and counted eligible beneficiary-months as relevant for the measure until death or December 31, 2017, to capture the time during which a beneficiary had the opportunity to receive a given low-value service. We then calculated service use within each health system, defined as the number of eligible beneficiary-months of those who received a given service at least once divided by all eligible beneficiary-months in 2016-2017.We operationalized 41 claims-based, low-value care definitions described in prior research and in the Milliman MedInsight Health Waste Calculator, a stand-alone, proprietary software program that identifies potentially inefficient services based on recommendations from the Choosing Wisely campaign and professional medical societies .18 The cDetermining eligibility for each measure required analysis of data from the lookback (2015) and study (2016-2017) periods, including beneficiary age, sex, health care service receipt, medical diagnoses, and/or prescription receipt. For example, for the measure “injection for low back pain,” the measure denominator included eligible beneficiary-months after 2 or more diagnoses of low back pain (excluding radiculopathy) 7 or more days apart in 2016 and 2017. The measure numerator included eligible beneficiary-months for all beneficiaries in the denominator who also received a low-value injection  on or after the second low back pain diagnosis date. We grouped measures into the following 5 clinical categories: laboratory testing, imaging, cardiopulmonary and neurologic testing, procedures, and drugs. See the eMethods and eTables 1 and 2 in We defined the following health system characteristics: size , specialty mix , insurance product ownership , Accountable Care Organization (ACO) status , profit status , and teaching status . Attributed beneficiary characteristics included proportion with Medicaid-Medicare full dual enrollment and proportion of non-White race (Master Beneficiary Summary File); race was treated as a binary measure (White and non-White race). Area-level characteristics included US Census region (based on system headquarters), area-level standardized, risk–adjusted, per capita health care spending , and area-level hospital market concentration .Statistical analysis was conducted from January 26 to July 15, 2021. We estimated low-value care use by health systems for each of the 41 measures and averaged across measures within each clinical category. We tested for correlation (Pearson) in individual health systems between and within these clinical categories. We then created a composite measure using the 28 most common measures among health systems (defined by ≥11 beneficiaries receiving the service in at least 50% of systems). The composite score is the mean of proportions for the 28 measures, standardized to reflect distance from the all-systems mean value. We mapped systems with color coding reflecting the composite score to explore geographical patterns associated with low-value care intensity.In sensitivity analyses, we calculated the composite score in 3 additional ways. First, we repeated the main composite score method for all 41 measures. Second, to address the possibility that some health systems may not have the capability to provide certain low-value services such that the main composite measure may give these systems undue “credit” for avoiding these services, we reestimated the main composite score using 23 of the 28 measures that required no specialized capability . Third, we reestimated the main composite score excluding 3 measures for which lookback periods of more than 1 year would be ideal to determine measure eligibility but were not available in our data. We used Spearman rank correlation to compare these sensitivity composites with the main composite score.To explore attribution and accountability for low-value care use, we used a National Provider Identifier–to–health system linkage file based on IQVIA to calculate the measure-specific and overall proportions of low-value service recipients for whom a National Provider Identifier on the service claim was associated with the system to which the beneficiary was attributed.Finally, to examine health system characteristics associated with low-value care use, we used bivariate analyses to report mean composite scores for each characteristic level and then performed multivariable linear regression to estimate mean composite scores adjusted for the other characteristics.P values were 2-sided, and P < .05 represented statistical significance.We created claims measures and ran descriptive, validation, regression, and correlation analyses using SAS, version 9.4 (SAS Institute Inc) and Stata, version 16.1 (StataCorp LLC). To create the figures, we used Python in Google Collaboratory and Tableau Public. Reported Our analysis included 556 systems serving a total of 11 637 763 beneficiaries; system-attributed cohorts ranged from 250 to 379 949 beneficiaries   use of each of the 41 low-value services ranged from 0% (0.01%) to 28% (4%) of eligible beneficiaries (eTable 4 in r = 0.14-0.46). The highest correlations were between laboratory testing and procedures (r = 0.46) and between laboratory testing and cardiopulmonary and neurologic testing (r = 0.43). Exploring within-category service correlation, we found significant correlations between 10 of 15 pairs of laboratory test measures , 28 of 66 pairs of imaging test measures (19 positive and 9 negative), 8 of 15 pairs of cardiopulmonary and neurologic testing measures (7 positive and 1 negative), 18 of 55 pairs of procedures (17 positive and 1 negative), and 7 of 15 pairs of drug measures (5 positive and 2 negative) , North Memorial Health Care (Minnesota), and Rutland Regional Health Services (Vermont). Health systems with the greatest relative use of low-value services were El Camino Hospital , Eisenhower Medical Center , and Emerson Hospital Health System (Massachusetts) , no major teaching hospital , more non-White patients , headquartered in the South or West , and serving areas with more health care spending .In bivariate analyses, health systems differed in low-value care use based on size, specialty mix, ACO status, teaching status, percentage of non-White–attributed beneficiaries, region, and area-level per capita health care spending . In mult24 Absolute values suggest the level of potential waste, while comparison reveals the broad range of low-value care use; health systems in the lowest range of low-value care use demonstrate what can be achieved. The identification of health system characteristics  that are associated with low-value care use suggest a focus for future research on the causes of and remedy for overuse.Building on past measurements of low-value care at the national and regional levels, we measured low-value care at the actionable level of individual health systems.25 This finding corroborates prior work and validates inclusion of these services in many Choosing Wisely lists and as targets for intervention.26 We found a substantial number of positive, significant within-system correlations between and within clinical measure groups, consistent with prior work showing modestly positive associations between service categories within Veterans Affairs medical centers and within provider organizations .28 Correlations between cardiac screening measures and between head imaging measures raise the possibility that system factors, such as screening protocols or capital investment in imaging technology, may contribute to system-specific practice patterns. At the same time, some heterogeneity in low-value care provision within health systems was not surprising given the diverse drivers of these services and is consistent with prior studies showing limited correlations between quality measures.31 Each health system may perform well in some aspects of low-value care and poorly in others based on numerous factors that create unique low-value care “fingerprints.”Our results highlight potential contributors to system-level patterns of low-value care. Laboratory testing and prescription drugs were the most frequently used low-value services, which is not surprising given the broad base of clinicians ordering these services.24 showing that each additional primary care physician per 1000 residents in HRRs was associated with a substantial decrease in a low-value care composite of 20 measures and evidence that primary care physicians account for a small share of low-value spending, as well as studies linking primary care with lower overall health care spending.33 We did not find a significant association between having an ACO contract and low-value care use, similar to prior research showing only modest reductions.21 A 2012-2015 survey of ACO executives found that just 32% of ACOs had implemented strategies to reduce the use of low-value services.34 We found that health systems without a major teaching hospital used more low-value care, consistent with work by Chalmers et al35 comparing hospital-specific low-value services between teaching and nonteaching hospitals. Our finding of greater use of low-value care among systems serving more non-White patients is consistent with literature revealing that patients in minority racial and ethnic groups are more likely to receive care from lower-quality health systems compared with White patients.38 We found that health systems with greater use of low-value care were clustered in the South and West, consistent with extensive work on low-value care.39 Systems in higher-spending areas provided more low-value care, on average, mirroring research demonstrating a correlation between regional low-value care spending and total spending.19 This finding also highlights the relationship between large systems and regional practice pattern measures that reflect the sum of their constituents.Health system characteristics associated with low-value care use echo prior work and suggest directions for future research. We found that having more primary care physicians was associated with less low-value care use, building on the work by Zhou et al27 Our focus on system-level measurement is particularly important because an increasing number of individuals in the US receive care from system-affiliated clinicians each year,40 and we found that most beneficiaries receiving these low-value services received them from health system physicians. This finding could have both positive and negative implications for efforts to curb low-value care.41 On the positive side, health systems can leverage efficiencies of scale to apply quality metrics across all affiliated clinicians; track performance outcomes; streamline and disseminate new evidence, programs, or technologies to improve care delivery ; and foster a culture of health care resource stewardship.43 On the negative side, even though hospitals are financially disincentivized to perform low-value inpatient services by the diagnosis-related group payment system, large health systems that offer many hospital-based procedures have higher commercial prices and incentives to refer patients to other system-affiliated clinicians and services. This situation may result in increases in marginal services,46 such as low-value magnetic resonance imaging studies, as a recent study demonstrated among physicians newly employed by health systems.46Health system organizational features are likely key drivers of low-value care through policy setting, workflows and protocols, culture, and compensation models.47 If systems differ systematically in the completeness of diagnoses recorded, this may bias our conclusions. Although we expect that the low-value care patterns observed among large cohorts of fee-for-service Medicare enrollees likely reflect practice patterns of the systems studied, subpopulations as well as practices and hospitals within each system may have distinct care patterns. We limited our study to 556 large health systems that collectively served about 40% of all fee-for-service Medicare beneficiaries aged 65 years or older in our study period, but our findings may not be generalizable to other systems or independent practices. Because we limited the main composite measure to the 28 most common services to reduce the influence of rare events, its interpretation is limited to the scope of measures included. These measures represent a narrow share of all low-value care and an even smaller share of care quality overall. The composite score is most valuable at the extremes of the range: identifying relative outliers. Finally, all results represent care patterns in our study period and may not reflect current practice.This study has important limitations. Although we use widely vetted claims-based definitions of low-value care, claims data do not contain the nuanced clinical details necessary for more precise value measurement and risk misclassifying appropriate services as low-value. To mitigate this risk, we used the most specific service definitions available. Known variation in the quality and quantity of physician coding practices may influence measures that rely on diagnostic codes.48 For policy makers and payers, future repeated measurements may demonstrate how systems are changing over time and may reveal interventions associated with the increase or decrease of low-value services. Health systems might use this report to inform the internal measurement of low-value care. If internal measurements confirm our findings, they could illuminate factors associated with specific low-value services and inform strategies to address them. For example, because laboratory testing is among the most frequently used low-value services and is a common component of standard order sets, health system leaders could revise such tools to ensure that they promote high-value care. In addition, state or regional quality improvement organizations could use such results to establish a health system learning community focused on reducing the most frequently used low-value services in that region.49This system-level assessment of recent low-value practice patterns may enable cross-system comparisons and empower a range of stakeholders with actionable metrics. Research has shown that multicomponent interventions are most effective at reducing low-value care.50 Even with its imperfections, the transparent and actionable measurement of low-value care is a critical step toward improving the quality and affordability of US health care.Health systems could use system-level data on low-value care to develop incentive schemes to reduce unnecessary care, educate their workforce, or link hiring and retention decisions to use of low-value services. Health plans might include specific targets for the reduction of low-value care in contract negotiations or create networks of “high-value” clinicians. System-level reporting may also help to inform patients about specific low-value services and where they may be at greater risk of receiving them. Future measurement might emphasize other important facets of low-value care, such as cost, potential for direct harm, and potential for care cascades.


Short- to mid-term functional outcome in spontaneous cervical artery dissection is favorable, but the concomitant psychosocial impact is underreported. We aimed to determine these possible sequelae, with a special focus on sex differences, in our cohort of spontaneous cervical artery dissection subjects. During a standardized prospective in-house follow-up visit we, among other values, evaluated functional outcome , psychosocial measures  and health-related quality of life (WHO-QoL-BREF and SF-36-questionnaires). 145 patients participated in the long-term prospective follow-up. Median follow-up time was 6.5 years and excellent functional outcome (mRS ≤ 1) was achieved in 89.0% subjects. 87.6% returned to work and 17.6% married patients had a divorce during follow-up. Even though relevant baseline-/discharge characteristics and functional outcome did not differ between the sexes, women were less likely to return to work compared to men  and divorce rate was considerably higher in women . Health related quality of life did not differ significantly between the sexes, but women consistently reported lower values. Even though functional outcome is beneficial in most patients, measures to prevent poor psychosocial outcome should be considered in the long-term care of patients with spontaneous cervical artery dissection, especially women. As sCeAD is one of the main reasons for ischemic stroke at a young age, it is not surprising that subjects with all-cause cerebral ischemia before the age of 55 share these generally favorable neurological and functional outcomes6. Still, psychosocial outcome parameters, such as return to work, depression and anxiety proportions are impaired in all-cause young ischemic stroke survivors14. Interestingly, even though these data exist in all-cause young ischemic stroke patients, psychosocial outcomes have mostly been neglected in sCeAD4. The little available data suggest that a sCeAD diagnosis may have profound effect on the psychological state of patients but outcome analyses to date have focused on isolated metrics, such as functional parameters or health-related quality of life, individually15. Furthermore, analyses on sex-differences in psychosocial outcomes are scarce at best in both patients with all-cause or sCeAD related ischemic stroke16. Therefore, our goal was to provide evidence on long-term psychosocial sequelae after sCeAD within our single center cohort study (ReSect-study) with a special focus on sex differences.Functional outcome in spontaneous cervical artery dissection (sCeAD) is considered favorable on a short- and medium term18. In short, electronic medical files of patients treated at the Department of Neurology at the Medical University of Innsbruck due to suspected sCeAD between July 1996 and January 2017 were collected and underwent chart review. Subjects were included if intramural hematoma was evident in T1-weighted fat-saturated MRI imaging of cervical vessels and no timely association to high impact trauma existed. All eligible patients were invited to a standardized in-house clinical follow-up visit at least one year after sCeAD event, which included a physical examination as well as psychosocial outcome assessment through questionnaires and detailed history taking by experienced stroke neurologists.Details on the ReSect-Study were published previously20. Return to work was defined as restart of the occupational activity (including stay-at-home parent or caregiver) prior to the index event. The variable was scaled as (1) return to the work in full or reduced capacity (i.e. reduced working hours or in need of assistance for duties previously capable of doing by oneself) or (2) no return to work. The divorce rate was calculated for subjects that were married at baseline.Clinical and functional status of patients were evaluated using the National Institutes of Health Stroke Scale (NIHSS) at hospital admission and the modified Rankin Scale (mRS) at hospital admission,—discharge and follow-up. Excellent functional outcome was defined as mRS ≤ 1. Long-term psychosocial outcome included social reintegration (return to work rate and divorce rate) as well as health related quality of life (HRQoL) measured by WHO Quality of Life-BREF and the 36-Item Short Form Survey (SF 36) questionnairesDescriptive statistical analysis was performed using IBM SPSS Statistics (Version 26.0). Differences in categorical variables were examined by Chi-squared test and in continuous variables by the means of Mann–Whitney-U or T-test. A binomial logistic regression model was created to identify independent risk factors for poor functional and social outcome.This study was approved by the local ethics committee of the Medical University Innsbruck. All patients that took part in the ReSect-study signed appropriate informed consent. All methods were carried out in accordance with relevant guidelines and regulations.In total, 272 sCeAD patients were eligible for inclusion and 145 of these participated in the in-person follow-up evaluation of the ReSect-Study , three men and two women, died during the survey period between 1996 and 2017. Demographics, baseline characteristics and follow-up parameters of all ReSect subjects are given in Table .8%, threA total of 127 (87.6%) sCeAD patients had returned to work and 19 of 108 (17.6%) married patients had a divorce during follow-up Fig. .Figure 3Males predominated in our cohort and were older than women at initial event . Other baseline characteristics, especially measures of physical functioning at baseline , as well as functional outcome at discharge  and follow-up  did not differ between sexes  and divorce rate after sCeAD was considerably higher in women   was evident in 90% of patients after a median follow-up of 6.5 years. Social reintegration parameters overall were in line with the functional metrics as almost 90% of patients returned to work. This is much higher than the 60% reported for all-cause young ischemic stroke survivors13. Furthermore, only one in six subjects had a divorce during the follow-up period. Even though literature to date reports equal outcomes in sexes after sCeAD33, we observed a pronounced gap between men and women with females being significantly less likely to return to work and more likely to get divorced after sCeAD  after sCeAD and had information on divorce proportions but not on other outcomes in long-term unmarried cohabitating relationships. Lastly, it would have been rewarding to present a control group of subjects suffering from other cause ischemic stroke.Limitations include that not all subjects completed the HRQoL questionnaires. Even though baseline and follow-up characteristics between those assessed within the clinical visits and those evaluated via chart review did not differ Table , we cannIn conclusion, even though functional outcome is beneficial in most sCeAD patients there is a need for measures to prevent poor psychosocial outcome, especially in women.