Background: We aimed to identify the associated risk factors of type 2 diabetes mellitus (T2DM) using data mining approach, decision tree and random forest techniques using the Mashhad Stroke and Heart Atherosclerotic Disorders (MASHAD) Study program.Study design: A cross-sectional study. Methods: The MASHAD study started in 2010 and will continue until 2020. Two data mining tools, namely decision trees, and random forests, are used for predicting T2DM when some other characteristics are observed on 9528 subjects recruited from MASHAD database. This paper makes a comparison between these two models in terms of accuracy, sensitivity, specificity and the area under ROC curve. Results: The prevalence rate of T2DM was 14% among these subjects. The decision tree model has 64.9% accuracy, 64.5% sensitivity, 66.8% specificity, and area under the ROC curve measuring 68.6%, while the random forest model has 71.1% accuracy, 71.3% sensitivity, 69.9% specificity, and area under the ROC curve measuring 77.3% respectively. Conclusions: The random forest model, when used with demographic, clinical, and anthropometric and biochemical measurements, can provide a simple tool to identify associated risk factors for type 2 diabetes. Such identification can substantially use for managing the health policy to reduce the number of subjects with T2DM . WHO predicts the prevalence of T2DM in Iran to be 6.8% in 2025, and this translates to 5215000 citizens of Iran3.Type 2 diabetes mellitus (T2DM) is a major public health problem and its mortality is increasing worldwide4 and Mashhad cohort states this prevalence as 14% 5.The results of Tehran cohort show the prevalence of type T2DM in Iran is 11%st century7. Diabetes has its roots in interactions between genetic, environmental and behavioral characteristics9. Cardiovascular diseases particularly are responsible for 80% of deaths due to T2DM10. Dominant possible risk factors in the development of T2DM are ethnicity, obesity, unhealthy diet, lack of physical activity, insulin resistance, and family history of diabetes11. Heart disease, stroke, blindness, kidney disease, and amputations are associated with diabetes 12. It is therefore essential to identify and diagnose individuals that run a high risk of T2DM 613.T2DM is one of the most serious challenges for developing countries in the 2114. One reason for not using classical statistical method is the number of predictors which the classical methods cannot select them conveniently. These two models, decision tree, and random forest are two of classification models and there are not so many studies in this regard.In recent decades, different researchers in Iran have used data mining methods such as decision tree, neural network, support vector machine, random forest to predict the associated risk factors of T2DM16. Data mining can discover new factors and also find relationships among factors that can reveal patterns and develop predictions based on new factors associated with T2DM18.Data mining is a new collection of statistical methods used to characteristics significantly associated with T2DMThere are not many studies regarding associated risk factors of T2DM using data mining algorithms in Eastern Iran until yet. In this study, we developed the predicting model to identify associated risk factors of T2DM as a supplement in screening and public health in Eastern Iran.19.The MASHAD study started in 2010 and will continue until 2020. The city of Mashhad is located in the north-eastern part of Iran. The total population of Mashhad was estimated using the national Iranian census of 2006 so the sample size was determined accordingly. Participants were enrolled from three regions of Mashhad. Each region was divided into nine sites centered at Mashhad Healthcare Center divisions. Overall, 9528 subjects were enrolled as a part of MASHAD study This protocol was approved by the Ethics Committee of MUMS, and an informed written consent was obtained from every participant.19. Biochemical parameters included: fasted serum triglycerides (TGs), total cholesterol (TC), HDL-cholesterol and LDL-cholesterol, fasting blood glucose (FBG) and hs-CRP were measured as previously described19. Diagnosed T2DM was identified based fasting blood glucose (FBG) ≥126 mg/dl 20.Demographic characteristics such as age, gender, marital status, education, cigarette smoking habit, physical activity level (PAL), family history of diabetes (FHD) and depression score were collected from all the subjects. The Beck’s depression inventory-II (BDI-II) was used to evaluate the depression. Anthropometric information including weight, height, waist and hip circumference were obtained. Systolic and diastolic blood pressures were measured as described earlier The final data contains 9528 records and 18 variables, divided into 17 predictor variables and one outcome or target variable. The target variable has two possible states, namely occurrence of T2DM or no occurrence of T2DM. Demographic characteristics included age, gender, body mass index (BMI), marital status, level of education and biochemical markers, physical activity level (PAL), cigarette smoking habits, family history of diabetes (FHD) and depression score were considered as predictors -2.23. Decision tree algorithms develop splitting criteria at internal nodes to from the tree. The split of a node attempts to minimize the impurity of the node. If a split is unable to achieve any improvement in terms of reducing impurity, the node is not split and is declared as a leaf node. If a split is able to reduce impurity, then the split providing the maximum reduction in impurity is selected and two branches are formed, forming two new nodes. The popular splitting criteria are information Gain, Gini index and gain ratio. CART is one of the decision tree algorithms that construct a binary tree using Gini index for selecting the splitting variable at every internal node. The Gini index at a node D is given byA decision tree is a non-parametric method named according to the nature of target variable. It is called a classification tree if the target variable is categorical and a regression tree if the target variable is continuous. The purpose of a decision tree is to develop a predictive model in terms of predictor variables. The tree is formed by successively dividing data according to one of the predictor variables. A decision tree consists of three types of nodes: root node, internal nodes, and leaf nodesi is the probability that an observation in D belongs to the class Ci and is estimated by |Ci, D|/|D| 25. The sum is taken over them possible classes. The tree begins with all observations forming the root node and successive splits determine the order of importance of the predictor variables.where pRandom forest is an ensemble learning method. It generates many classification trees by selecting subsets of the given dataset and selecting subsets of predictor variables randomly, finally aggregating the results of all models to obtain a random forest. Multiple classification trees are obtained from bootstrap samples in order to arrive at the final “majority” classification rules. The tree training parameters used in the present study are (i) ntree=500, the number of trees generated (ii) ntry=17, the number of predictor variables used in each tree, and (iii) node size=5, the minimum number of observations in a leaf node. Supervised machine learning algorithms divide the data into two parts, namely training data and test data.26.One of the most important features of random forest and decision tree is the output of the variable importance. Variable importance measures the degree of association between a given variable and the classification result. Random forest and decision tree have four measures for the variable importance: raw importance score for class 0, raw importance score for class 1, decrease in accuracy and the Gini indexStatistical analyses were performed using R packages rpart (for decision trees), random Forest (for random forest) and caret. The complete sample contained 1361 individuals with T2DM and the remaining 8167 individuals without T2DM. The present study adopted a 10‐fold cross validation method to evaluate decision tree and random forest model. The 10‐fold cross validation method involves randomly separating the acquired data sets into 10 data sets that are equal in sample size. The decision tree and random forest models are constructed on the basis of a training data set. The rest of the nine data sets were used as testing data for verifying model effectiveness. Ten repeated empirical tests were conducted, where each subset was used as the test data. The bootstrap (500 replications) optimism-corrected area under the receiver operating characteristic curve (ROC) was estimated using R software.The decision tree developed on the training data was used to obtain the splitting criteria for different nodes and was then applied to observation in the test data. The resulting tree is used to measure sensitivity, specificity, and accuracy of the model. If values of these measures are high for training data and lower for test data, it is considered as a case of overfitting. These measures must be obtained on training data as well as on test data in order to establish validity of the model. The models reported in this paper have been validated and results on test data are reported here.27.Models are evaluated by constructing the confusion matrix for test data. In addition, accuracy, sensitivity, and specificity are also measured for each model. Accuracy, sensitivity, and specificity of a classification model are defined as follows Accuracy=(TP+TN)/(TP+FP+TN+FN)Sensitivity=TP/(TP+FN)Specificity= TN/(FP+TN)Here TP, TN, FP, and FN are truly positive, true negative, false positive and false negative respectively.The receiver operating characteristic (ROC) curve is the plot that displays the full picture of trade-off between the sensitivity and (1- specificity) across a series of cutoff points. Area under the ROC curve is considered as an effective measure of inherent validity of a diagnostic test.P <0.001). The mean BMI of diabetic patients was 28.78 ±4.4 and for non-diabetic persons was 27.76±4.7. The results of the independent t -test showed that the BMI in diabetics was significantly higher than non - diabetic people (P <0.001). The mean PAL of diabetic individuals was lower than non-diabetic individuals .Anthropometric and biochemical features are summarized in Based on the results of the random forest model, TG, hs-CRP, SBP, LDL, TC, FHD, age, BMI, and PAL were the most important risk factors related to T2DM . In a suBased on the results of the decision tree model, FHD, age, TG, SBP, hs-CRP, BMI, and DBP were the most important risk factors related to T2DM. P <0.001).Sensitivity (95% CI) of decision tree and random forest model are, respectively, 64.5%  and 71.3% , and their specificity (95% CI) rate are 66.8%  and 69.9%  respectively, and their accuracy (95% CI) are 64.9%  and 71.1% . We used the area under curve ±standard error (95% CI) to compare these two models. The related value in the case of decision tree amounted to 68.6 ±1.39 (65.8-71.3) and 77.3±0.001  for the random forest model . The decWe developed a prediction model based on cross-sectional study to predict risk factors of T2DM according to decision tree and random forest models.4.The random forest model showed that TG, hs-CRP, SBP, LDL, TC, FHD, age, BMI, and PAL were strongly associated with T2DM. The decision tree model found FHD, age, TG, SBP, hs-CRP, BMI, and DBP were strongly associated with occurrence of T2DM. Putting the two results together, TG, FHD, hs-CRP, SBP, and BMI are common associated risk factors of T2DM in the two models. In a cohort study by using a decision tree, TG, family history of T2DM, BMI, SBP, education level and occupation were the associated risk factors of T2DM30. RF creates multiple classification and regression (CART) trees, each trained on a bootstrap sample of the original training data and searches across a randomly selected subset of input variables to determine the split31. The variables such as family history of diabetes, age, triglycerides, LDL-cholesterol, body mass index, and physical activity level have already been identified as important associated risk factors of diabetes34. The present study has found hs-CRP as an important associated risk factor of T2DM, but it has not been reported so far 33.Decision tree algorithm is a classification model based on different predictor variables and is widely being used in medicine30.The results of our study showed that family history of diabetes and triglycerides were the most important risk factors related to T2DM in the decision tree and random forest models. In other studies also, family history of diabetes and TG were the most important associated risk factors for T2DM38.Decision trees are one of the easiest tools to decision systems and easy to understand. Decision trees can easily convert to if-then rules. Programs based on these rules can be made and used on personal computers for decision analyses, used easily with physicians and health care personnel to conclude the outcomes 3139. On the other hand, sensitivity of C4.5 algorithm was higher than random forest, but specificity of random forest was higher than decision tree (C4.5)39. The reason for being difference between sensitivity of them is using different algorithm.In this study, comparison of decision tree and random forest models showed that sensitivity and specificity values of random forest were higher than decision tree which was inconsistency with previous studies 40.The ROC curve is a technique to visualize, organize, and choose classification based on the performance of the classification. The area under the curve (AUC) is an index of which model performs better and has a high level of accuracy. This index, which compares the performance of true positive and false positive of two different decision extremes, is often used to evaluate the predictive accuracy of classification models39. Random forest model is an accurate model for investigation of novel predictor markers, which is in line with previous31.In the current study, the AUC of random forest of testing dataset was significantly higher than decision tree which was consistent with previous studies The strength of the study lies in its large sample size that makes it applicable to general population. One potential limitation of this study is that it is based on a cross-sectional data and cannot obtain results obtained from longitudinal or cohort data.Random forest models can provide good prediction models due to their efficacy and sensitivity and specificity. According to random forest model, TG and hs-CRP are the most important associated risk factors for T2DM. This study has also identified some new risk factors associated with T2DM indicating the need for further evaluation of clinical applicability of this model.This study was financially supported by the Biochemistry of Nutrition Research Center of Mashhad University of Medical Sciences, Mashhad, Iran.The authors declare that there is no conflict of interest.This study was supported by Mashhad university of medical sciences.Based on the RF model TG, hs-CRP, SBP, and FHD are the most important associated risk factors for T2DM.  Based on the DT model FHD, TG, age, and hs-CRP are the most important associated risk factors for T2DM.  RF model demonstrated a better discriminatory power compared with DT model.


Postherpetic neuralgia (PHN) is one of the most common types of chronic neuropathic pain, which seriously affects quality of the life because of pain severity and poor response to the currently available treatments. Ju Re Ba Du therapy as a form of acupuncture therapy which is proved to be effective in RCTs and very suitable for patients, has been used in Postherpetic neuralgia in patients for a long time, therefore a systematic review is necessary to provide available evidence for further study.The following databases will be searched from their inception to October 2020: Electronic database includes PubMed, Embase, Cochrane Library, Web of Science, Nature, Science online, VIP medicine information, and CNKI . Primary outcome: pain intensity assessed on a visual analogue scale (VAS); Additional outcomes:1.Quality of life assessed by the 36-Item Short-Form Health Survey (SF-36);2.Self-Rating Anxiety Scale (SAS);3.Self-Rating Depression Scale (SDS);4.Sleep quality measured by the Pittsburgh Sleep Quality Index (PSQI).Data will be extracted by two researchers independently, risk of bias of the meta-analysis will be evaluated based on the Cochrane Handbook for Systematic Reviews of Interventions. All data analysis will be conducted by data statistics software Review Manager V.5.3. and Stata V.12.0.The results of this study will systematically evaluate the effectiveness and safety of Ju Re Ba Du therapy intervention for people with Postherpetic neuralgia.The systematic review of this study will summarize the current published evidence of Ju Re Ba Du therapy for the treatment of Postherpetic neuralgia, which can further guide the promotion and application of it.This study is a systematic review, the outcomes are based on the published evidence, so examination and agreement by the ethics committee are not required in this study. We intend to publish the study results in a journal or conference presentations.https://osf.io/r6y9b)September 29, 2020 osf.io/r6y9b. ( There is no large-scale epidemiological investigation on HZ in China, but according to the results of some small-scale investigations, the incidence of HZ in China is close to that of other countries.,4 As the most serious complication of HZ, the incidence of Postherpetic neuralgia (PHN) also increases with age, and its pain symptoms are obvious, lasting from several months to several years, which seriously affects the quality of life of patients and brings serious economic burden and public health problems to the society.Herpes zoster (HZ) is a common and frequently-occurring disease among middle-aged and elderly people. The global incidence is increasing year by year, reaching 25% to 30%, and tends to increase with age. The average age of onset of HZ is 51.7 years old. Neuropathic pain, such as postherpetic neuralgia, is a chronic disease like hypertension and diabetes, and requires long-term treatment to improve or control symptoms.Postherpetic neuralgia (PHN) is one of the most common types of chronic neuropathic pain, which seriously affects quality of the life because of pain severity and poor response to the currently available treatments. The main strategies for PHN management are medication and invasive interventional therapies; however, these approaches have many adverse effects, so it is important to find another effective and safe treatment for PHN. but the therapeutic effect is not satisfactory. Scholars at home and abroad have carried out many studies on the treatment, and the exploration has attracted the attention from many disciplines.There is no consensus on the specific treatment drugs and diagnosis. At present, the clinical treatment is mostly empirical treatment, mainly including physical therapy, drug treatment, traditional Chinese medicine treatment, nerve block and so on, Therefore, this research intends to adopt the method of system valuation and meta-analysis of the Ju Re Ba Du therapy for Postherpetic neuralgia to evaluate the efficacy and safety.In recent years, acupuncture and moxibustion has been widely used in clinical and experimental studies of PHN, and its effectiveness has been fully proved. As a form of acupuncture and moxibustion, Ju Re Ba Du therapy has been used to relieve symptoms in Postherpetic neuralgia patients, but its effectiveness and safety have not yet reached a definitive conclusion.22.1The protocol of the systematic review has been registered.https://osf.io/r6y9b). This systematic review protocol will be conducted and reported strictly according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement guidelines, and the important protocol amendments will be documented in the full review.Registration: Open Science Fra network (OSF) registration number: September 29, 2020 osf.io/r6y9b. , which main treatment of Postherpetic neuralgia is Ju Re Ba Du therapy. The language of the trials to be included only Chinese or English.2.2.21.Repeated publications2.Review of literature and cases3.Animal studies4.Incomplete literature5.Non-randomized controlled trials2.3The types of subjects included patients diagnosed with Postherpetic neuralgia, regardless of their degree and possible complications. All patients were treated with Ju Re Ba Du therapy.2.4Interventions included treatment with Ju Re Ba Du therapy. The control group only received conventional western medicine treatment. The routine treatment of each RCT may not be identical, but the use of Ju Re Ba Du therapy is the only difference between intervention and control.2.52.5.11.Pain intensity assessed on a visual analogue scale (VAS)2.5.21.Quality of life assessed by the 36-Item Short-Form Health Survey (SF-36);2.Self-Rating Anxiety Scale (SAS)3.Self-Rating Depression Scale (SDS)4.Sleep quality measured by the Pittsburgh Sleep Quality Index (PSQI).2.62.6.1We will search the following electronic databases from their inception to October 2020: Electronic database includes PubMed, Embase, Cochrane Library, Chinese Biomedical Database, VIP medicine information, and CNKI . Fig. . The res2.6.2The following MeSH terms and their combinations will be searched: 1) Ju Re Ba Du therapy; (2) RCT OR RCTs; (3) Postherpetic neuralgia. (Table  Ju Re Ba2.72.7.1There will be two researchers (SH and ZP) carry out the selection of research literature independently using Note-Express software. We will first make the preliminary selection by screening titles and abstracts. Secondly, we will download full text of the relevant studies for further selection according to the inclusion criteria. If there is any different opinion, two researchers will discuss and reach an agreement. If a consensus could not be reached, there will be a third researcher (JW) who make the final decision. The details of selection process will be displayed in the PRISMA flow chart.2.7.2Two researchers (SH and ZP) will read all the included text in full, and independently extract the following information:(1)general information, including trial name and registration information;(2)trial characteristic, including trial design, location, setting, and inclusion/exclusion criteria;(3)the characteristics of the participants, including age, race / ethnicity, course of illness, etc.(4)details of intervention, including acupoints, time of intervention, course of treatment, time of single treatment, etc.(5)details of comparison interventions;(6)out- comes as described under type of outcome measure section.If we couldn’t reach an agreement, a third researcher (JW) would make the final decision. One researcher (ZL) would contact the corresponding author by telephone or e-mail for more information when the reported data were insufficient or ambiguous.2.7.3 The quality of each trial will categorized into ‘low’, ‘unclear’, or ‘high’ risk of bias according to the following items: adequacy of generation of the allocation sequence, allocation concealment, blinding of participants and personal, blinding of outcome assessors, incomplete outcome data, selected reporting the results and other sources of bias .All the included studies will be evaluated based on the guidelines of Cochrane Handbook for Systematic Reviews of Interventions.2.7.4P value of < 0.10 was considered to indicate the exist of reporting biases and small study effects.Reporting biases and small-study effects will be detected by funnel plot and Egger test if there are 10 more studies included in this Meta- analysis. For Egger test, 2.7.5We used Revman 5.3 software provided by the Cochrane collaboration to analyze the data. Binary outcomes will be summarized using risk ratio (RR) with 95% confidence interval (CI) for relative effect. Continuous outcomes will be summarized by using weighted mean difference (WMD) with 95% CI. We will use random-effect model (REM) for meta-analysis in this review according to research recommendations.2 and I2 statistical tests. Where P value ≥0.1and I2 ≤50%, there is no obvious statistical heterogeneity among the studies. On the contrary, where P value < 0.1or I2 > 50% indicates a considerable heterogeneity. Meta-analysis will be performed when the statistical heterogeneity is acceptable , otherwise, subgroup analysis will be applied to explore the influence of potential factors on the outcome measures. We will conduct sensitivity analyses by omitting studies one by one in order to probe the impact of an individual study. If a meta-analysis cannot be performed, we will conduct descriptive analysis instead.Statistical heterogeneity will be assessed by X2.7.6This is a meta-analysis study based on previously published data, so patient and public involvement will not be included in this study.2.7.7Ethical approval will not be required as this is a protocol for systematic review and meta-analysis. The findings of this study will be disseminated to a peer-reviewed journal and presented at a relevant conference.2.7.8 To achieve transparency and simplification, the quality of evidence is divided into 4 levels in GRADE system: high, medium, low and very low. We will employ GRADE profiler 3.2 for analysis.The quality of evidence for this study will be assessed by “Grades of Recommendations Assessment, Development and Evaluation (GRADE) standard established by the World Health Organization and international organizations.3 Studies have shown that the global incidence of HZ increases year by year.,17 Postherpetic neuralgia (PHN) is the most serious complications in HZ, is characterized by severe pain, its incidence of 5% to 30%, when patients older than 85 years old was 50%, and the higher incidence of pain and sustainable for several months to several years, the serious influence the patient's quality of life, brings the serious economic burden to society, caused a public health problem.,19Zoster (HZ) is caused by the human body being infected with Varicella Zoster virus (VZV). The virus is latent in the neurons of the ganglion, and as the host grows older or the immune function is reduced, the virus is reactivated. The main clinical manifestations were ganglionic clustered red acromial herpes and neuralgia.,21 2014 book “evidence-based acupuncture clinical practice guidelines herpes zoster (revised edition)” is now the disease only acupuncture clinical scheme, although the guide lists the different recommended level of acupuncture and moxibustion treatment, but the plan is not fixed, clinicians may, in accordance with the personal experience a bias when choosing different treatment methods, which can lead to clinical curative effect is not stable, differences of health economics. Therefore, it is necessary to form a more unified and effective acupuncture intervention program through the study of optimized program, so as to facilitate clinical promotion. The treatment regimens adopted in this study not only clearly list different treatment methods in different periods, but also specify the treatment sequence and treatment duration. Therefore, the optimized regimen can reduce the bias in the selection of treatment methods, narrow the differences in health economics, and have more clinical promotion value.Acupuncture and moxibustion is a common intervention method of HZ and PHN. Meta-analysis showed that acupuncture and moxibustion had better efficacy in terms of cure rate, removal time and pain improvement, no matter in the acute stage, crusting stage or post-neuralgia stage. Th17 has pro-inflammatory properties, which can stimulate the synthesis of various inflammatory factors and induce local reactions. Treg cells are T cells with active inhibitory effect and anti-inflammatory properties. Th17/IL-17 plays an important role in the body's immune defense and is a key factor for the occurrence and development of autoimmune diseases. However, silencing NLRP3 can inhibit the expression of inflammatory cytokines IL-1, IL-23 and chemokine CXCL1, thereby upregulating the Treg/Th17 cell ratio. Studies have shown that NLRP3 inflammasome can regulate the activation of spinal microglia cells and participate in the neuropathic pain process in rats.,25 HZ/PHN pain also belongs to neuropathic pain. At present, there are few studies on HZ/PHN and NLRP3 inflamriome, and only 1 paper in 2011 showed that VZV virus can trigger the formation of NLRP3 inflamriome. Pain, as a symptom that most affects the quality of life in Patients with HZ/PHN, is closely related to the SP/NK-1R axis. SP is widely distributed in various tissues of the body and is an important neurokinin related to the regulation of harmful information. It plays an important role in pain transmission, inflammation, immune regulation and other aspects.,28 It often binds to nK-R1 receptors, thus causing cell excitement, increased sensitivity, and induced immune function, stimulating the secretion of inflammatory factors, increasing vascular permeability, and vascular dilation, thus causing pain.–32 Therefore, it is found that NLRP3 inflammasome may play a role in acupuncture treatment of HZ/PHN.HZ and PHN's pathogenesis is complex, involving many aspects, inflammatory corpuscle is an important component of the innate immune system, including NLRP3 inflammatory corpuscle by nucleotide combination structure of oligomerization domain  members of the family of receptors NLRP3, protein ASC and cohesion in radix asparagi protease precursor of caspase - 1, ASC for linking NLRP3 and scaffold protein procaspase - 1,Procaspase-1, on the other hand, can shear the proinflammatory factor IL-1, the precursor of IL-18, and cause the proinflammatory factor to mature and release, thus triggering the inflammatory response.Ju Re Ba Du therapy for postherpetic neuralgia can significantly improve the pain symptoms of patients with significant clinical effects, and is worthy of further promotion and application in clinical practice.In summary, this systematic review will be an evaluation of the efficacy and safety of Ju Re Ba Du of postherpetic neuralgia. On this basis, a better processing method can be established to provide a reliable basis for its wide application.Conceptualization: Shijie Huang, Zhengqi Pan.Data curation: Shijie Huang, Zhengqi Pan, Tingting Ma.Formal analysis: Shijie Huang, Zhengqi Pan.Investigation: Jie Wu, Zimeng Li.Methodology: Jie Wu.Project administration: Shijie Huang.Resources: Zimeng Li.Software: Xinyun Zhu, Tingting Ma.Supervision: Jie Wu.Validation: Xinyun Zhu.Writing – original draft: Shijie Huang, Zhengqi Pan.Writing – review & editing: Shijie Huang, Zhengqi Pan.


Due to the high complication rate of panniculectomies, preoperative risk stratification is imperative. This study aimed to assess the predictive value of the 5-item modified frailty index (mFI-5) for postoperative complications in the elderly following panniculectomy.A retrospective cohort study of the American College of Surgeons National Surgical Quality Improvement Program database for patients over the age of 65 years who underwent a panniculectomy between 2010 and 2015 was conducted. The mFI-5 score was calculated for each patient based on the presence of diabetes, hypertension, congestive heart failure, chronic obstructive pulmonary disease, and dependent functional status, and an mFI-5 score of 2 was used as a cutoff. Multivariate logistic and linear regression analysis was used to determine the validity of the mFI-5 as a predictor of postoperative complications.P = 0.03), overall complications , and significantly longer hospital length of stay . mFI-5 score of 2 or more was an independent risk factor for wound complications  and overall complications .A total of 575 patients were analyzed. Patients with an mFI-5 score of 2 or more  had significantly higher rates of wound complications (19.5% versus 12.8%; Frailty, as measured by the mFI-5, holds a predictive value regarding outcomes of wound complications and overall complications in elderly patients after panniculectomy. The mFI-5 score can be used to identify high-risk patients before surgery. It is estimated that 228,000 bariatric procedures were performed in 2017, and up to 74% of patients desire subsequent body contouring procedures post-bariatric surgery, including panniculectomy.3 Despite its popularity, panniculectomy is associated with high overall complication rates ranging from 23% to 44%.5 Complications most commonly include seroma, hematoma, tissue necrosis, and wound dehiscence. Although most complications can be managed conservatively, postoperative morbidity remains a concern.6Abdominoplastic surgeries are among the most common surgical procedures performed by plastic surgeons in the United States, with 157,492 procedures performed in 2018.7 Several studies have identified possible risk factors for post-panniculectomy complications, including preoperative body mass index (BMI), mass of the resected pannus, and hypertension.8 However, many of these risk factors have not been shown to be predictive across different populations.9 This indicates the need for a more comprehensive approach to risk stratification.Due to the high complication rate of panniculectomies, risk stratification of patients before surgery is important. In particular, a patient after bariatric surgery may often have nutritional deficiencies, residual medical comorbidities, and a complex body habitus, which may negatively affect postoperative outcomes.10 The modified frailty index (mFI-5) is a 5-factor index designed to stratify patients based on the level of frailty and has been shown to be an effective predictor of mortality and postoperative complications in several subspecialties, including plastic surgery.11 The mFI-5 comprises the following variables: hypertension requiring medication, diabetes mellitus, chronic obstructive pulmonary disease (COPD) or a diagnosis of pneumonia within 30 days, functional status , and congestive heart failure (CHF).11 These comorbidities have been identified as contributing factors to increased frailty, which predisposes patients to adverse outcomes following invasive surgery. The aim of this study was to evaluate the efficacy of the 5-factor mFI as a predictor of postoperative complications in patients who have undergone panniculectomy.Frailty, or the measure of physiologic reserve and resistance to stressors, has been recognized as a predictor of healthcare outcomes.12 A retrospective review of the database was performed for the following current procedure terminology (CPT) code: 15830 . Cases recorded with a primary abdominoplasty CPT code  were not included in our study.For the purpose of this study, the Participant Use Files database from the years 2010–2015 was reviewed. The database is compliant with the Health Insurance Portability and Accountability Act and is exempt from institutional review board (IRB) review. The American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) is a prospective, risk-adjusted, outcomes-based registry that records demographic, perioperative, and 30-day postoperative de-identified patient information.11 using the ACS-NSQIP database. These 5 factors include history of diabetes, hypertension, CHF, COPD, and dependent functional status. Each of these factors accounts for 1 point, and the minimum value for the mFI-5 is 0, while the maximum value is 5. mFI-5 score was analyzed as a binary categorical variable to compare groups with a low mFI-5 score of <2 with those with a high mFI-5 score of ≥2.14 Patient preoperative demographics, clinical factors, and medical comorbidities at the time of surgery were analyzed. Primary outcomes of the study included wound complication and overall complication. Wound complication was a composite outcome that consisted of superficial incisional surgical site infection, deep incisional surgical site infection, or wound dehiscence. Overall complication was a composite outcome that included developing any of the following: wound complication, reoperation, readmission, mortality, postoperative bleeding or transfusion requirement, deep venous thrombosis, pulmonary embolism, myocardial infarction, urinary tract infection, or sepsis. Secondary outcomes included operative time and hospital length of stay (LOS).As a retrospective cohort study, database review was performed for the selected CPT code, and cases with missing information on age, gender, weight, height, functional status, diabetes status, and history of COPD, CHF, or hypertension were excluded from the analysis. mFI-5 score was the risk factor of interest in this study, and only patients aged 65 years or older were included. We relied on the mFI-5 described by Subramaniam et al2 or Fisher’s exact test (n < 10) for categorical variables and student’s t test for continuous variables. Potential confounders were controlled for using multivariate logistic and linear regression analyses. Variables included in our regression models included the following: mFI-5 score, age, BMI, gender, history of smoking, bleeding disorder, steroid use, American Society of Anesthesiology (ASA) classification, race, surgical team specialty, and wound classification. Statistical significance was defined as P value <0.05. All data analyses were performed using SPSS version 23.0 .Continuous variables are reported as “mean ± SD,” while categorical variables are reported as frequencies and percentages within their corresponding groups. Univariate analysis was performed using χReview of the database identified 7030 patients who underwent abdominal panniculectomy, and 575 of these were over the age of 65 years and eligible for inclusion in the study. Descriptive statistics of patient preoperative variables and characteristics are included in Table P < 0.001) and higher percentage of men . There were significant differences between both groups in the ASA class (P < 0.001), racial distribution (P = 0.001), surgical team specialty distribution (P = 0.02), and distribution of wound classification (P = 0.03)  patients had a score <2 and 154 (26.8%) had a score of 2 or more. Patients with an mFI-5 score of 2 or more had a significantly higher BMI  and overall complications , as well as significantly longer hospital LOS   when controlling for potential confounding variables. BMI  was also a significant independent risk factor for wound and overall complications, respectively , 1.26; 95% confidence interval (CI), 1.08–2.20; P < 0.001) ,30 and pulmonary comorbidities (including COPD)30 have all independently been identified as risk factors for postoperative complications.In the 575 patients included in this study, the overall complication rate of 23.3% and wound complication rate of 14.6% are comparable to those cited in the panniculectomy literature of 23%–44%.32 In addition, patients with increased mFI-5 scores were more likely to be men, which has been previously identified as an independent risk factor for complications after abdominoplasty.29Increased mFI-5 also correlated with several previously established risk factors for increased morbidity and mortality following panniculectomy. Patients with increased mFI-5 were significantly more likely to have an ASA class >3, which has been identified as an independent risk factor for morbidity following panniculectomy.34 The increased rate of postsurgical complications in obese patients is thought to arise from associated comorbidities, as well as physiologic changes, such as chronic low-level inflammation and impaired drug clearance.36 Although the relationship between frailty and BMI is unclear, there is new literature exploring the contribution of BMI to frailty and their prognostic values for perioperative planning following bariatric surgery.38 This study adds to the growing evidence that supports the validity of the mFI-5 as a valid risk stratification tool to predict morbidity and mortality following surgery.We have found that increased BMI both correlates with increased mFI-5 and functions as an independent risk factor for wound and overall complications, as well as increased hospital LOS. This further supports the external validity of the mFI-5 as a predictor of morbidity following panniculectomy. Several studies have shown that higher preoperative BMI is an independent risk factor for the development of postoperative complications following panniculectomy.39 However, the mFI-5 is only one measure of perioperative risk and must be weighed against the improvement in a patient’s health when deciding the appropriateness of surgical intervention. Patient-reported outcome measures in post-bariatric patients indicate a substantial improvement in the quality of life following body-contouring surgery.40 Understanding additional risks incurred due to increased frailty could better inform expectations for the recovery process and improve preoperative surgical counseling.The mFI-5 as a perioperative risk stratification tool can be used for quality improvement in plastic surgery by contributing to the domain of patient safety.Using the NSQIP database allows for a robust, retrospectively collected sample data. However, there are also several limitations inherent to the NSQIP database. Data availability is limited to 30 days postoperatively, and as a result, the incidence of complications may be underestimated. Findings are also limited to the 713 hospitals that are enrolled in the NSQIP database, which may differ from hospitals that do not participate in national data collection programs. Although these results may not be reflective of the overall patient population nationally, this is the largest study applying the mFI-5 to panniculectomy procedures to date. Finally, this is a retrospective study of the collected data while the proposed utility of the mFI-5 in preoperative risk stratification would be prospective. A prospective registry can therefore be maintained to assess the utility of the mFI-5 in practice.This study querying the NSQIP database for patients undergoing panniculectomy shows that frailty, as measured by the mFI-5, holds predictive value regarding outcomes of wound complications and overall complications. As such, the mFI-5 can be used by the surgeon preoperatively to enhance risk stratification and identify high-risk patients before panniculectomy. Future studies evaluating the prospective application of the mFI-5 before panniculectomy and its ability to improve perioperative evaluation are needed.This study was supported by American Diabetes Association Pathway Award 1-16-ACE-08 to Dr. Ceradini and the National Institute of Health Clinical and Translational Science Awards at NYU.


The aim of this study was to develop a model that could be used to forecast the bleeding risk of ITP based on proinflammatory and anti-inflammatory factors. One hundred ITP patients were recruited to build a new predictive nomogram, another eighty-eight ITP patients were enrolled as validation cohort, and data were collected from January 2016 to January 2019. Four demographic characteristics and fifteen clinical characteristics were taken into account. Eleven cytokines  were used to study and the levels of them were detected by using a cytometric bead array (CBA) human inflammation kit. The least absolute shrinkage and selection operator regression model was used to optimize feature selection. Multivariate logistic regression analysis was applied to build a new predictive nomogram based on the results of the least absolute shrinkage and selection operator regress ion model. The application of C-index, ROC curve, calibration plot, and decision curve analyses were used to assess the discrimination, calibration, and clinical practicability of the predictive model. Bootstrapping validation was used for testing and verifying the predictive model. After feature selection, cytokines IL-1, IL-6, IL-8, IL-23 and TGF-β were excluded, cytokines IFN-γ, IL-4, IL-10, IL-17A, IL-22, TGF-β, the count of PLT and the length of time of ITP were used as predictive factors in the predictive nomogram. The model showed good discrimination with a C-index of 0.82  in training cohortn and 0.89  in validation cohort, an AUC of 0.795 in training cohort, 0.94 in validation cohort and good calibration. A high C-index value of 0.66 was reached in the interval validation assessment. Decision curve analysis showed that the bleeding risk nomogram was clinically useful when intervention was decided at the possibility threshold of 16–84%. The bleeding risk model based on IFN-γ, IL-4, IL-10, IL-17A, IL-22, TGF-β, the count of PLT and the length of time of ITP could be conveniently used to predict the bleeding risk of ITP. The destruction of the platelets and an increasing risk of bleeding have a close connection with immune disorder in T cells in ITP patients2. However, T helper (Th) cells compose a broad group of immune cells that includes T helper 1 (Th1) cells, T helper 2 (Th2) cells, T helper 17 (Th17) cells, and T regulatory (Treg) cells. Th1/Th2 and Th17/Treg cell imbalances have been confirmed in ITP patients4. Additionally, the imbalances between these T cells result in an inflammatory state, which contributes to vascular endothelial injury and platelet destruction in ITP patients. In addition, the differentiation of Th1 cells is induced by the cytokine IFN-γ, the differentiation of Th2 cells is induced by the cytokine IL-4, the differentiation of Th17 cells is induced by the cytokine IL-17, and the differentiation of Treg cells is induced by the cytokine TGF-β. The activities of these T cells are also regulated by the cytokines IL-1, IL-6, IL-8, IL-10, IL22, IL-23, TNF-α and so on8. Therefore, the expression of cytokines might influence the severity of platelet destruction and vascular endothelial injury. In other words, alterations in the above cytokines may increase the risk of bleeding in ITP patients. Moreover, the risk of bleeding is controlled by a complex regulatory network that is built from a wide diversity of interacting molecular components. Nevertheless, there is still little information on the relevance of these cytokines to the risk of bleeding in ITP patients. Hence, it is necessary to determine the relationships between these cytokines and the risk of bleeding by using a model based on multiple cytokines.Immune thrombocytopenia (ITP) is a hematological disorder characterized by a decrease in platelet numbers with or without potential bleeding at multiple sitesIn this study, a cytometric bead array (CBA) human inflammation kit was used to detect the expression of IFN-γ, IL-1, IL-4, IL-6, IL-8, IL-10, IL-17A, IL-22, IL-23, TNF-α and TGF-β. Then, a predictive model was established by combining the expression levels of these cytokines with clinical information from patients. The model was used to determine the relationships between cytokines and bleeding risk. In addition, it is meaningful for clinicians to estimate the bleeding risk of ITP patients.9: (1) Finding thrombocytopenia during a routine blood count at least twice. Blood flm should be examined to exclude pseudothrombocytopenia and to check for morphological abnormalities of the blood cells. (2) Splenomegaly occurs infrequently in ITP patients. (3) Bone marrow examination is recommended. A bone marrow exam of ITP patients will show a normal or increased megakaryocyte count, with a decreased thromocytogenic megakaryocyte count. (4) Exclusion of secondary thrombocytopenia. The co-morbidity of patients were HT and DM, and there were no patients with other diseases such as strock, heart, and hyperlipidemia. There were no patients used drugs such as antiplatelet, anticoagulant, ant-lipid. The patients enrolled didn’t take any other medication other than glucocorticoid (Prednisone) for treatment of ITP such as thrombopoietin and rituximab. All participating patients provided written informed consent and completed questionnaires including personal information.Research approval was obtained from Xi Yuan Hospital, China Academy of Chinese Medical Sciences’ Ethics Committee (2015XLA108-2), and all participants provided informed consent in accordance with the relevant regulations and guidelines. Patients in training and validation cohorts were recruited from the Xiyuan Hospital, China Academy of Chinese Medical Sciences and Dongfang Hospital, Beijing University of Chinese Medicine, between January 2016 and January 2019, and they came from all over China. All the patients we enrolled were diagnosed according to Chinese guidelines for treatment of adult primary immune thrombocytopenia11.Two milliliter of venous blood was collected from each volunteer, then 200 μl serum were separated by density gradient centrifugation for cytokines analysis. The levels of cytokines  were detected by using a cytometric bead array (CBA) human inflammation kit according to the manufacturer's instructions  and analyzed by AimPlex Bead-based multiparametric flow cytometry . Briefly, AimPlex Bead-based Multiplexed Immunoassays for Flow are similar in principle to a sandwich ELISA, with each bead population conjugated with a specific capture antibody to trap the protein of interest, such as a cytokine, in a sample. The amount of the analyte captured is detected via a biotinylated antibody that recognizes a secondary epitope in the protein, followed by streptavidin-PE treatment. The fluorescence intensity of PE on the beads is quantified on a flow cytometer. The concentration of a protein of interest in a sample can be obtained by comparing the fluorescence signals of the sample to those of a standard curve generated from serial dilutions of a known concentration of the analyte12, no bleeding group was 0 score, the others was bleeding group, which score was above 0.The evaluation of bleeding was based on the patient history of bleeding and physical examination during the first week of consultation. The examination covered 9 anatomical sites including the skin, oral cavity, nose, gastrointestinal tract, urinary system, gynecological tract, lungs, intracranial and conjunctiva. Then, patients were divided into two groups according to ITP-BAT scorehttps://www.R-project.org).R software was used for statistical analysis , 95% confidence interval (CI) and P-value were calculated for all features. The statistical evaluations were all two-sided. Demographic characteristics with a P-value ≤ 0.05 were included in the model, and variables associated with disease or treatment characteristics were also included. All potential predictors were applied to develop a predictive model for the risk of bleeding by using the cohort15.The least absolute shrinkage and selection operator (LASSO) method was used to select the optimal predictive features among risk factors for bleeding in patients with ITP because it is fit to constrain high-dimensionality data16. Then, the discrimination performance of the bleeding risk nomogram was quantified with the area under the receiver operating characteristic (ROC) curve and Harrell’s C-index. Subsequently, bootstrapping validation  was conducted to calculate a relatively corrected C-index for the bleeding risk nomogram18. However, the clinical usefulness of the bleeding risk nomogram was detected by decision curve analysis, which quantified the net benefits at different threshold probabilities in the validation dataset19. However, the method of calculating net benefit was to subtract the proportion of all false-positive patients from the proportion of true-positive patients and to weigh the relative risk of intervention against the negative consequences of needless intervention20.Calibration curves were used to assess the calibration of the bleeding risk nomogram. A significant test statistic would indicate that the model was not perfectly calibrated9/L. All of the members were divided into bleeding and nonbleeding groups according to ITP-BAT score, no bleeding group was 0 score, the others was bleeding group, which score was above 0. The results for the expression of cytokines are shown for four groups, which were created by grouping by percentile . Additionally, patients were divided in three groups according to the platelet count based on consensus-based recommendation for target platelet counts for surgery or medical therapy in adults21. All patients data, including demographic and clinical characteristics, in the two groups are reported in Table There were 100 patients of training cohort and 88 patients of validation cohort enrolled in this study, included persistent (3–12 months) and chronic (> 12 months) patients with the platelet count range of 12–78 × 10The 19 features , which have unique patterns of effector cytokine secretion2. Th1, Th2, Th17 and Treg cells are differentiated from Th0 cells. Th1 cells secrete the specific cytokine IFN-γ, Th2 cells secrete the specific cytokine interleukin-4 (IL-4), T helper 17 (Th17) cells secrete the specific cytokine IL-17A, and Treg cells secrete the specific cytokine TGF-β. Breg cells are another kind of immunoregulatory cell that secretes the specific cytokines IL-10 and IL-2227.However, ITP is considered to be a consequence of complex immunoregulation disorder events in T cells, B cells and related cytokines. Previous studies showed that the development of ITP was connected with immunoregulation disorder caused by Th1/Th2 and Th17/Treg cell biases and abnormal secretion by BregsGenerally, cytokines can be divided into two types depending on their function. One type is proinflammatory, and the other is anti-inflammatory. The expression of proinflammatory and anti-inflammatory factors tends to be balanced under physiological conditions; otherwise, immune disorders occur.28.Additionally, cytokines are derived from immune cells, and the secretion and function of immune cells are regulated by related cytokines. In other words, the occurrence and development of ITP is due to the overexpression of proinflammatory factors and low expression of anti-inflammatory factors, according to researchSince cytokines are so important in the onset of ITP, cytokines should be incorporated into bleeding models. However, previous hemorrhagic risk assessment models did not take cytokines into account. In this study, a relatively accurate predictive tool was used to evaluate bleeding risk in patients with ITP. The internal validation and external validation of the tool showed good discrimination and calibration capabilities; in particular, our high c-index measured by interval validation and AUC indicate that our model can be used widely and accurately to assess bleeding risk.29, platelet function30, platelet activation31, and the immature platelet fraction32. However, these models, such as the ITP-BAT33, WHO bleeding scoring system34, Buchanan hemorrhage score35, and Khellaf hemorrhage score36, are based on only clinical hemorrhagic symptoms. It is difficult to assess the risk of bleeding in a patient if he or she bled previously, but the symptoms have disappeared or no positive signs of bleeding are found on physical examination. At this time, the risk of bleeding is underestimated, which affects treatment choice. Additionally, low platelet counts do not always indicate a high bleeding risk; therefore, if treatment is based only on platelet counts, overtreatment may occur, resulting in wasted medical resources and forcing patients to take unnecessary medical risks. In addition, platelet function detection methods include optical turbidimetry, the impedance method, thromboelastography, Plateletwork tests and flow cytometry37,38. However, these methods have some defects, such as poor replicability, a requirement for a high platelet quantity and expensive instruments. In fact, predicting the bleeding risk in individual patients is difficult, and the inclusion of various factors and multifaceted interventions may be the most effective solution. Therefore, it is very necessary to find an index that can comprehensively evaluate the hemostatic state of ITP patients while also being convenient and inexpensive. Similar to previous studies, our study showed that a low platelet count and long course of disease increased the risk of bleeding. In contrast to previous studies, this study took into consideration the expression of cytokines.Regarding the risk of bleeding in ITP patients, a previous studies mainly focused on the effect of platelets, including the platelet countIn this study, 65% of ITP patients exhibited bleeding, including in the purpura, muscle, nose, retinal and gingival. Then, we obtained eight key factors that forecasted the risk of bleeding in ITP patients by risk factor analysis. This nomogram suggested that patients with high expression of IFN-γ, IL-17A, and IL-22; low expression of IL-4, IL-10, and TGF-β; a low platelet count, and a long course of disease had an elevated risk of bleeding. This result basically agreed with clinical feature findings. Therefore, it is helpful to take into account cytokines when predicting bleeding risk. This model will assist physicians in accessing the bleeding risk of ITP patients and taking interventions in time, preventing unnecessary measures in low-risk situations and avoiding delays or discontinuity in treatment when the most appropriate time appears. We also believe that this model will be beneficial in the evaluation of drug efficacy since improvements in patients with bleeding symptoms are as important as improvements in platelet levels.There are also several limitations of our current study. First, our study did not enroll newly diagnosed patients (less than 3 months) and the platelet level as 80–< 100 were not included, so the bleeding risk for these patients cannot be evaluated. Second, children and teenagers were not included, so the difference between pediatric and adult patients was not be taken into consideration. Third, the cohort was not representative of all Chinese patients with ITP since patient race and region of origin were not considered. These factors need to be externally evaluated in a broader population of ITP patients.


Accumulating evidence has shown that valproate has the greatest teratogenic potential for increasing the risk of major congenital malformations, such as neural tube defects, cleft palate, and neurodevelopmental disability. Although valproate is a pharmacological option for acute mania and is used as a stabilization drug for patients with bipolar disorder, some global guidelines state that valproate should not be used for girls or women of childbearing age with bipolar disorder. We investigated patterns in psychiatrists' prescription of valproate for bipolar female patients of childbearing age in Japan.From March to May 2018, we conducted a questionnaire survey among psychiatrists from all prefectures in Japan on psychiatric practice as it relates to major depression and bipolar disorder throughout women's life. The questionnaire had two parts: (1) assessment of participating psychiatrists' backgrounds and attitudes toward patients and (2) their patterns of prescription of psychotropics for female patients with mood disorders across generations and periods of pregnancy. Each question item had four response options: “not at all,” “rarely,” “sometimes,” and “frequently.” We examined patterns of prescription for childbearing-aged women  and pregnant women.n = 23 (7.5%); rarely, n = 69 (22.5%); sometimes, n =116 (37.8%); and frequently, n = 99 (32.2%); childbearing-age, older adults: not at all, n = 13 (2.7%); rarely, n = 67 (13.8%); sometimes, n = 185 (38.1%); and frequently, n = 220 (45.4%)]. The proportion of general hospital psychiatrists who answered “not at all” or “rarely” to the frequency of their valproate prescriptions was higher than that of psychiatrists working in other medical facilities (χ2(3) = 18.2, p < 0.001).In total, 571 psychiatrists  responded appropriately to the questionnaire, including 320 who examined at least one or more late adolescence/young adulthood bipolar women. Approximately 70% of psychiatrists answered that they frequently or sometimes prescribed valproate for bipolar women of childbearing age [late adolescence/young adulthood: not at all, Most psychiatrists frequently or sometimes prescribe valproate for women of childbearing age in Japan. Bipolar disorder frequently emerges in the late teens and young adults –3 and itPharmacological treatment plays a crucial role in the continuous management of patients with bipolar disorder. According to several worldwide guidelines –14, pharAlthough valproate is one of the pharmacological options for the treatment of acute mania and the stabilization in patients with bipolar disorder, accumulating evidence has shown that valproate, among other pharmacological treatments for bipolar disorder, has the greatest teratogenic potential as it increases the risk of major congenital malformations, such as neural tube defects, including spina bifida {odds ratio [OR] = 12.7 }, and cleft palate [OR = 5.2 ] , 17, andOur recent study that investigated the top 11 most frequently prescribed anticonvulsants and lithium using the National Insurance claims database in Japan from April 2014 to March 2015, showed that valproate was the most prescribed major anticonvulsant for female outpatients of childbearing age, and was prescribed slightly lesser compared to same-aged men. However, this data preceded the publication of the above-mentioned guidelines . The resThe aim of this study was to identify psychiatrists' patterns of prescribing valproate for bipolar female patients of childbearing age (in their late adolescent years and late forties), compared to those who are pregnant, in Japan. We also investigated the relationship between characteristics and reproduction-related interviews in the consultation of psychiatrists and their frequency of valproate prescription. To assess this, this study was performed through a questionnaire survey about psychiatric practices in major depression and bipolar disorders throughout the stages of a woman's life; this was conducted among psychiatrists in all prefectures in Japan from March to May 2018. This study also examined the frequency of prescription of other mood stabilizers , antipsA cross-sectional study was conducted among psychiatrists who belong to the following associations: Association of Japan Psychiatric Clinics: Tokyo branch and Chiba branch, Association of Japan Psychiatric Hospitals: Ibaraki branch, Chiba branch, and Tokyo branch, JSGHP; Japanese Society of General Hospital Psychiatry in 2018.A total of 1414 medical institutions  were chosen from the list of cooperation associations and 4,816 questionnaires were mailed through the postal service between March and May 2018. There were 571 respondents . Psychiatrists who did not see patients of childbearing age (18 to 49 years) were excluded. Of 571 respondents, 320 (56.0%) and 497 (87.0%) psychiatrists met the eligible criteria for evaluating the prescription pattern among female bipolar patients in late adolescence/young adulthood (ages 18–24 years) and in early or middle age, respectively. Of 571 respondents, 571 (100.0%) psychiatrists met the eligible criteria for evaluating the prescription pattern of female bipolar patients who were pregnant.At the beginning of the questionnaire, we declared the objectives of the study and our commitment to confidentiality. Therefore, completing and mailing back the questionnaire were considered to reflect written informed consent regarding participation in this study. The questionnaire survey of this study was performed anonymously. The study's protocol was approved by the ethics committees of the Graduate School of Medicine and School of Medicine, Chiba University (20 November 2017).The questionnaire was used to assess participating psychiatrists' backgrounds, attitudes toward patients, and prescribing behavior. Regarding psychiatrists' backgrounds, participants were asked about their demographic data, including sex, age, years of clinical experience, hospital facility, and the number of patients with depression and bipolar disorder they treat per month. Regarding attitudes toward patients, the psychiatrists rated how often they focused on problems specific to life stages when counseling their patients. The question was followed by four response options: “not at all,” “rarely,” “sometimes,” and “frequently.” In this study, we focused on measurements of psychiatrists' attitudes toward patients' fertility by asking how often psychiatrists inquire about the following: any menstrual disorder, whether patients wanted to bear children, methods of contraception, and feelings of rejection for medication during pregnancy and breast-feeding periods. In their prescribing behavior, psychiatrists were requested to indicate how often they prescribed 16 types of medicine, depending on the patients' life stages. The life cycle was divided into 7 stages depending on the age: ages 0–11 corresponded to childhood, ages 12–17 to pubescence, ages 18–24 to late adolescence/young adulthood, ages 25–49 to early middle age, ages 50–59 to late middle age, ages 65 and over to late adulthood, and the pregnancy period. The number of the questions was 326 in total, consisting of 35 questions on the psychiatrists' backgrounds including sociodemographic data, 67 on their attitudes toward patients, and 224 on prescribing behavior .The primary outcome was to identify the psychiatrists' patterns of prescribing valproate to female bipolar patients of childbearing age, compared to those who are pregnant, and the relationship between the psychiatrists' characteristics and reproduction-related interviews and patterns of valproate prescription. The secondary outcomes were to clarify the frequency of the prescription of other mood stabilizers , antipsychotics, and antidepressants in bipolar women of childbearing-age and in pregnant women.χ2) tests for categorized variables to analyze categorical data between the current and previous affiliations of psychiatrists  and the frequency of prescription of valproate to female bipolar patients in late adolescence/young adulthood. Categorical data were compared using the χ2 tests followed by a residual analysis for multiple comparison .We first performed chi-squared ; rarely, n = 69 (22.5%); sometimes, n =116 (37.8%); and frequently, n = 99 (32.2%). Regarding the prescription of valproate to childbearing-aged, older female patients with bipolar disorder the answers were the following: 13 psychiatrists responded not at all, n = 13 (2.7%); rarely, n = 67 (13.8%); sometimes, n = 185 (38.1%); and frequently, n = 220 (45.4%). On avoiding prescription of valproate to pregnant patients: 387 psychiatrists responded “not at all” (75.3%) while 126 responded “rarely”, “sometimes”, and “frequently” (24.7%).In the questionnaire, the psychiatrists' answers regarding the prescription of valproate to late adolescence/young adulthood female patients with bipolar disorder were the following: 23 psychiatrists responded not at all, n = 15 (5.8%); rarely, n = 54 (17.5%); sometimes, n =125 (40.6%); and frequently, n = 111 (36.2%). Regarding the prescription of lithium for childbearing-aged, older female patients; 7 psychiatrists responded not at all, n = 7 (1.4%); rarely, n = 42 (8.7%); sometimes, n = 181 (37.3%); and frequently, n = 255 (52.6%). Regarding lithium prescription to pregnant patients: 382 psychiatrists responded “not at all” (74.0%), while 134 responded “rarely,” “sometimes,” and “frequently” (26.0%).Regarding the prescription of lithium to late adolescence/young adulthood patients with bipolar disorder, 15 psychiatrists responded not at all, In addition, regarding the prescription patterns of other medications, including other mood stabilizers  and antipsychotics for pregnant women, more than half (55.6%) the psychiatrists responded, “not at all” or “rarely” Table 2.χ2-test showed that there were significant differences in the current affiliation of psychiatrists and valproate prescriptions to female bipolar patients in late adolescence/young adulthood, as shown in χ2(3) = 18.2, p < 0.001]. The Haberman-type residual analysis showed that psychiatrists working for general hospitals reported that they prescribed valproate less frequently  than that of psychiatrists working for other medical facilities and hospitals. Additionally, the psychiatrists working for general hospitals answered “frequently,” regarding valproate prescription, were associated with significantly negative standardized residuals, which was less than psychiatrists working for other medical facilities or hospitals. However, in their previous affiliations, there were no differences between general hospitals and other medical facilities in terms of valproate prescriptions.The r = 0.13, p < 0.05). Conversely, in childbearing-aged, older adults (25–49 years) and pregnant women, there is no relationship between the two variables. There is no relationship between the contents of reproduction-related medical interviews and valproate prescriptions in childbearing age or pregnancy. In addition, there were no relationships between reproduction-related medical interviews and prescriptions of other psychotropics in childbearing age or pregnancy.We calculated the partial correlations, controlling for sex, between years of clinical experience, valproate prescription, and psychiatrists' attitude toward bipolar patients of childbearing age and in pregnancy Table 4.In this study, three important findings about psychiatrists' prescription patterns of valproate for female bipolar patients of childbearing age in Japan deserve to be mentioned. First, this study demonstrates that 70% of psychiatrists responding to the questionnaire sheet answered that they frequently or occasionally prescribed valproate for women of childbearing age in Japan, although most answered that they did not prescribe for women who were pregnant. Specifically, 32% of psychiatrists answered that they frequently prescribed valproate for women aged 18 to 24 years, and 38% occasionally prescribed it for same-aged women. Thirty percent of psychiatrists answered they rarely prescribed valproate, or that they did not at all prescribe it. Second, psychiatrists affiliated with general hospitals answered that they tended to refrain from prescribing valproate for childbearing-aged women compared to those affiliated with other medical facilities such as psychiatric hospitals or private clinics. Third, the frequencies of reproduction-related medical interviews were not correlated with the tendency to prescribe valproate to women of childbearing age, although years of psychiatric experience was positively correlated with this.http://pmhguideline.com/, in Japanese only); stating that valproate should not be prescribed to women of childbearing age. However, the society is small due to the small number of psychiatrists. Additionally, there were only 20 regular members at that time, and midwives constituted a large proportion of the regular members of the society (over 100 people). In other countries, such as Finland  those who refrained from prescribing valproate to childbearing-aged women, and (2) the other ones who prescribed it with caution. It is difficult to interpret the positive correlation between years of psychiatric experience and the tendency to prescribe valproate to childbearing-age women. This result is limited because of the lack of surveillance about real-world childbearing-aged women prescription of valproate, and the lack of assessment of each psychiatrist's valproate prescription dosage. Therefore, senior psychiatrists with enough clinical experience may have the opportunity to examine childbearing-aged women with more refractory bipolar disorder than junior ones. Further studies are needed to clarify this.Regarding other psychotropics, especially lithium and carbamazepine prescriptions, the NICE 192 guideline cautions against use on girls and childbearing-aged women ; howeverAdditionally, the present study shows that most psychiatrists are rather reluctant to prescribe medications including lamotrigine and some atypical antipsychotics with efficacy and/or relative safety in the treatment and prevention of mood episodes in pregnant women with bipolar disorder in Japan. Among major mental disorders, bipolar disorder is the highest risk group with regard to relapse and readmission during the postpartum period . WesseloWe acknowledge that there are several limitations to this study. First, we were unable to measure the real-world valproate prescriptions (including the dosage) of the responding psychiatrists. Given that some female patients with bipolar disorder who consulted their responding psychiatrist were expected to have various comorbidities, intellectual disabilities, and complications , valproate prescription may be adequate for them if prescribed with caution. Second, the response rate of 11.8% was low, although 571 psychiatrists responded to this questionnaire in Japan. The biggest possible reason for the low response rate in this survey was the large amount of questions in the questionnaire  since it was developed to investigate overall psychiatric practices in major depression and bipolar disorder throughout the stages of the women's lives. Hence, it was likely that lots of questions caused difficulties for almost 90% candidates to complete the questionnaire sheet. Third, the items about reproduction-related medical interviews were relatively scarce in the questionnaire; hence, this may have influenced the correlations between the answers and frequencies of valproate prescriptions.This study demonstrates that most psychiatrists frequently or occasionally prescribed valproate for women of childbearing age in Japan, although almost all answered they did not prescribe valproate to pregnant women. Specifically, 70% of psychiatrists answered that they frequently or sometimes prescribed valproate for women aged 18 to 24 years. Considering that fetal exposure to valproate in the first trimester must be avoided, and that many pregnancies are unplanned or unintended, psychiatrists should not prescribe valproate to girls and women of childbearing age with bipolar disorder.t.hashimoto1109@gmail.com.The datasets analyzed in this article will be made available by the authors, and to any qualified researcher without undue reservation. Requests to access the datasets should be directed to TH, The studies involving human participants were reviewed and approved by the ethics committees of the Graduate School of Medicine and School of Medicine, Chiba University. The patients/participants provided their written informed consent to participate in this study.TH, MamiT, YaS, TakaT, TakeT, HW, MN, and MI contributed substantially to the conception and design of this study. MasuT, TH, and MamiT contributed to organizing the data, formal analysis, writing an original draft. AK, TakeT, KH, and MI contributed to the management of this study as supervisors. SK, AK, SE, YuS, TN, JH, and KH contributed substantially to facilitating data curation and study procedure as investigators. All authors contributed to the interpretation of data, revision of the manuscript, and approved of the submitted manuscript.This work was supported by the Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research C Grant Number 17K10265. The members of this grant were TH, HW, MN, MI, YS, TakaT, and TakeT.TH reported personal fees from research support of a clinical trial that the signant health  company manages. TakaT has received personal fees from Otsuka, GlaxoSmithKline, MSD, Dainippon-Sumitomo, Janssen, Eisai, Eli Lilly, Daiichi-Sankyo, Shionogi, and Tanabe-Mitsubishi. KH has received lecture honoraria for Dainippon-Sumitomo, Janssen, Meiji Seika, MSD, Otsuka, Takeda, and Tanabe-Mitsubishi, and has served as a consultant for Dainippon-Sumitomo, MSD, and Meiji Seika. HW has received speaker's honoraria for MSD and Eli Lilly. TakeT has received honoraria from Otsuka Pharmaceutical Co., Ltd. and Mochida Pharmaceutical Co., Ltd as potentially influencing but not directly related to this work. MI received consultant fees from Janssen, Elilly, Otsuka, and Meiji Seika Pharma and reports honoraria from Janssen, Eli Lilly, Otsuka, Meiji Seika Pharma, Astellas, Dainippon Sumitomo, Ono, Mochida, MSD, Eisai, Daiichi-Sankyo, Novartis, Teijin, Shionogi, Hisamitsu, and Asahi Kasei.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.


Mangifera indica L.), one of the five most important fruit crops worldwide in terms of production. In this study, we characterize pollen–pistil interaction in mango, paying special attention to three key players known to be involved in the directional pollen tube growth of porogamous species such as starch, arabinogalactan proteins (AGPs), and γ-aminobutyric acid (GABA). Starch grains were observed in the style and in the ponticulus at anthesis, but their number decreased 1 day after anthesis. AGPs, revealed by JIM8 and JIM13 antibodies, were homogenously observed in the style and ovary, but were more conspicuous in the nucellus around the egg apparatus. GABA, revealed by anti-GABA antibodies, was specifically observed in the transmitting tissue, including the ponticulus. Moreover, GABA was shown to stimulate in vitro mango pollen tube elongation. The results support the heterotrophic growth of mango pollen tubes in the style at the expense of starch, similarly to the observations in porogamous species. However, unlike porogamous species, the micropyle of mango does not show high levels of GABA and starch, although they were observed in the ponticulus and could play a role in supporting the unusual pollen tube growth in chalazogamous species.Most flowering plants show porogamy in which the pollen tubes reach the egg apparatus through the micropyle. However, several species show chalazogamy, an unusual pollen tube growth, in which the pollen tubes reach the embryo sac through the chalaza. While ovary signals for pollen tube growth and guidance have been extensively studied in porogamous species, few studies have addressed the process in chalazogamous species such as mango ( In flowering plants, after pollen germination, the pollen tubes face a long journey before the two male sperms meet the female gametophyte or embryo sac, which at maturity usually contains seven cells , embedded in the sporophytic tissues of the ovule. Pollen germination and pollen tube growth take place within the carpel. Pollen grains first land on the stigma where they germinate and pollen tube growth takes place along the style, until reaching the ovarian cavity, the locule, at the base of the style. At this point, most flowering plants show porogamy in which pollen tubes reach the egg apparatus through the micropyle in order to achieve the double fertilization process typical of angiosperms . The mic2+ gradients  have been observed along the pollen tube pathway in a number of unrelated porogamous species. AGPs are extensively glycosylated hydroxyproline (Hyp)–rich proteins  that hav2+-permeable channels of the pollen tube cell walls  has also been reported in the ovule, specifically in the tip of the inner integument that forms the micropyle . Its prell walls . Severaletophyte .Casuarina by Treub in 1891 [cited in Juglans regia  (Carya (Ticodendron (Casuarina (Alnus (Gymnostoma (Pistacia (Mangifera indica (mango) (Pistacia vera), a ponticulus develops bridging the growth of the pollen tube from the style to the ovule  , Carya   . In the  (mango) . In the he ovule . HoweverMango is one of the most important fruit crops, ranking fifth worldwide in production after bananas, apples, grapes, and citrus . Mango iM. indica L.) of the cultivars “Kent” and “Keitt” located in a mango collection at the IHSM-La Mayora-CSIC-UMA , at latitude 36°45′N, longitude 4°4′W, and altitude 35 m above sea level. Hermaphrodite and male flowers were collected in the morning, from 08:30 to 10:30 h, before anther dehiscence. Then, these flowers were placed on wet white paper inside a Petri dish in the laboratory, where anther dehiscence occurred between 11:00 and 15:00 h. These flowers were used in the pollination experiments in which one hermaphrodite flower, which had been previously emasculated by removing the stamens, was pollinated by touching the stigma with the open anther of a single male or hermaphrodite flower  at pH 7.3, left overnight at 4°C, dehydrated in an acetone series, embedded in Technovit 8100 (Kulzer), polymerized at 4°C, and sectioned at 2 μm. Sections were placed in a drop of water on a slide covered with 2% 3-aminopropyltrietoxysilane and dried at room temperature . Differein vitro protocol previously developed for mango that used a medium with 23 PEG 8000, 10% sucrose, 0.2 g/L MgSO47H2O, 0.1 g/L KNO3, 0.1 g/L H3BO3, and 0.7 g/L Ca(NO3)24H2O 24H2O . To thisO3)24H2O , which it test, P < 0.05. RFUs were collected from at least three biological independent replicates. The statistical analysis of pollen germination and pollen tube elongation was performed with an analysis of variance, and Duncan multiple-range test was used to separate means (P ≤ 0.05). The percentages of pollen germination were arcsine–square root transformed for the Duncan multiple-range test. Statistical analyses were performed with the SPSS 15.0 statistical software.The immunofluorescence signal was measured as relative fluorescence units (RFUs) using the Fiji program , and sigTo define the unusual pathway of the pollen tubes in mango, we first evaluated the pistil anatomy of the hermaphrodite flower, with special attention to the transmitting tissue, and pollen tube growth. The mango pistil consists of a single carpel, showing a recognizable suture line that extends from the horseshoe-shaped stigma to the base of the style . A transAfter pollination, a fast hydration and germination of the pollen grains on the stigma were observed. Pollen tubes were observed in the upper part of the style 4 h after pollination, reaching the ovule 24 h after pollination. We observed pollen tubes growing along the stylar transmitting tissue close to the vascular bundles . At the Fertilization requires synchronization of the male gametophyte with the receptivity stage of ovule development. As chalazogamy has been related to a not fully developed ovule , we firsn = 15, n = 27) and 100% (n = 13), respectively, of the analyzed ovules showed almost no starch grains (n = 17) from flowers at anthesis (n = 19) of the analyzed ovules from pollinated flowers 1 day after anthesis . AGPs that reacted to JIM8 and JIM13 were specifically detected in the distal part of the nucellus, next to the micropyle, and in direct contact with the embryo sac, in the ovules from flowers in anthesis . The analysis of variance of RFUs between the transmitting tissue and the surrounding cells in the style was slightly higher (P = 0.09) of the limit of significance in anthesis  but was significantly different 1 day after anthesis in unpollinated flowers . However, high levels of anti-GABA signal were not observed in the single integument of the ovule , in which the ponticulus has been shown as a funicular protuberance in close proximity to the stylar base  (Actinidia deliciosa)   , kiwifruliciosa) , or applmestica) . Starch in vitro pollen tube elongation in mango, as has been reported in others species (via modulating Ca2+-permeable membrane channels was reported in Nicotiana (A. cherimola (Arabidopsis (Interestingly, similar to the observation in the stylar transmitting tissue, we also observed GABA detected by anti-GABA in the ponticulus. Moreover, GABA stimulates  species . Thus, oicotiana . Howeverherimola  and Arabbidopsis , it has A. cherimola (Malus domestica (Amaranthus hypochondriacus (Arabidopsis, where the mature embryo sac shows a single nucellus cell layer between the megasporophyte and the epidermal cells, AGPs detected by JIM8 and JIM13 were observed in the tip of the inner integument and embryo sac (Besides the signal from the micropyle in porogamous species, the signals from the embryo sac are key for pollen tube growth and guidance . To our herimola , Malus domestica , and Amandriacus . In the bryo sac . SimilarIn summary, in contrast to the observation in porogamous species, immunolocalization did not reveal AGPs and GABA in the micropyle of the mango ovule, . AGPs, dThe original contributions presented in the study are included in the article/JL, VP, MH, and JH conceived the study. JL, VP, MH, and JH designed the experiments and wrote the manuscript. JL and VP performed the experiments. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Despite the availability of BCG vaccination data and the mathematical models needed to compare these data with COVID-19 cases, our results show that, in geographic areas where BCG coverage was low, 50% presented a high prevalence of COVID-19 cases that were young; thus, low-coverage years were more affected.The Bacillus Calmette–Guérin (BCG) is a well-known vaccine with almost a century of use, with the apparent capability to improve cytokine production and epigenetics changes that could develop a better response to pathogens. It has been postulated that BCG protection against SARS-CoV-2 has a potential role in the pandemic, through the presence of homologous amino acid sequences. To identify a possible link between BCG vaccination coverage and COVID-19 cases, we used official epidemic data and Ecuadorian Ministry of Health and Pan American Health Organization vaccination information. BCG information before 1979 was available only at a national level. Therefore, projections based on the last 20 years were performed, to compare by specific geographic units. We used a Mann–Kendall test to identify BCG coverage variations, and mapping was conducted with a free geographic information system (QGIS). Nine provinces where BCG vaccine coverage was lower than 74.25% show a significant statistical association (χ SARS-CoV-2 was accidentally discovered in China towards the end of 2019, when a cluster of unidentified viral pneumonia cases occurred in Hubei province. During the initial outbreak, the virus was discovered to cause an acute respiratory syndrome, named coronavirus disease 2019 (COVID-19), mostly affecting the elderly  and individuals with underlying health conditions . At the Mycobacterium bovis, is widely used to eradicate tuberculosis (TB), and was among the most broadly used vaccinations in the neonatal and young in the twentieth century [Along with the spread of the pandemic, most countries imposed a policy of social distancing and other regulations to mitigate COVID-19 . However century . Further century , in the  century . Other mA recent study suggested that enduring innate immune memory is conferred by myeloid cells  in response to microbial molecules, metabolic products, or cytokines. Macrophages, which increase effector function (“activation”), are primed for short-term responses (“priming”) or become unresponsive (“tolerance”) . MicrobiAdditionally, bioinformatic analysis of BCG antigens showed that bacterial proteins Rv0934, Rv3763, Rv3875, and Rv2997 share common informational properties with the spike protein of SARS-CoV-2, suggesting immunological cross-reactivity which could induce a specific adaptive immune response against SARS-CoV-2. If experimentally confirmed, this theoretical finding could serve as a basis for the design of a BCG vaccine with improved protection against COVID-19 [Moreover, the BCG vaccine has shown a complete response in urothelial bladder cancer in up to 70% of cases. The recommendation of the European Association of Urology and the American Urological Association is to apply the BCG vaccine to patients with intermediate-to-high risk after transurethral resection . Its effCurrently, the BCG vaccine is provided to the entire population, in most countries with a high TB incidence . At presIn Ecuador, BCG vaccination is still mandatory and free of charge for all newborns , but a bEcuador is located in Northwest South America and straddles both the line of the Equator and the Andes Mountains that cross the country from the north to the south and divide it into three continental regions , and there is also one insular region . Geographically, Ecuador comprises 221 cantons that are grouped into 24 provinces and divided into 4 regions, i.e., Coast (7 provinces), Highland (10 provinces), Amazon (6 provinces), and Galapagos Archipelago.https://public.tableau.com/profile/direccion.nacional.de.vigilancia.epidemiologica.msp#!/vizhome/COVID19ecu_MSP_DNVE/COVID-19MSP. These data can be disaggregated to the canton level and are updated daily. Data were mapped by using political division by province.Data on the COVID-19 situation in Ecuador are available online at t-test were performed by using SPSS software version 24, where appropriate.Information on BCG vaccination coverage was obtained primarily from the Ecuadorian Ministry of Health and the Pan American Health Organization. Unfortunately, detailed records at the cantonal level are available only from 2000. Data from between 1990 and 2000 are available only at the province level; until 1979, there were data only at the country level. The BCG vaccination rates 40 years ago (1980), at the province level, were estimated by using a retrospective projection based on the last 20 years with the information available. In addition to the free geographic information system (QGIS) map, a monotonic trend based on the Mann–Kendall statistic was calculated for the BCG coverage, in order to identify changes over time. The Mann–Kendall non-parametric statistical test is used to determine the time trend throughout the annualized vaccination coverage. The test does not require that the data fit any particular distribution. For each spatial unit, the statistic makes combinations of each pair of observed values over time and counts the number of pairs that increase or decrease over time and calculates the relative frequency of increases minus the relative frequency of decreases. Mann–Kendall values range from −1 to +1. When a value approaches +1, it means that there is a monotonic upward trend; when it approaches −1, the trend is downward; and a value of 0 indicates no trend. Statistical analysis: Descriptive analysis, Pearson’s chi-square test, and Student’s n = 5130, 6%), 20 to 49 years , 50 to 64 years , and 65 years and older , there were more cases per 100,000 inhabitants in people aged 50–64 . When prThe BCG vaccination coverage in Ecuador that was reported in 1979 was 28%, and it increased in 1980 to 76%. Between 1981 and 1991, BCG coverage ranged from 82% to 99%. After that period, BCG vaccination coverage was stable at around 99% until 2012. Since then, over the last eight years, nationwide coverage has dropped to around 90%.The analysis from 221 cantons where data were available showed that, in the last seven years, there were 67 cantons (30.6%) with BCG vaccine coverage lower than 50% on average, where nearly one million people live. BCG vaccination coverage between 50% and 89% was found in 90 cantons (41.1%), which have around seven million inhabitants, and only 62 cantons (28.3%) had BCG vaccination coverage over 90%, which included around nine million people, and almost half of these cantons corresponded to urban areas. Additionally, we identified 11 cantons where the BCG coverage was invariably under 50% during the last seven years, with a total of 111,263 inhabitants. The Mann–Kendall analysis showed that BCG vaccine coverage has decreased in 42 cantons during the last seven years, and it has increased in just 14 cantons .Although, as mentioned above, Ecuador currently has 24 provinces, in 1980, there were four fewer provinces: two on the Coast and two in Amazonian regions. In our retrospective projection for the BCG vaccine in 1980, 9 out of 20 provinces had less than 80% coverage. Of those, four were in the Highlands, three on the Coast, one in the Amazon, and one in the Galapagos Archipelago .p= 0.31) was double that of younger people aged 20 to 49 years old , whereas, in younger people aged 20 to 49 years old, it was r = −0.196 . However, we did not find a statistically significant association with other age groups or when using the incidence rate. Furthermore, 15 cantons with a low BCG coverage (<50%) presented a high COVID-19 prevalence, over 50 cases per 10,000 inhabitants, with a total population of 6.5 million inhabitants.Comparing provinces with the age distribution of cases  with BCG retrospective projection coverage below 74.25% shows a significant statistical association  , as wellum bovis . A recenRS-CoV 2 . This rePrevious clinical trials found that BCG revaccination in adults is safe, well-tolerated, and able to boost the immune response of Th1, Th17, and memory NKT-like and NK cells ,29. HoweA recent paper found that the type of BCG policy  had a significant effect on COVID-19 deaths per million, with a stronger policy being associated with lower COVD-19 mortality . AdditioOur findings are consistent with an ecological study in 173 countries, including Ecuador, analyzing BCG vaccine coverage (%), using morbidity and mortality as outcomes, which we obtained on an open source basis . That stOn the other hand, another recent study reviewed evidence for a potential biological basis of BCG cross-protection from severe COVID-19, and refined the epidemiological analysis to mitigate effects of potentially confounding factors , demonstrating that every 10% increase in the BCG index was associated with a 10% reduction in COVID-19 mortality . Moreoveclinicaltrials.gov). It is important to note that the BCG vaccine leads to lymphadenitis and disseminates disease as a relatively frequent side effect in up to 3% of the population, so whether a large-scale vaccination strategy should be planned in order to achieve a greater advantage  should be considered [These data could suggest a protective effect of BCG vaccination, but such studies cannot provide definitive proof of causality, owing to several inherent biases, including the quality of raw data COVID-19 reports , differensidered .If the effectiveness of the BCG vaccine against COVID-19 is proven, we are extremely concerned about the impact that this will have in the event of a vaccine shortage, which is what happened at the beginning of the pandemic with other drugs, for instance, hydroxychloroquine for patients with rheumatoid diseases . CurrentUnfortunately, the main limitation of this study was the fact that detailed data on BCG vaccination coverage in Ecuador were only available from 1990 and, for the previous decade, the only information accessible was the national consolidates requested by the Pan American Health Organization . As our Our analysis found that the 15 cantons that currently have a low BCG coverage (<50%) presented a high COVID-19 prevalence, over 50 cases per 10,000 inhabitants, with a total population of 6.5 million inhabitants. However, from our retrospective analysis, it was not possible to demonstrate a relationship between childhood BCG vaccination and actual SARS-CoV-2 prevalence. We were also unable to establish a statistically significant association between cantons where BCG vaccination reductions happened in recent years. Apart from the impact in the form of a reduction in COVID-19 deaths based on BCG vaccination rates from 60 years ago, we believe that our findings in relation to the BCG vaccination coverage reductions in the majority of cantons provide an important index for the Ecuadorian social phenomena during the last decade, such as the important external immigration process and reductions in the size of the health budget ,44. Our Although, before the COVID-19 pandemic, Ecuador allocated 2.78% of its gross domestic product  to health coverage , it was