Long distance transportation is a welfare concern because it may cause sickness  or death . Commercial transportation in Europe is regulated by the Council Regulation (EC) No. 1/2005 which regulates the maximum journey in the different species. After this time animals must be unloaded for resting, watering and feeding at control posts (CPs) where Official Veterinarians (OVs) have to check their health. This study analyzed the surveillance reports filled by OVs at a CP in Southern Italy from 2010 to 2015. A total of 1391 trucks stopped at the CP, transporting a total of 111,536 animals. The average mortality and morbidity rates were 0.025% and 0.010%. Cases of mortality and/or morbidity were reported for only 11 out of the 1391 trucks (0.8%). In a truck transporting lambs, 14 dead on arrival (DOA) were recorded, and this represented 93% of all DOAs. This is the first study reporting the results of surveillance practices conducted by OVs on animals travelling from North Europe to a CP in Southern Italy in compliance with EC 1/2005. Further studies should be conducted comparing the implications of long distance transportation at different CPs along different routes.p < 0.05). Overall, this study provided statistics based on official surveillance reports, suggesting that small space allowance during long haul transportation of sheep/goats may affect their health and welfare.Statistics on animal transport and its implications for health and welfare are limited. This study documented the animals transiting through a control post and their welfare outcomes measured by mortality rate and the prevalence of animals considered unfit for further transport . Reports filed by the director of the control post and Official Veterinarians from 2010 to 2015 were analyzed. A total of 60,454 (54.2%) sheep/goats, 45,749 (41.0%) cattle, and 5333 (4.8%) pigs travelled in 225 (16.2%), 1116 (80.2%) and 50 (3.6%) trucks, respectively. Trucks coming mainly from France (71.3%), Spain (14.0%), and Ireland (7.4%) went mainly to Greece (95.4%), which was also the most common nationality of the transport companies (44.6%). Cases of mortality and/or morbidity were reported for only 11 out of the 1391 trucks (0.8%). The average mortality and morbidity rates were 0.025% and 0.010%, with maximum values for transport of lambs . Species of animal being transported and space allowance were associated with the measured welfare outcomes ( Millions of animals are transported daily all over the world. The movement of livestock across the borders of Member States of the European Union is monitored using the Trade Control and Expert System (TRACES) and reported in the Activity Report . For exaOne of the determining risk factors is journey duration ,14. ConsSurveys on farm animal transport have been performed to explore the epidemiological basis of transport-related health and welfare issues worldwide. For instance, the mortality due to road transport has been calculated for beef cattle in North America (0.01%) , fatteniWith the permission of the OVs and the director of the control post, surveillance reports from 2010 to 2015 filed by the director of the control post (CP) IT CE 07/PS Bitritto  in compliance with Council Regulation (EC) No. 1255/1997 (Article 5(h)), and double-checked by OVs from the Local Health Authority  were used for this manuscript.The CP  measured approximately 8000 square metres during the data collection period and could contain up to 1200 bovines, 1850 sheep/goats, 500 pigs and 150 horses. It had a lorry wash, three hay barns, five animal houses and many different pens, equipped with watering and feeding points, bedding and resting facilities. The animal houses had adequate natural and artificial light to ensure proper inspection of the animals by the OVs.At the control post, three OVs working for the Local Health Authority , including one of the authors (DT) checked the animals during loading, and immediately before they left the CP, to assess their fitness for transport following the criteria set out in Annex 1 of Council Regulation (EC) No. 1/2005. Depending on the day of arrival, the animals were checked by one of the three OVs. However, those three OVs had each received official EU training to assess fitness for transport and used the same check list to compile the surveillance reports. In compliance with the (EC) No. 1/2005, the OVs judged animals as unfit to continue the journey when they were either seriously injured or presented clinical signs of a pathological process, including severe lameness , prolapse, severe open wound, and respiratory and gastro-enteric disease . Such animals were either humanely euthanized, slaughtered in the local slaughterhouse or treated depending on the severity of the case after the assessment performed by the OVs.The following parameters were recorded in the surveillance reports: date and time of arrival, species, number of animals transported per truck (NATT), country of provenance, TRACES code, truck registration number, transport company, number of dead on arrival (DOA), of those which died at the control post (DCP), of animals judged unfit to continue travel (UFT), day and time of departure, and country of final destination. Since these reports were official documents, there were no missing data.Based on the surveillance reports, the dates were expressed as month and season. Using the category which was reported in each TRACES, the category of transported animals was added to the dataset. The categories were chosen in compliance with Council Regulation (EC) No. 1/2005, for bovines and sheep/goats the following categories were used: small calves (100 kg), medium-sized calves (200 kg), heavy calves (325 kg), heavy cattle (550 kg), very heavy cattle (>700 kg), lambs, and sheep/goats; whereas for pigs, the following categories were applied: light fattening (110–120 kg), heavy fattening (130–150 kg), and breeding (>200 kg).2/animal. It was not possible to calculate the space allowance in kg/m2 because the surveillance reports did not report the total weight of the truck load. The space allowance per animal was also added to the dataset. It was calculated by dividing the available floor space in each truck by the number of animals transported in the truck and expressed as mThe transport companies were aggregated by country of origin  and the total number of dead animals was calculated as the sum of DOA and DCP.®, Sydney, Australia) with year, season, month, species, category, country of provenance, nationality of transport company and country of destination as categorical variables, while NATT, DOA, DCP, total dead, UFT and space allowance were used as numerical variables.Descriptive statistics of the data collected were obtained using an online statistical software .Chi-square tests were conducted to determine the association between factors . Trucks with DOA, DCP, and/or UFT were considered as having a welfare problem. A univariate logistic regression model was developed with welfare problem as a binary outcome  and year, season, month, species, category, provenance, destination, nationality, and space allowance as predictive variables. ificance . The afoP threshold was set at 0.05. Data are expressed as least square means ± standard error (SE).The effect of year, season, month, provenance, destination, transport company nationality, species and category on NATT was determined using a General Linear Model (GLM) procedure. Tukey’s HSD (honestly significant difference) test was used as a post-hoc test. Statistical analyses were performed using SAS version 9.4. A total of 1391 trucks, mainly trailers and semi-trailers, stopped at the control post over the study period, transporting a total of 111,536 animals. The frequency and the percentage of all the trucks transiting across the control post based on category, provenance, nationality of transport company and destination are shown in The majority of the trucks transported bovines, while heavy cattle  was the category most frequently transported. The most common provenances were France, Spain, and Ireland, even though the transport companies were mainly from Greece, which was also the most common final destination. There was a decrease in the number of trucks and transported animals after 2012, in particular for bovines and sheep. The transport of bovines tended to drop in August, while the transport of sheep/goats peaked in July and October .The maximum number of DOAs (14) was recorded in a truck transporting a total of 214 lambs from Spain, and this represented 93% of all DOAs. 2/animal for bovines, from 0.20 to 0.50 m2/animal for sheep and at least 235 kg/m2 for pigs of 100 kg. In our study, the minimal values of space allowance resulted in lamb transportation .Space allowance varied accordingly with the species, and was within the range reported in CE 1/2005, which regulates a space allowance from 0.30 to 1.60 mThe mortality and morbidity rates were 0.025% and 0.010%, respectively, with maximum values for transport of lambs . Cases of mortality and/or morbidity were reported for only 11 out of the 1391 trucks (0.8%). 2 = 40.25; p < 0.001), season , month , provenance , nationality of transport company  and destination  (There was an association between species transported and year (X< 0.001) .2 = 841.29; p < 0.001), season , month , provenance , nationality of transport company  and destination . There was a higher number of trucks transporting sheep/goats in June, July and August, i.e., in summer, than in other months and seasons of the year. Sheep/goats came mainly from Spain and Hungary.Consequently, there was also an association between category transported and year  (p < 0.05).In the univariate logistic model, species of animal being transported and space allowance were the only predictive variable which proved to be associated with a welfare problem (Xctively) . Trucks ctively) . In the p = 0.072) on NATT. While the number of transported animals per truck varied significantly depending on species , the effect of the other predictive variables associated with species resulted in significant differences on NATT  on a stopover at a control post in Southern Italy using reports of OVs. This study reports on the number of animals which died and were judged unfit to continue travel by OVs in compliance with Section 5 of Annex 1 of Council Regulation (EC) No. 1/2005, thus providing the first report containing statistics on farm animal movements from Northern Europe to the Balkan Peninsula. Considering that only 0.8% of the shipments was associated with one of the considered welfare issue , and that the Eurobarometer  reports The animals on the stopover at our CP were mainly travelling to Greece, a country which relies heavily on meat and dairy imports . AnimalsIn our study, the majority of the trucks came from France, transporting mainly bovines. This was expected, since France exports millions of stocker calves to other EU countries each year for fattening . Spain wMortality is often used as an animal welfare indicator, since it is beyond doubt that such deaths are preceded by a period of suffering and poor welfare . In relaThe mortality in pigs was 0%, lower than reported by Averós et al. in 2010 . HoweverThe morbidity rate was lower than reported in literature ,16,29 anIn this study, sheep/goats were found to be more likely affected by transport than the other types of animals. It has been reported that sheep usually cope better with transport than other species , though 2/animal, which vary according to the category of animal transported. The range for lambs goes from 0.20 to 0.30 m2/animal. However, it has been shown that stocking density should not be expressed in square meters per animal but in square meters per 100 kg to allow lambs to lie down and cope better with transport stress, and that lamb health and welfare may be affected by high stocking density [Overcrowding has been identified as a risk factor for transport-related health and welfare issues and it was the most frequent infringement observed during vehicle inspections carried out between 2001 and 2010 at the border between France and Italy . Council density . Our datJourney duration was confirmed as the most important risk factor in the development of transport-related diseases . SurprisSeason and month have been identified as risk factors for transport-related mortality ,30,33. HThe NATT was affected by many of the factors studied, in particular by species and category. There was a high average number of transported animals among vehicles transporting sheep/goats or younger—and consequently less heavy—animals. Since species and category were associated with other factors such as provenance, nationality of the transport company and destination, the effects of these latter factors on the number of transported animals per truck should be interpreted as a consequence. For instance, since sheep/goats came mainly from Spain and Hungary, all trucks coming from those countries contained higher than average numbers of transported animals. The association between species and category with season or month could, however, reflect consumer trends  and the typical commercial life of each animal. For instance, lambs are usually born in spring, weaned after three months and are then shipped to feed lot or slaughter; this could explain why we found that the transport of sheep/goats was positively associated with summer months.2/100 kg. Notwithstanding these limitations, this is the first paper reporting statistics based on surveillance reports filed by official veterinarians for livestock transported from Northern Europe to the Balkan Peninsula and transiting through a control post in Southern Italy.Our data should be considered preliminary because this study was limited by a number of factors. Firstly, due to the small number of events (welfare problem occurred) the logistic model was likely to suffer from small-sample bias. Secondly, the surveillance reports were cumulative by truck, which made it impossible to perform a proper risk analysis based on single transported animals. Thirdly, as the assessment of welfare outcomes was related only to death and severe pathology, in compliance with Section 5 of Annex 1 to Council Regulation (EC) No. 1/2005, many minor injuries and pathologies were not recorded and consequently were not analyzed in this manuscript. Finally, as previously discussed, our dataset was unable to include the health condition of the animals before the journey, the exact duration of the journey and stocking density expressed in mThis was the first study documenting farm animal movements from Northern Europe to Greece transiting through a control post and their subsequent welfare outcomes as assessed by OVs. Only 0.8% of the trucks reported a case of mortality or morbidity and lambs transported with minimal space allowance proved to be at higher risk of poor welfare. Due to the aforementioned limitations, our findings should be considered preliminary. Further studies should be carried out in more CPs and in other countries to confirm our data. Further studies should also assess welfare on individual transported animals to identify risk factors and to analyze welfare outcomes among transported animals, comparing the welfare assessment based on official reports versus the scientific welfare assessments suggested in more recent literature.


Diabetic mellitus erectile dysfunction (DMED) refers to erectile dysfunction (ED) secondary to diabetes. As people's lifestyle changes and the population ages, the incidence of DMED continues to increase. Many clinical trials have proven that Chinese medicine has a significant effect in the treatment of DMED. In this systematic review, we aim to evaluate the effectiveness and safety of traditional Chinese medicine (TCM) for DMED.We will search PubMed, Cochrane Library, AMED, EMbase, WorldSciNet; Nature, Science online and China Journal Full-text Database, China Biomedical Literature CD-ROM Database, and related randomized controlled trials included in the China Resources Database. The time is limited from the construction of the library to February, 2019.We will use the criteria provided by Cochrane 5.1.0 for quality assessment and risk assessment of the included studies, and use the Revman 5.3 and Stata13.0 software for meta-analysis of the effectiveness, recurrence rate, and symptom scores of DMED.This systematic review will evaluate the efficacy and safety of TCM for treating Diabetic mellitus erectile dysfunction. Because all of the data used in this systematic review and meta-analysis has been published, this review does not require ethical approval. Furthermore, all data will be analyzed anonymously during the review process trial. With the improvement of people's living standards, the incidence of diabetes, especially type 2 diabetes, has been increased, and the complications it has brought cover multiple organs of the human body.,5 ED is 1 of its common complications. Surveys in the United Kingdom, the United States, and other countries have shown that the incidence of erectile dysfunction in the normal population is 0.1% to 18%. However, the incidence of ED in diabetic patients has increased nearly 3-fold compared with the normal population, and tends to be in younger population. Studies have shown that the number of people with ED in diabetes has reached 71% in the past 10 years. Diabetes ED patients often have severe symptoms and are a type of refractory ED, which seriously affects the quality of lives of the diabetic patients.Diabetic mellitus erectile dysfunction (DMED) refers to erectile dysfunction (ED) secondary to diabetes. It is characterized by persistent or repetitive penile erection and insufficient hardness or sufficient time to be satisfied. The phenomenon of completing sexual activity. Some scholars believe that diabetes causes penile artery blood supply and venous closure to cause ED. Apart from this, age, duration of diabetes, control of blood glucose, smoking, hypertension, atherosclerosis, adverse drug reactions, and psychological factors are all closely related to the development of DMED. DMED not only seriously deteriorates the quality of life of patients, the harmony between family and society, but also causes greater physical and mental pain to male patients. The rapid development of material civilization and spiritual civilization is increasingly concerned by the society for the prevention and treatment of DMED. The current treatment methods for DMED can be roughly summarized as primary disease treatment, psychotherapy, and symptomatic treatment. These include basic treatment , psychotherapy, and medication .,12 However, due to the complexity of the pathogenesis of DMED, there is still no specific treatment for DMED in modern medicine. Studies have shown that PDE-5 inhibitors have side effects such as headache, facial redness, and gastrointestinal reactions during the treatment of DMED. Apart from these, in recent years, the studies have reported that some patients have prolonged drug onset time and drug resistance during the medication. Diabetes belongs to the category of “Xiaoke” in traditional Chinese medicine (TCM). Male erectile dysfunction belongs to the category of “yangshuo” in TCM. TCM believes that DMED lesions are mainly in the kidney, “yin deficiency with internal heat,” and “qi stagnation and blood stasis” is its main pathogenic factor.,16 With the application of TCM in the treatment of DMED, a unique diagnosis and treatment system, clinical efficacy is significant. Modern research shows that the effective active ingredients in Chinese medicine can not only promote the activity of sputum-glucosidase, but also improve the blood supply of peripheral blood vessels, so as to achieve the purpose of treatment.,18 TCM regulates body function as a whole through multifaceted and multitarget mechanism, and has unique advantages in the treatment of DMED.The pathogenesis of DMED is not fully understood. At present, most scholars believe that high glucose environment leading to central nervous system and pericardial nerve damage is an important cause of the disease.After a preliminary search and analysis of database resources, we found that randomized controlled trials (RCTs) of TCM for DMED are gradually increasing. However, due to the size and number in clinical trials, most clinical trials face problems with small sample size and evidence-based exploration. Therefore, we expect a meta-analysis of relevant studies on TCM treatment of DMED and a meta-analysis to assess its clinical efficacy and safety.2This is a systematic review and ethical approval was not necessary.2.1This systematic review protocol has been registered on PROSPERO as CRD42019117444 (hops://www.crd.york.cord.php?RecordlD=17444).2.22.2.1Take TCM or Chinese medicine combined with other effective interventions as main treatment, including RCTs of the control group (effective methods other than TCM). Language is limited in Chinese and English. Non-RCTs, quasi-RCTs, case series, case reports, and crossover studies will be excluded.2.2.2Men with a history of diabetes who match the Diagnostic Criteria for Diabetes: refer to the American Diabetes Association Diabetes Care Guidelines. The diagnosis is ED after diabetes, and the International Index of Erectile Function 5 (IIEF-5) score is <21. The course of ED is ≥3 months. The patient must be at least 18 years of age. The sexual partners of the patients are fixed. The group is well balanced when enrolled.2.2.32.2.3.1The drug composition, the dose-specific Chinese medicine preparation or the combined western medicine, is used as experimental interventions. Both prescription and Chinese patent medicines will be included. Other TCM treatments such as intravenous medication, acupuncture, and massage will be limited.2.2.3.2As for the control interventions, those who accepted simple western medicine can be used as a control intervention or those who did not get any treatment as a blank control would be adopted. However, once they had accepted the therapy of TCM, the trials will be rejected.2.2.4The primary outcome measurement will be assessed using the International Erectile Function Index.Healing: IIEF-5 score ≥22 points after treatment; significant effect: IIEF-5 score <22 points after treatment, score improvement ≥60%; effective: IIEF-5 score <22 points after treatment, points improved <60%, but ≥30%; invalid: IIEF-5 score <22 points after treatment, score improvement <30%.The secondary outcome measurement will be assessed according to the TCM syndrome scoring criteria.Healing: The clinical symptoms and signs of TCM disappear or disappear, and the syndrome score is reduced by ≥90%; markedly effective: the clinical symptoms and signs of TCM are obviously improved, the syndrome score is reduced by ≥60%; effective: TCM clinical symptoms signs and signs have improved, syndrome points reduced by <60%, but ≥30%; invalid: Chinese clinical symptoms and signs have not improved, or even worse, syndrome scores reduced by <30%. Integral variation formula (Nimodipine method [{pretreatment score − post-treatment score} ÷ pretreatment score] × 100%).2.2.52.2.5.1The electronic search database includes PubMed, Cochrane Library, AMED, EMbase, WorldSciNet, Nature, Science online and China Journal Full-text Database, China Biomedical Studies CD-ROM Database (CBM), and China Resources Database. The clinical research studies on the treatment of Diabetic mellitus erectile dysfunction with Chinese medicine published in domestic and foreign biomedical journals from the establishment of the library to February, 2019 was searched. Based on the standards of the Cochrane Collaboration Workbook of the International Evidence-Based Medicine Center, a manual and computer-based method is used to conduct related studies. The search terms include: Chinese medicine, traditional Chinese medicine, proprietary Chinese medicine, Chinese herbal medicine, Diabetic mellitus erectile dysfunction, impotence. According to the characteristics of different databases, comprehensive search using keyword was conducted. All search results are determined after multiple searches. We will follow the references included in the studies to incorporate relevant studies as much as possible to avoid missed detection. The search term in the Chinese database is the translation of the above word. The complete PubMed search strategy is summarized in Table 2.2.5.2The manual search mainly is used for searching relevant studies such as “Chinese Journal of Endocrinology and Metabolism,” “National Journal of Andrology,” and “Chinese Journal of Urology” before the database creation.2.2.6The data collection and analysis was conducted as follows:1.Applying the EndnoteX7 software to manage the included references. Two qualified evaluators independently screened the titles and abstracts of the selected studies, excluding duplicates and documents that did not significantly conform to the study.2.The second screening of the studies: Screening out unqualified studies such as case report, theoretical discussion, and nonconformance of interventions was done. After preliminary evaluation, the remaining studies to further screen out the unqualified studies such as ordinary control study, no control group, no random grouping, no outcome index, and data mine equivalent were carefully read.3.For the studies that could not be determined whether to be included or not, it was decided by 2 researchers. If the opinions were not uniform, third-party expert was asked. A clinical RCT was finally included in the study.4.Information and data extraction for the final included documents: The extracted data and information included the test methods of the study, the basic information of the included cases, the observation period, the intervention methods of the treatment group and the control group, the observation indicators, and the test results Fig. .2.2.7The studies quality assessment applies the bias risk assessment tool recommended by Cochrane to evaluate the quality of all included studies and the risk of bias. The assessment will include sequence generation, allocation concealment, blinding of participants, personnel and outcome assessors, incomplete outcome data, selective outcome reporting, and other sources of bias. The risk of high and low bias is expressed as “high risk” and “low risk,” respectively. The information provided in the study is inaccurate or does not provide sufficient information for the bias assessment to be expressed as “unclear risk.” The above content evaluation is independently evaluated by 2 researchers. If there are different opinions, the discussion will be conducted. If there are still differences, a third appraiser will be consulted. Otherwise, the Cochrane Professional Group will be consulted.2.2.8The meta-analysis in this study will use Rev Man5.3 and Stata13.0 statistical software. Heterogeneity tests will be used for the included experimental studies. The numerical variable will be expressed as the normalized mean difference with a confidence interval (CI) of 95%. The heterogeneity of each pair-wise comparison will be tested by chi-square test (test level α = 0.1). If there is no heterogeneity, a fixed-effect model will be used. If there is significant heterogeneity between a set of studies, we will use a random-effects model (REM) for meta-analysis. We will explore the reasons for the existence of heterogeneity from various aspects such as the characteristics of the subjects and the degree of variation of the interventions. The source of heterogeneity is further determined by means of sensitivity analysis.2.2.9If a result of a meta-analysis contains more than 10 articles and above, we will use a funnel plot to test the risk of publication bias. Quantitative methods such as Begg testing and Egger testing will be used to help assess publication bias in the application.2.2.9.1https://gradepro.org/).The Grading of Recommendations Assessment, Development and Evaluation (GRADE) method will be used to assess the quality of evidence for key outcomes. This assessment will be conducted through a Guideline Development Tool (GRADEpro GDT, 3,21 About half of the diabetic patients have ED. TCM believes that the etiology and pathogenesis of ED is related to spleen and kidney deficiency, and qi and blood block. TCM can play the role of strengthening the spleen and kidney, promoting blood circulation and collaterals, and at the same time, can improve the mood and achieve the purpose of treatment.–24 Modern research shows that the effective active ingredients in Chinese medicine can not only promote the activity of sputum-glucosidase, but also improve the blood supply of peripheral blood vessels, so as to play a corresponding therapeutic role.,26In recent years, with the changes in people's lifestyles and the aging of the population, the incidence of diabetes has increased. Diabetes has a variety of chronic complications, including heart disease, high blood pressure, stroke, and ED. Among them, diabetes is most closely related to ED and is the leading cause of organic ED.With the deepening of understanding of diabetes and its complications, the trials and clinical reports of TCM treatment of DMED have gradually increased. Whether it is syndrome differentiation or special disease, Chinese medicine has achieved good results in the treatment of diabetes ED. To the best of our knowledge, there has been no comparison of the efficacy and safety of TCM for the treatment of DMED in recent years. Therefore, we will compare the effectiveness and safety of TCM in the treatment of DMED by applying systematic evaluation and meta-analysis. The results of this study can provide a possible ranking for Chinese medicine treatment of DMED. We hope that the results will provide clinicians with the best options for treating DMED and provide them with research directions. Although we will conduct a comprehensive search in this study, languages other than Chinese and English will be restricted, which will lead to some bias. In addition, the relevant studies on TCM treatment of DMED are small and the overall quality is low, which may affect the authenticity of this study. Therefore, we hope that in the future, we will have a more rigorous and reasonable multicenter RCT to explore the clinical efficacy of TCM in the treatment of DMED, so that the conclusion is more objective and reasonable.Conceptualization: Jianwei Shang.Formal analysis: Xudong D. Yu, Jisheng S. Wang, Songli Wu.Funding acquisition: Xudong D. Yu, Jisheng S. Wang, Jianwei Shang.Investigation: Jisheng S. Wang.Project administration: Jisheng S. Wang.Supervision: Xudong D. Yu, Jisheng S. Wang, Yaosheng Zhang.Validation: Xudong D. Yu, Jisheng S. Wang, Jingyang Lim.Writing – original draft: Xudong D. Yu, Jisheng S. Wang, Guang Zuo, Fuxing Ge.Writing – review & editing: Xudong D. Yu, Jisheng S. Wang, Xin Wang, Fuxing Ge.


Vulvar cancer (VC) is a rare disease of which recurrence poses management problems due to patients’ advanced age and comorbidities, and to the localization of the disease. Palliative treatments, allowing local disease control in patients previously treated with multimodal therapies or with comorbidities, are lacking. In this study we tested electrochemotherapy (ECT) on recurrent VC refractory to standard therapies to assess the tumor response and to define the selection criteria for patient’s candidate to ECT. This is a multicenter observational study carried out in five Italian centers. Data about patients and tumor characteristics, treatment, toxicity, and clinical response were recorded. In all procedures, intravenous bleomycin was administered according to European Standard Operative Procedure ECT (ESOPE) guidelines. Sixty-one patients, with a median age 79 years (range: 39–85) and mainly affected by squamous cellular carcinoma (91.8%), were treated with ECT. No serious adverse events were reported. Patients were discharged after three days . Two months after ECT, the clinical response rate was 83.6% and was not related to age, body mass index, International Federation of Gynecology and Obstetrics (FIGO) stage, number of treated nodules, or previous treatments. ECT is a safe procedure with a favorable cost-effectiveness ratio and should be considered as a treatment option for local disease control in patients unsuitable for standard therapies. Vulvar cancer (VC) is a rare disease accounting for 5% of all gynecological malignancies, with a higher incidence in older women. However, during recent decades, the incidence of VC has increased in young women due to the spread of Human Papilloma Virus (HPV) infection. Squamous cellular carcinoma (SCC) represents the most common histotype (90% of cases), even if other less-frequent histological types  are reported . PatientElectrochemotherapy (ECT) represents a new therapeutic option that, during the last few years, has expanded to different clinical settings. In fact, the procedure was initially introduced to treat small metastatic cutaneous tumors not amenable to surgery or radiation therapy . HoweverIn 2013, we published the first report on the use of palliative ECT on nine patients with VC who relapsed after multimodality treatments and were unsuitable for standard therapies. The results were encouraging, with a 62.5% complete response (CR) rate and relevant symptoms relief  . ECT wasThe aim of this multicenter observational study is to report on the oncological outcome as a local control of the disease, achieved with the use of ECT in the treatment of VC refractory to standard treatment in a larger patients’ series. The secondary endpoint is to identify prognostic factors in order to improve patients’ selection for ECT.This observational study was partly retrospective and partly prospective. The study was approved by the Ethical Committee of Bologna (EC code 42/2013/O/OssN), and patients signed the consensus to data analysis. Women with relapsed VC treated with ECT were included in the analysis. Patients’ data was collected using a web-based electronic Case Report Form (e-CRF). The collected information was anonymized, encrypted, and finally sent to the remote server for data storage. In this way, data about patients and their treatment characteristics were registered in a standardized and consistent way among different centers and physicians. In particular, the following data were recorded: Age, BMI, comorbidities, histologic type, stage, previous medical, surgical, and radiotherapy treatments and number, site, and size of recurrences. If available, a photographic image of the lesions before and after ECT was collected and uploaded. From our clinical records, we selected a cohort of patients with VC relapse treated with multimodal therapies as a control group. These women were submitted to palliative therapies before the introduction of ECT in our department.ECT was performed according to ESOPE guidelines . ElectriStatistical analysis was performed using IBM SPSS Statistics 25.0 software . Continuous variables are presented as medians and ranges. Categorical data are expressed as percentages and absolute rates. Statistical comparisons of response rates were performed using contingency tables and the Pearson chi square test. Survival curves were calculated using the Kaplan–Meier method. The log-rank test was used to compare survival curves. Local progression-free survival (LPFS) was calculated from the date of treatment to the one of relapse/progression in the treated area, or to the last follow-up.Sixty-one patients affected by VC refractory to standard therapies were treated with ECT and included in the analysis . The patients’ median age and Body Mass Index (BMI) were 79 years (range: 39–85) and 25 (range: 15–39), respectively. Their tumors’ histological types included SCC , Paget’s disease , and malignant melanoma . Considering all the treated patients, 37 presented single nodules (60.7%) and 24 (39.3%) presented multiple nodules. In total, 106 nodules were treated (range of number of nodules per patient: 1–7). The median diameter of the lesions treated was 20 mm (range: 1–110 mm). The topographic distribution of the nodules treated is shown in Forty-eight (79%) patients underwent ECT under general anesthesia, while 13 (21%) patients were treated under local anesthesia. All procedures were performed following ESOPE guidelines, and we were able to cover the whole area of the lesions in 105 treated nodules (99.1%). Intravenous bleomycin was administered in all cases. The linear electrode was used in 94 lesions (88.7%), the finger electrode in seven lesions (6.6%), whereas the hexagonal electrode was chosen for five lesions (4.7%). No other concomitant treatments were performed. No treatment-related serious adverse events were reported. Short-term toxicity was evaluated immediately after procedure and at the first follow-up; long-term toxicity was then evaluated at the subsequent follow-up. The most common local side effect was erythema (20%), hyperpigmentation (6%), skin ulceration (1%), and mild pain (24%) which was easily controlled by the administration of minor analgesics. Patients were discharged from the hospital after a median time of three days (range: 0–8 days).Response to therapy was assessed two months after ECT. Tumor response was evaluated for only 55 patients, because six patients did not reach the first follow-up evaluation; 4 of 6 patients died because of systemic disease progression and 2 of 6 were lost to follow-up. In all evaluable patients, the clinical overall response (CR+PR) rate was 83.6%. CR was recorded in 29 cases (52.7%), PR in 17 (30.9%), SD in 6 (10.9%), and PD in 3 (5.5%) . ConsideThe correlation between response to ECT per patient and different factors such as age, BMI, FIGO stage, and number of nodules was analyzed. Similarly, the correlation between response per nodule with size, previous treatments, and site localization was analyzed. None of the following parameters influenced the response to treatment: age; BMI; FIGO stage; number, site and dimension of nodules; and previous treatments .Considering all patients, the median follow-up was 142 days (range: 40–1524 days). Among the 29 complete responder patients, the median follow-up was 258 days (range: 40–1524 days). Six patients died of disease after a median time of 334 days (range: 78–586 days) and two patients experienced disease recurrence 136 and 192 days after ECT. Patients with disease recurrence were referred to palliative care. For the 17 partial response patients, the median follow up was 126 days (range: 40–1278 days). In this subgroup, five patients died after a median time of 101 days for systemic progression (range: 40–141 days), two patients were referred to other treatments (Imiquimod and nodulectomy), and five patients underwent a second ECT course with an additional response to treatment, including two patients with CR, two patients with PR, and one patient who died from systemic disease progression. Among the six patients with no change in tumor size (SD), the median follow-up was 88 days (range: 40–1440 days), with two patients who died after 168 and 192 days, respectively, for systemic disease progression. In p: 0.007). Overall survival for the entire cohort of patients is shown in The six-month local progression-free survival was 91% (C.I. 81–100%) and 73% (C.I. 41–100%) in responders and non-responding patients, respectively . Previous treatments were surgery in all patients, combined with radiotherapy (seven patients) or chemoradiation (two patients). Upon relapse, all patients were treated with chemotherapy (weekly paclitaxel). In four patients, the treatment was prematurely stopped due to toxicity and, in three patients, due to early progressive disease. Overall, 0/9 (0%) patients showed CR, 2/9 (22%) had PR, 4/9 (45%) showed SD, and 3/9 (33%) had PD. We did not perform actuarial analysis of local control and overall survival due to the small sample size.Our group was the first to test the local efficacy of ECT in refractory cases of VC, and here we present the largest study cohort on the use of palliative ECT in this group of patients. In this study, we analyzed only the outcome of the response to therapy. The present results add new evidence to previous reports ,15,16, sWe introduced ECT in the management of refractory VC for several reasons, mainly because of the many challenges related to the treatment of these neoplasms. These challenges include VC site , multifocality, and high incidence in elderly patients. An oncological radical resection can cause disfiguring consequences, while concurrent chemo-radiotherapy can produce significant acute toxicity and potential late complications . Tumor-rIn our experience, the procedure was at first tested in older patients and then progressively offered to all patients requiring a palliative treatment. Our study confirmed the efficacy of ECT, strengthening the results of previous studies. Importantly, in the management of skin cancers, ECT allows the local control of the disease of VC and, if needed, can be administered concomitant to, or following, other treatments such as radiotherapy or systemic therapies in patients with distant metastases.Identification of patient- and tumor-related predictive factors for responses to ECT should be important in selecting the best candidates for this procedure. Therefore, all clinical-related aspects that could influence ECT activity were studied.Surprisingly, no statistical difference in terms of response to treatment in relation to histology, site, dimensions, number of nodules, and previous treatments was found. Moreover, no difference was observed between patients with SCC (57) versus patients with other types of cancer histology . These results, within the limitation of some small subgroups in patients’ analyses, seems to confirm previous observations of a tumor response not related to tumor histology, as reported in literature for other tumors. In fact, several analyses reported that ECT is equally effective in basal cell carcinoma, malignant melanoma, skin recurrences from breast cancer, squamous cell carcinoma, Kaposi’s sarcoma, Merkel cell carcinoma, and other tumors .Furthermore, no correlation was found between the response to treatment and the tumor’s site. The possible explanation is that even though some areas are more difficult to treat than others for anatomical reasons when ECT was performed according ESOPE guidelines and all the tumor’s area was covered by the treatment, the tumor response could be the same regardless of distance from urethra, vagina, or anus.In literature, controversial data about the correlation between the response to ECT and the size and number of skin cancer nodules treated is reported. Some studies showed no statistical difference among the responses when tumors were grouped according to their size ,19, and No correlation was observed between responses and previous treatments; this means that the histological modification of tissues due to radiotherapy does not compromise the efficacy of ECT treatment. Probably, the real mechanism underlying the ECT response is little understood. High drug concentrations led to cellular toxicity and death. ECT combines the ability to exert a cytotoxic effect on cancer cells with the capability to generate an anticancer immune response. In fact, some studies demonstrated that the local effect of ECT is reduced in immunodeficient mice with a lack of T lymphocytes, and interleukin-2 or other pro-inflammatory cytokines . This imBLM and cisplatin (CDDP) are the most-used drugs for ECT . Based oIt is very difficult to compare our group of patients treated with ECT with others submitted to palliative treatments in our Institution, because before the introduction of ECT most patients in this setting received only supportive therapy. Therefore, we were able to find only a very small group of patients treated with chemotherapy. Despite the small sample size of our control group, the superiority of ECT in terms of clinical response seems evident. Obviously, a more reliable comparison would require the design and execution of a prospective comparative trial.Based on these results, and considering the poor alternatives available for the management of VC, we suggest ECT as a new therapeutic tool for patients unsuitable for surgery, for chemo-radiotherapy-resistant lesions, and for patients with severe comorbidities and/or of advanced age who have already received the available standard treatments. Therefore, Oncological Societies should consider this therapy as one of the options in palliative care for vulvar cancer.The procedure is easy and quick to perform (25–30 min) and is associated with a short hospital stay. For these reasons, and due to the low price of the drugs used, ECT has a favorable cost-effectiveness ratio. The side effects are minor, and most patients require only low dose of analgesics to treat ECT-related pain. Moreover, unlike radiotherapy, it is possible to repeat several ECT cycles without precluding other types of treatment.Further studies are required to (i) evaluate the risk of pulmonary complications produced by BLM in the case of multiple ECT, (ii) analyze the possibility of severe ulcers mainly after the treatment of large tumors, and (iii) identify the best candidates for this promising treatment.Finally, based on the significant correlation between clinical response and local control, further trials investigating the possibility of improving the response rate are justified. These studies could be based on the combination of ECT with other therapies.


Background: Health literacy (HL) is an important factor in improving health inequalities in poor and marginalized groups. Assessing comprehensive HL is critical. In this study, we validated the use of a comprehensive short-form HL survey tool (HLS-SF12) and examined the determinants of HL among people in rural areas. Methods: A cross-sectional study was conducted in July 2019 on 440 people residing in mountainous areas in Vietnam. Health literacy was measured using the HLS-SF12. Personal characteristics were also collected. We analyzed data using confirmatory factor analysis, internal consistency analysis, and regression analysis. Results: The questionnaire demonstrated a good construct validity with satisfactory goodness-of-fit indices and item-scale convergent validity. The tool was reliable and homogeneous with Cronbach’s alpha = 0.79, with no floor/ceiling effects. People who were married had lower HL  = −5.69, −0.56; p = 0.017) compared with those who never married. Higher education attainment , a better ability to pay for medication , and a tendency to view health-related TV/radio more often  were associated with higher HL. Conclusions: The HLS-SF12 is a valid survey tool for the evaluation of HL in rural populations. A number of personal characteristics were strongly associated with HL. The commitment of the 2030 Sustainable Development Goals (SDGs) agenda is that “no one will be left behind” . EducatiNon-communicable diseases (NCDs) are a heavy burden to Vietnam and the world ,6. The sHealth literacy is an important component of public health practice, and it is needed to enable effective health promotional activities/programs and behavior changes ,20,21,22It is critical to assess HL and its associated factors that may provide helpful evidence when it comes to appropriate health resource allocation. This could improve the effectiveness of health promotion programs and further reduce health inequalities in the context of SDGs. However, a valid, comprehensive survey tool has not been available for use in rural settings. Our aim in this study was to validate the comprehensive short-form health literacy questionnaire (HLS-SF12) and examine the potential determinants of HL among people living in rural areas.A cross-sectional study was conducted in July 2019 at two community health stations at the Nham commune in the A Luoi district and the Thuong Long commune in the Nam Don district, both of which are located in the Thua Thien Hue province, Vietnam. These two communes are located in poor mountainous areas in the center of Vietnam, with limited resources, such as health stations, for providing adequate examinations and medication to residents.The sample size required for conducting the structural equation model  was recommended to be 10 times the item number . A samplResidents visiting community health stations were recruited during the medical tours of Hue University of Medicine and Pharmacy in July 2019. The people selected were those who resided in two mountainous communes in two districts in a central province in Vietnam, were aged 18 and above, did not have any emergency conditions or any mental health problems, and were able to listen and understand the local language or dialect. A total sample of 440 people was collected and analyzed.Index = (mean − 1) × (50/3), where Index is the specific index calculated, mean is the mean of all participating items for each individual, 1 is the minimal possible value of the mean , 3 is the range of the mean, and 50 is the chosen maximum value of the new metric. Thus, an index value is obtained where 0 represents the lowest HL and 50 the highest HL [The short-form health literacy questionnaire (HLS-SF12), which has been validated in the general population of six Asian countries , was useghest HL . The indThe following variables were also assessed; age, gender, ethnicity, marital status, education, occupation, the ability to pay for medication, social status, tendency to view health-related TV/radio, and community involvement.The medical students who were utilized as interviewers were trained in data collection by a senior researcher. A four-hour training session took place on the university campus. A daily meeting was conducted in order to improve the quality of the data collected. The research team  had a two-hour meeting with the medical tour team (volunteer doctors and nurses) and local volunteers regarding the surveys in order to organize the health check and interview sessions. The face to face interviews were conducted at the community health stations using printed questionnaires. Local volunteers helped with interpretation for participants who used the local dialect (about one fifth to one fourth of participants). A consent form was obtained by each participant, while adequate time (15–30 min) was allowed for answering all the questions.The study was approved by the Institutional Ethical Review Committee of Hanoi School of Public Health, Vietnam (No. 379/2019/YTCC-HD3). All participants were asked for their permission and signed consent forms before their participation.2/df ratio). This method has also been used in a previous study [p-value < 0.05. Data were analyzed using the IBM SPSS Version 20.0 , and AMOS version 22.0 [Firstly, descriptive analysis was conducted to examine the distribution of study variables. The frequency and percentage, mean and standard deviation were reported. The independent-samples T-test and one-way ANOVA test were used to compare the distribution of health literacy between categories of participants’ characteristics, appropriately. Secondly, to evaluate the validity of the HLS-SF12, we used confirmatory factor analysis (CFA), with a maximum likelihood algorithm estimation to assess the construction of the questionnaire, focusing on three domains of health including health care, disease prevention, and health promotion . The goous study . In addius study . Thirdlyus study , and theus study ,42. Fourus study . Finally NY, USA).The average age of the study participants was 40.8 ± 13.4 years old; 43.6% were men, 85.9% were an ethnic minority, 87.7% were married, 19.1% were illiterate, 77.7% had participated in agroforestry work, 73% had difficulty paying for medication, 38.1% were perceived to have low social status, 40.2% never or rarely viewed health-related television or radio, and 52.7% were never or were rarely involved in community activities . The ove2/df = 4.34) was also adequate , disease prevention (0.62–0.73), and health promotion (0.63–0.73), respectively . The intp = 0.017) as compared with those who had never married. In comparison with illiterate people, those with higher education attainment at Elementary School , Secondary School , High School , or Vocational/University  had higher HL scores. Compared with people with the ability to pay for medication at the very difficult level, those with the ability at the levels of fairly difficult , fairly easy , and very easy  had higher HL scores. Compared to people who had never viewed health-related TV/radio, those who viewed rarely , sometimes , and often  had higher HL scores  = −5.69, −0.56, L scores .The HLS-SF12 was shown to have satisfactory construct validity with a good model-data-fit . In addiThe internal consistency of the HLS-SF12 was at an adequate level  in the current study population, which was slightly lower than previous studies conducted in Europe   and AsiaIn the current study, age was negatively associated with HL in the unadjusted model. However, the association was attenuated in the adjusted model. This indicates that age might not be a predictor of HL in rural areas, even though age was one of the important factors of HL in populations in Austria, Bulgaria, Greece, Poland, and Spain, and was partially important in the Netherlands ,37,48, tEducation attainment and the ability to pay for medication were both highly associated with HL in both the current study and in previous studies in Europe ,48 and AHealth literacy was not significantly different between ethnic minority groups in the current study. This was also found in a previous study in the Netherlands . In addiThe current study has some limitations. Firstly, the study was conducted during the medical tour that provided free health checks and medication for people who live in rural areas. Therefore, the sample collected might not be representative for the whole population, and the external validity of the survey tool may be limited. Secondly, the findings of the current study were based on a sample of 440 people who resided in two mountainous communities in Vietnam. Therefore, the association between personal characteristics and health literacy may not be generalizable to all rural areas in Vietnam. In addition, the associations found in a cross-sectional study can only raise the phenomenon or hypothesis for future study; the causality cannot be generated. Future study with a larger study population in rural areas is required to explore the health literacy levels and the associated factors that might contribute to potential interventions in order to improve the quality of healthcare services and people’s health in rural areas in Vietnam and across the Globe and to achieve SDGs by the year 2030 .The HLS-SF12 was shown to be valid as a comprehensive survey tool to measure health literacy in a rural setting. Marital status, education, the ability to pay for medication, and the tendency to view health-related television/radio were all determinants of health literacy. The findings may be helpful for effective public health interventions to enhance people’s HL and reduce health inequalities in rural areas.


Specialists of human genetic diagnostics can be divided into four groups: Medical Geneticists (MDG), Genetic Nurses and/or Counsellors (GN/GC), Clinical Laboratory Geneticists (CLG) and Laboratory Genetics Technicians (LGT). While the first two groups are in direct patient contact, the work of the latter two, of equal importance for patient care, are often hidden as they work behind the scenes. Herein the first study on the rights and duties of CLGs is presented. We present the results of a survey performed in 35 European and 18 non-European countries with 100 participating specialists. A national CLG title is available in 60% of European countries, and in 77% of the surveyed European countries a CLG can be the main responsible head of the laboratory performing human genetic tests. However, in only 20% of European countries is a lab-report valid with only a CLGs’ signature - even though the report is almost always formulated by the CLG, and an interpretation of the obtained results in a clinical context by the CLG is expected in nearly 90% of European countries. Interestingly, CLGs see patients in 30% of European countries, and are also regularly involved in student education. Overall, the CLG profession includes numerous duties, which are quite similar in all regions of the world. Strikingly, the CLG’s rights and responsibilities of leading a lab, or signing a report are regulated differently according to country specific regulations. Overall, the CLG is a well-recognized profession worldwide and often working within a multidisciplinary team of human genetic diagnostics professionals. Human genetic diagnostics is an emerging field, as knowledge of underlying causes of inherited and acquired diseases continues to grow exponentially . This exIs there a national CLG-title in your countryThe survey  led to inconclusive answers and/or responses indicated the question was outside of the knowledge of the interviewed participants  and thus were excluded in the final evaluation.Evaluable survey replies , and in only 27% of the countries must an MD be head of the laboratory. Within Europe this accounts for 23% of the countries, i.e., in 77% of the European countries, a CLG may be head of a human genetic diagnostic laboratory. This statistic was reflected not surprisingly by the response of a Dutch colleague to question 5b regarding MD involvement: ‘Certainly a CLG can be responsible head of a lab without an MD at his/ her side. How could an MD be responsible for a lab?'.A human genetic report is only valid with a signature of an MD in 1/3 of the 53 countries. In Europe, the signature of an MD is required for validity in only 1/5 of the countries. Interestingly, in some countries instead of an MD, LGTs or a second CLG co-sign the report. The validity of a report must be considered in the context of reimbursement by health insurance companies for services rendered. In USA, a report with MD signature is reimbursed at a higher rate by insurance companies, with reports of lower reimbursement for an identical report with CLG signature only. Conversely, e.g., in Netherlands and Switzerland it is logically concluded that the CLG signature is the reason for reimbursement of laboratory diagnostics given these tests are completed under their aegis.In greater than 95% of the countries, the final report goes to the MD who requisitioned the diagnostic test. In fewer than 5% of countries, the patient or family receives the report. In addition, in some countries GNs/GCs or midwifes are recipients of reports.In all European countries excluding Turkey, and only 5 non-European countries is the final report written by the CLG.Further tests can be suggested by a CLG in all European countries excluding Russia and Turkey, and 6 non-European countries. In nearly 90% of the countries, the expertise of CLGs is well-recognized and reflected in their ability to requisition additional tests to optimize patient care.Result interpretation in a clinical context within the report is expected in 31/35 European and 12/18 non-European countries.CLGs see and/or counsel patients alone or with MDs in nearly 30% of European and 22% of non-European countries. In some countries participation in MD-lead genetic counselling is obligatory during CLG education.CLGs teach at universities in all European countries, excluding Denmark, and in all non-European countries excluding Iraq. In addition, teaching activities are dependent on the employer (private or public).The responses of CLGs in the 53 countries were as follows:https://www.ebmg.eu/666.0.html as national contacts for ErCLG related questions were included, and for non-European countries, either registered affiliated ErCLGs and/or heads of corresponding human genetic societies were contacted. However, for 28 countries only one contacted representative participated. In general, the more people who replied for each country, the more comprehensive the country specific data became for data evaluation. This can be contributed to the fact that a CLG knows best their working environment, and may hold additional information regarding working conditions of one or more laboratories within the country. Differences from general rules are expected between countries, given local specificities are abundant. Local specificities were evident in this survey when contradictory answers were given by participants in the same country, and when multiple respondents reported detailed comments on their country. However, conflicting questions and their corresponding responses were thus excluded from final evaluation. The results of this survey have been made freely available on EBMG webpage (https://www.ebmg.eu/910.0.html). New data on further countries and/or corrections on information described herein are welcome and will be uploaded when sent to Thomas.Liehr@med.uni-jena.de.The present survey is, to the best of our knowledge, the first performed on the working conditions of CLGs. The responses were collected from 100 CLGs (Table https://www.eshg.org/fileadmin/eshg/EBMG/CLG/Direttiva_36_EN.pdf) and the “Proposal for modernising the Professional Qualifications Directive” = EU Directive 2013/55/EU (https://www.eshg.org/fileadmin/eshg/EBMG/CLG/Direttiva_55_EN.pdf), as at present (i.e. March 2018) Finland, France, Greece, Italy, Hungary, Latvia, Poland, Slovenia and UK have national state recognized CLG titles. Efforts to (re-)achive this kind of national recognition, are currently underway in Portugal, Spain, Cyprus, and Denmark. The EBMG is in preparation of starting this EU CLG recognition process (https://www.ebmg.eu/668.0.html).Herein, for the first time, the differences in working conditions of CLG in non-European and European countries are reported. In general, the profession is more widely recognized in Europe, North-America, and Australia, rather than other parts of the world. Accordingly, working conditions of European, Canadian, US-American and Australian CLGs are more regulated. Their duties and influence on diagnostics are well-defined given their nationally (Tab. 1) or on European level  certifiaAs the majority of the surveyed countries not only allow a CLG to be head of the laboratory performing human genetic tests, but also to sign responsibly without an MD demonstrates the already high acceptance rate of the CLG profession. Examples from Norway, Poland, Pakistan, Romania, Russia, Serbia, Switzerland, and Ukraine suggest that even more responsibility in reporting can be successfully shared with LGTs, too.Undoubtedly, reports of human genetic laboratory diagnostics primarily go via the requesting MD to the patient or family nearly everywhere in the world. However, it is noteworthy that GNs/GCs or midwifes  can alshttps://www.eshg.org/fileadmin/www.eshg.org/documents/QAGuidelineseng.pdf).CLG is a well-recognized profession worldwide. CLGs have clearly defined rights and duties within a multidisciplinary team composed of four professions: MDGs, GNs/GCs, CLGs and LGTs, working diligently to provide patients reliable diagnoses with a focus on ‘precision medicine’ for all . In AustSupplementary data 1


Our objective in this study was to find determinants of high-school dropout in a deprived area of Japan using longitudinal data, including socio-demographic and junior high school-period information.We followed 695 students who graduated the junior high school located in a deprived area of Japan between 2002 and 2010 for 3 years after graduation . Multivariable log-binomial regression models were used to calculate the prevalence ratios (PRs) for high-school dropout, using multiple imputation (MI) to account for non-response at follow-up.The MI model estimated that 18.7% of students dropped out of high school in approximately 3 years. In the covariates-adjusted model, three factors were significantly associated with high-school dropout: ≥10 days of tardy arrival in junior high school , daily smoking  and severe problems, such as abuse and neglect . Among students with ≥30 days of tardy arrival in addition to daily smoking or experience of severe problems, ≥50% high-school dropout rates were observed.Three determinants of high-school dropout were found: smoking, tardy arrival, and experience of severe problems. These factors were correlated and should be treated as warning signs of complex behavioral and academic problems. Parents, educators, and policy makers should work together to implement effective strategies to prevent school dropout. The first grade of junior high school corresponds to seventh grade in the United States, and the third grade of high school corresponds to the 12th grade. In recent decades, more than 95% of junior-high-school students continued to high school in Japan5; however, approximately 2 percent of high school students drop out of school within a year.6 More than 50% of high-school dropouts started their first job as non-standard employees, while this percentage was approximately 20% for high school graduates who had not attended college.7Educational attainments, usually achieved in youth, are strongly associated with later employment, type of occupation, and income in adulthood and also affect health status.8 Adolescents’ behaviors can deviate from social or legal norms, and these behaviors —such as tobacco, alcohol, or drug use, truancy, and unsafe sex— tend to co-occur, resulting in poor academic outcomes, such as school dropout.9,10 These risky behaviors are also correlated with their environment, such as the socioeconomic status of their households or place of residence .1 Previous studies of children and adolescents in the United States found that growing up in deprived neighborhoods is associated with many social problems, such as delinquency, low intelligence quotient, and dropping out.11,12 A previous study in Japan reported that more than 30% of students with the lowest education level (lowest quintile of entrance examination average score) dropped out of high school, while less than 3% with the highest education level (highest quintile) dropped out.13 However, this study did not account for other factors, including information from junior high school. Empirical longitudinal studies that examined the risk factors for high-school dropout have mostly been conducted in the United States or Europe and are scarce in Japan,9,14–16 especially in deprived areas. Our objective in this study was to find determinants of high-school dropout in a deprived area in Japan using longitudinal data, including socio-demographic and junior high school-period information. This will provide insights into preventive measures for policy-makers and other professionals.Adolescence is an important period for human development, but it is also often a rebellious period.17 According to the 2005 census, the ward is one of the most deprived areas, with a higher unemployment rate (22.4%) than the overall Japanese total (approximately 5%). Providing continuous support for all students, including vulnerable students, was considered to be very important, not only during compulsory attendance at junior high school but also after graduation. To take care of the students and to conduct this study, we utilized our personal and organized relationship within the junior high school and its neighboring area organizations, including junior high school, local volunteer groups, local NGOs, and local government. Information on students’ socio-demographic and school life-related factors were collected between 2000 and 2013 from various school records, repeated questionnaires, or personal communication . For example, total days of tardy arrival were calculated from the attendance record of the junior high school. Daily smoking was determined using the lifestyle guidance record by the teacher and personal communications between teachers and students. These data were integrated as explanatory variables: sex (boy or girl), calendar period of junior high school graduation , household economic status , family structure (living with mother and father or living with either or neither parent), nationality (Japanese or other), total days of tardy arrival in junior high school during the 3-year period , total days of absence in junior high school during the 3-year period , smoking status at 9th grade (daily smoker or non-daily smoker), experience of severe problems (such as abuse and neglect), and achieved academic level (level of entrance examination difficulty of the high school the student attended was used as a proxy variable for the student’s achieved academic level). Experience of severe problems was collected according to student’s records from areal case-conference by municipality or NGOs. Severe problems included child abuse, neglect, bullying, poverty, and risky behaviors, such as arson, violence, and robbery. Most cases had many problems concurrently, such as economic problems, aggravated interpersonal relations, mental and physical illness, and trouble in school, family, and life. Once a student was recognized as a case (for case-conference) if his/her comprehensive problems were complex, classifying his/her problem to one reason was not appropriate. However, the most critical reason (based on the conference consensus) was recorded for the dataset. Thus, experience of severe problems was not classified by each specific reason but just by the most important one and then dichotomized (0 or 1).The target population of the study were all students in a junior high school located in a northern part of Nishinari ward, Osaka, Japan.18,19 Tardy arrival was defined as arrival at the school gate after 8:30 am. Achieved academic level was defined using the Japanese high school “deviation value” system.20 Japanese high schools are highly stratified, and the within-stratum rank of students was highly correlated with their academic performance.21 A trial exam is given as a guide to make it easier for students to decide on which schools’ test to take when matriculating on to high school; this shows “deviation value”. Four categories for the value  were used. The information was collected for students who graduated junior high school between 2002 and 2010 and was used as baseline data for the longitudinal assessment.The standard definition of public assistance in Japan is available elsewhere.22 To increase the response rate for the follow-up survey, questionnaires for the survey were sent with a letter that students had written to their 3-year older selves (these letters may capture the attention of students). These letters were prepared immediately before junior high school graduation. At every chance during junior high school, students were informed that participation was voluntary and their answers were confidential. When students graduated junior high school, their consent to the follow-up survey was obtained. As no student declined to participate in the study, data from all students who graduated junior high school was analyzed in the study (baseline response rate = 100%). Analysis of the research was approved by the institutional review boards of Osaka City University (2012).Study design with the time course is shown in Figure n = 731) who consented to participate in the study and graduated junior high school between 2002 and 2010, 95.1% (n = 695) attended high school, 3.4% (n = 25) were working (regular employment), and 1.5% (n = 11) were undecided (including part-time employment or students who had failed an entrance examination for high school). The eligible 695 high school students were followed up for evaluation of their high-school dropout .Of students .24,25 Univariable and multivariable analyses were used to document the crude and adjusted relationship between independent explanatory variables and high-school dropout. Characteristics of subjects who participated in the study may differ from those who did not respond at follow-up survey . Log-binomial regression models were used to calculate the prevalence ratios (PRs) and 95% confidence intervals (CIs) for high-school dropout, as the outcome occurred in more than 10% of the sample.p survey . To accoormation . Second,P < 0.05 was considered statistically significant. All analyses were performed using SAS version 9.3 .Probability values for statistical tests were two-tailed, and Characteristics of the study subjects and the high-school dropout rates are shown in Table The correlation matrix among explanatory variables used in this study is shown in Table Results of the univariable and multivariable analyses for high-school dropout are shown in Table Focusing on the three variables which were significantly associated with high-school dropout in the adjusted models, dropout rate according to combinations of total days of tardy arrival, smoking status, and experience of severe problems are shown in Figure 6 Compared with these figures, a considerably higher high-school dropout rate was observed in the current study. However, compared with the figure from high school with the lowest education level (lowest quintile of entrance examination average score) (≥30%)13 as stated in the introduction, the dropout rate in the current study was lower, even in the monotone imputation model (26.6%).The MI model estimated that 18.7% of students dropped out of high school during a period of approximately 3 years in a deprived area in Japan. During the study period (2000–2013), the high-school dropout rates within 1 year among all high-school students (corresponding 10–12th grade in the United States) ranged between 1.5% and 2.6% in Japan.Three factors were significantly associated with high-school dropout consistently in all multivariable models : ≥10 days of tardy arrival in junior high school, daily smoking, and experience of severe problems. According to the numerical values of the PRs . The earlier people start smoking , the greater the harm. Although the smoking rates of junior-high-school students have been rapidly declining in Japan in the past 20 years,29 we need to prevent smoking in junior high school. In the USA 2013 Youth Risk Behavior Survey, the prevalence of current cigarette smoking was 10.2% among ninth graders.30 Previous epidemiologic research suggests that school dropout has been linked to mental health conditions, substance use, chronic health problems, and criminal behavior.16 However, longitudinal studies where smoking evidently preceded school dropout are scarce. In the current study, we found that daily smoking is a leading indicator for future risk of high-school dropout. When we find a student who smokes, we should not only support him or her to stop smoking but should also treat smoking as a sign of increased risk of future school dropout and, therefore, of the need to provide preventive care for his/her school life. This speculation may be supported by previous studies that found that smoking was associated with other indicators of academic problems, such as boredom or lack of connection to school.31–33As for smoking, the observed rate of daily smoking in junior-high-school students in this study  was much higher than that of a previous study among representative Japanese junior-high-school students,Regarding experience of severe problems, the experience included various types of social issues  and was significantly associated with high-school dropout. This is unsurprising, as these events often reflect various difficult aspects of the student’s socioeconomic condition. However, the adjusted PR of the experience was slightly lower than that of daily smoking (1.66 vs 2.01 in the MI model). This may indicate that daily smoking should be treated in the same way as experience of severe problems.34,35 In the current study, academic level was not significantly associated with dropout after covariates-adjustment, consistent with a previous study using tardy arrival as a covariate.15 Regardless of academic achievement levels, for example, students who smoke and have a rebellious attitude 36 may defy school rules and teachers, so their school life may not be successful. Results highlight that these three determinants, which correlate with each other, may comprise a complex, maladaptive behavioral problem. Accounting for multiple risk factors and their interactions may be necessary to develop prevention programs.Adolescence is a period of human development marked by the co-occurrence of multiple risk taking behaviors, such as smoking. To date, in many previous studies, academic achievement level has been found to be associated with school dropout.37 Unmeasured factors may have biased the results. For example, information on the educational attainments of students’ parents was not available in the study. Finally, more research is needed to understand the socioeconomic, familial, and other school-related characteristics of adolescents. Despite these limitations, this study has the strengths of a prospective design, which allows examination of a detailed picture of high-school dropout, and the use of multiple covariate adjustments.There are several limitations to this study. The study was conducted in a specific geographic area, a socially deprived environment in Japan, limiting the generalizability of the findings. The estimated dropout rate may be an overestimate, especially for the monotone imputation model. Some participants were lost to follow-up or withdrew consent; however, the statistical analysis was robust to missing data.38 Three factors were statistically significant determinants of high-school dropout: ≥10 days of tardy arrival in junior high school, daily smoking, and experience of severe problems. These factors significantly correlate with each other. They should be treated as warning signs of complex behavioral and academic problems. Parents, educators, and policy makers should work together to implement effective strategies to prevent school dropout.High-school dropout will have a lifelong impact of lower income, difficulty finding employment, poorer health, and higher incarceration rates.


This article describes the discovery and use of the South American cinchona bark and its main therapeutic (and toxic) alkaloids, quinine and quinidine. Since the introduction of cinchona to Europe in the 17th century, it played a role in treating emperors and peasants and was central to colonialism and wars. Over those 400 years, the medical use of cinchona alkaloids has evolved from bark extracts to chemical synthesis and controlled clinical trials. At the present time, the use of quinine and quinidine has declined, to a large extent due to their toxicity. However, quinine is still being prescribed in resource-limited settings, in severe malaria, and in pregnant women, and quinidine made a limited comeback in the treatment of several cardiac and neurological syndromes. In addition, the article presents more recent studies which improved our understanding of cinchona alkaloids’ pharmacology. The knowledge gained through these studies will hopefully lead to a wider use of these drugs in precision medicine and to design of new generation, safer quinine and quinidine derivatives. In 1928, C.J.S. Thompson described in the British Medical Journal a “picturesque story” of the discovery of the cinchona bark qualities. The story, according to this paper, was about an Indian who became stricken with fever and drank the water of the lake into which a cinchona tree has fallen, fell asleep, and awoke to find that his fever was cured ,4.The European story of the discovery of the tree involves the Spanish Countess of Cinchón, the wife of the Viceroy of Peru, who, in the 1630s, was ill with intermittent fever in the palace of Lima. When the news on her fever reached Don Francisco Lopez de Canizares, the Corregidor of Loxa (who had himself been cured of fever by means of the same drug), he provided a packet of the powdered bark to the countess’s physician, Dr. Juan de Vega. Upon treatment, the countess rapidly recovered and ordered the preparation and distribution of a large amount of the powder to those suffering from fever, and brought it back with her when she returned to Europe, in 1640. The preparation thus became known as the “countess’s powder” ,5,6.Although today malaria is considered a disease of tropical regions, in the 17th century it was common in many parts of Europe, killing peasants, kings and popes. The term Malaria itself was Italian, describing “bad air”  that was believed to cause certain types of fever ,8.It was the English apothecarian Robert Talbor who provoked public awareness of cinchona, in the late 17th century. Robert Talbor exploited the fears of the public and used cinchona as a secret remedy, not disclosing that the central ingredient was the Peruvian bark. He successfully treated the King of England Charles II and the young son of Louis XIV using the mysterious powder . Louis Xyara chucchu cara chucchu. Yara means tree, cara the bark, chucchu cold of the fever, so to say the tree of intermittent fever” [In 1735, the Academy of Science of Paris decided to send a delegation that included the botanist Joseph de Jussieu to South America to define the curve and shape of the earth and to study the plants of South America. Joseph de Jussieu named the Cinchona tree in French “l’arbre des Fièvres” (the Fever Tree) . He wrott fever” . Unfortut fever” . In 1742Cinchona calisaya  and efficacy of the treatment was not clear [During World War 1, quinine was the only antimalarial drug available. The allied armies obtained their quinine supply from cinchona plantations in the Asian Dutch colonies, whereas naval blockade led to shortages of quinine among the German and the Austro-Hungarian forces . The proot clear ,21. In 1Quinine acts against the asexual erythrocytic forms of the four species of human malaria parasites. It is not active against liver stage parasites . Ronald The effect of quinidine on cardiac function was first reported in 1914 by Wenckebach , who wasQuinidine is a class IA antiarrhythmic which primarily blocks sodium channels but is also a broad-spectrum potassium channel blocker B. As a cQuinine has been used as a medicine before 1938, when the US Food, Drug, and Cosmetic Act was enacted. Therefore, it was not initially regulated by the US Food and Drug Administration (FDA). Quinine has a low therapeutic index, with a total oral daily dose for adults of about 2–8 g. The target plasma concentration of unbound quinine for treating malaria is roughly 0.8 µg/mL  . At fullQuinine can additionally cause severe immune-mediated systemic reactions . A systeThe most common adverse effect during quinidine therapy is diarrhea, usually occurring within the first several days of quinidine therapy. Diarrhea-induced hypokalemia may potentiate the arrhythmia torsades de pointes due to quinidine. Similar to quinine, quinidine therapy has been associated with immunological reactions, including thrombocytopenia, hepatitis, bone marrow depression, and lupus syndrome. These effects may occur at therapeutic plasma quinidine concentrations. At elevated plasma levels, quinidine also can produce cinchonism, which can be managed by dose reduction .Quinidine is more cardiotoxic than quinine. Marked QT interval prolongation and torsades de pointes may occur in 2–8% of patients treated with quinidine. High plasma concentrations of quinidine achieved by very high doses formerly used to try to convert atrial fibrillation to normal rhythm have been associated with marked sodium channel blockade and increased risk of ventricular tachycardia. This dosing approach is not being used today, and quinidine-induced ventricular tachycardia is unusual . QuinidiPlasmodium falciparum infections acquired in areas with chloroquine resistance. The treatment is given for seven days for infections acquired in Southeast Asia and for three days for infections acquired in Africa or South America. The combination is aimed to enhance quinine efficacy, shorten the duration of therapy and limit toxicity [P. vivax infections (mainly in Papua New Guinea or Indonesia) may be treated with quinine sulfate plus doxycycline or tetracycline. A combination of quinine sulfate and clindamycin is recommended for pregnant women diagnosed with uncomplicated malaria caused by chloroquine-resistant P. falciparum infection [Today, the treatment of choice for patients with severe malaria is artesunate. However, quinine is used in resource-limited settings and in the treatment of pregnant women ,19,41. Atoxicity ,42. Persnfection . Howevernfection . Since 1Quinine has been prescribed for idiopathic muscular cramps or restless leg syndrome , becauseP. falciparum malaria and in 2006 it removed over-the-counter quinidine products for nocturnal leg cramps from the market [In 2005, the FDA approved quinine for the treatment of uncomplicated e market . The labe market  and a Rie market . Quininee market . Howevere market ,40. Quine market . The maje market . In tradAtrial fibrillation is currently being largely treated by agents more effective or safer than quinidine. Hence, quinidine’s overall use as an antiarrhythmic drug has declined substantially . HoweverA fixed-dose preparation of quinidine sulphate and dextromethorphan hydrobromide (Nuedexta) is the first treatment approved in the United States and in Europe for pseudobulbar affect, a neurological disorder characterized by uncontrollable episodes of exaggerated emotional expression that occurs in adults with neurological damage conditions . In thisN-methyl-d-aspartate and nicotinic α3β4 receptors [In 2015, a phase II randomized controlled study demonstrated that patients with Alzheimer’s disease who received the quinidine/dextromethorphan combination had lower occurrence and severity of agitation than patients who received a placebo . The comeceptors .KCNT1 mutations were discovered in autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) [KCNT1 mutations cause a gain of function in the potassium channel, and in vitro studies in Xenopus oocytes demonstrated that quinidine reverses the increased channel function in a concentration-dependent manner [KCNT1 mutations, although therapeutic effectiveness was inconsistent [KCNT1 gain-of-function appeared to provide a clear target for quinidine. This has motivated a small crossover, add-on trial in which quinidine was given to six adults with ADNFLE carrying KCNT1 mutations [50: 0.9 µM) and its unbound therapeutic plasma concentration–Cu , which predicted effective P-gp inhibition at the human BBB. In that study, quinidine did not completely inhibit BBB P-gp. However, the authors suggested that it has the potential to produce clinically significant CNS drug interactions with other P-gp substrate drugs that have a narrow therapeutic window.Recently, quinidine has been evaluated as a precision therapy for people with seizures associated with mutations of the potassium channel KCNT1. (ADNFLE)  and in m(ADNFLE) . KCNT1 mt manner . Severalnsistent ,60,61,62utations . Unfortuutations . The intutations . This inutations . Quinidiutations  and is putations . Recentlutations . QuinidiThe history of cinchona and its alkaloids provides examples of the role of politics, religion and war in the introduction of medications into the clinic. Cinchona alkaloids, likely recognized hundreds of years ago by the natives of Peru as a toxin, are still being used, both for approved indications and off-label. Besides therapeutic successes, the use of cinchona alkaloids has been associated with life-threatening adverse reactions. Improved labeling and education of prescribers and the public, together with better understanding of the pharmacology of cinchona alkaloids, may minimize such adverse reactions . At the