Medical cannabis formulations with cannabidiol (CBD) and delta‐9‐tetrahydrocannabinol (THC) are widely used to treat epilepsy. We studied the safety and efficacy of two formulations.We prospectively observed 29 subjects (12 to 46 years old) with treatment‐resistant epilepsies  were treated with medical cannabis  for ≥24 weeks. The primary outcome was change in convulsive seizure frequency from the pre‐treatment baseline to the stable optimal dose phase.There were no significant differences during treatment on stable maximal doses for convulsive seizure frequency, seizure duration, postictal duration, or use of rescue medications compared to baseline. No benefits were seen for behavioral disorders or sleep duration; there was a trend for more frequent bowel movements compared to baseline. Ten adverse events occurred in 6/29 patients, all were transient and most unrelated to study medication. No serious adverse events were related to study medication.Our prospective observational study of two high‐CBD/low‐THC formulations found no evidence of efficacy in reducing seizures, seizure duration, postictal duration, or rescue medication use. Behavioral disorders or sleep duration was unchanged. Study medication was generally well tolerated. The doses of CBD used were lower than prior studies. Randomized trials with larger cohorts are needed, but we found no evidence of efficacy for two CBD:THC products in treating epilepsy, sleep, or behavior in our population. Treatment‐resistant epilepsy (TRE) affects 30% of epilepsy patients and can progressively impair neurological function and quality of life, and cause injury and death from sudden unexpected death in epilepsy (SUDEP), and cause other seizure‐related (e.g. drowning), and indirect (e.g. metabolic disorder from medication side effects) consequences. Among young adult TRE patients, epilepsy kills 3%–6% per decade.Cannabis (marijuana) species contain more than 500 compounds, including the cannabinoids Δ‐9‐tetrahydrocannabinol (THC) and cannabidiol (CBD). THC is the main psychoactive compound and CBD is the main non‐psychoactive compound in cannabis. Other cannabinoids and non‐cannabinoid molecules in cannabis have diverse biologic activities.Cannabinoids, terpenes, flavonoids, and other compounds vary by strain and may work independently, antagonistically, or synergistically to produce different beneficial and adverse effects.This prospective observational study was conducted at The Center for Discovery (TCFD), a residentially based program located in upstate New York. IRB approval was obtained to collect and analyze data on patients who were certified for medical cannabis (MC). For patients who lacked intellectual capacity, legal guardians consented. Per the New York State (NYS) Medical Marijuana Program, a registered physician completed the NYS Department of Health (NYSDOH) Medical Marijuana course and registered with the NYSDOH Medical Marijuana Program. Per DOH guidelines, enrolled patients were then certified by the registered physician to receive a NYS‐approved MC formulation . After certification, patients were registered with NYS and designated TCFD as a facility caregiver. The program has a high ratio of clinical staff to patients and routinely collects detailed data on all residents on neurological, medical, and behavioral/lifestyle factors.Twenty‐nine patients were observed over > = 24 weeks  after a 90‐day pre‐intervention baseline phase. Inclusion criteria included enrollment in the residential program, treatment‐resistant childhood‐onset epilepsy, and all of the following: (a) TRE failure to control seizures despite an appropriate trial of two or more anti‐seizure medications (ASMs) at therapeutic doses; (b) video‐EEG characterization of current seizures; and, (c) during pre‐baseline, > = 1 convulsive  seizure per month over a 90‐day consecutive period  (Table Patients failed an average of 10 ASMs. All 29 patients had convulsive seizures. Five patients were treated with a vagus nerve stimulator (VNS): one active and four explanted or inactive. Six patients used the ketogenic diet, and four patients had neurosurgery.Psychiatric medications, if used, were stable for 4 weeks prior to enrollment. The one patient with an active VNS had stable settings for a > = 90 days. Exclusion criteria included any of the following: (a) epilepsies associated with progressive or neurodegenerative diseases ; (b) epilepsies associated with an inborn error of metabolism ; and, (c) felbamate initiated within the past 6 months. Others were excluded if they had extended hospital stays with interruption of MC therapy.The MC certified for all patients was ClaraCeed, a low‐THC/high‐CBD (1 T:20C) and/or ClaraCeed Ultra, (1 T:50C) tablet that, as per protocol, included a maximal dose of 6 mg THC/per day regardless of ratio. Ingredients included: Microcrystalline Cellulose, Dicalcium Phosphate, Silicon Dioxide, Magnesium Stearate, Talc, Sodium Starch Glycolate, Fractionated Coconut Oil MCT. All patients in this analysis were started on 2 mg/day of THC at a low‐THC/high‐CBD preparation of MC ratio of 1 T:20C ratio. In all cases, patients included in the study analysis were increased by 2 mg/day of THC every 7 days up to a maximum dose of 6 mg/day of THC of the 1:20 tablet. Patients were on 3 mg of THC and 60 mg of CBD twice daily on study day 15. They were then observed by their clinical team on the dose determined to provide the best balance of efficacy and tolerability.If the efficacy after full titration of the 1 T:20C was incomplete by week 24, but study medication was well tolerated, patients were moved to a higher CBD:THC ratio, 1 T:50C, and titrated to a maximal dose of 6 mg/day of THC of the 1 T:50C (i.e. 6 mg/day of THC and 300 mg/day of CBD), as tolerated. Safety and tolerability evaluations were conducted at baseline, and every week for the first month or until maximal tolerated dose was achieved, then once a month through the end of the study period. Caregivers and clinical staff routinely document seizure frequency, duration, use of rescue medications, and recovery duration. All data were entered into the patient's record. Patients were also assessed for secondary factors including concomitant ASM levels a minimum of three times during the study period at weeks 1, 12, and 24. The concomitant ASMs remained stable for the first 4 weeks before study onset, except for Felbamate, which was required to be stable for 6 months before initiation. Figure Twenty‐nine patients were observed for a 90‐day pre‐intervention review for seizure frequency of at least one countable (i.e. convulsive) seizure per month. Pre‐intervention data included 90 days of seizure history. Mean monthly convulsive seizure frequency was 16.1 . Pre‐intervention baseline data for all subjects included primary and secondary outcome measures over 90 days before first dose of study medication included: (1) seizure type, (2) seizure frequency, (3) seizure duration, (4) rescue medication use, (5) postictal duration, (6) nightly sleep data, (7) bowel movement data, (8) maladaptive behavior data for patients who with behavioral problems and a behavioral intervention plan, (9) blood pressure, pulse and weight, (10) neurological examination, and (11) complete blood counts and comprehensive metabolic tests.During the study period, seizure type, date, time, duration, intervention use , and postictal duration were documented for each seizure. TCFD routinely collects data on sleep and bowel elimination for each 24‐hour period. All anti‐seizure medications administered including PRNs were recorded in the medical record. For patients with preexisting behavioral intervention plans, daily behavioral data were recorded; behavioral data were not recorded for those without a behavioral intervention plan. Vital signs, physical, and neurological examinations were conducted at each physician visit. In‐person physician visits were held once weekly from the first administration of MC through the achievement of the maximal dose, and bi‐weekly thereafter through a combination of in‐person and telephonic visits.All analyses were completed using the R programming language version 4.0Normality was assessed using histograms and measures of spread . Data were not normally distributed and required nonparametric analyses. Seizure data were analyzed using a Wilcoxon rank sum test due to the small number and non‐normal distribution.This study included three phases: 1) pre‐treatment; (2) titration; and, (3) stabilization on the maximal dose predetermined using a 1 T:20C formula. The pre‐treatment phase included 90‐days before the first dose of the study medication. The titration phase comprised the period from the first administration of the study medication until the maximal tolerated dose was determined. The stabilization on maximal dose phase included the first 90‐days following the maximal dose, unless the patient was removed from treatment before this time. The maximal dose was the most stable dose utilized during the study period on a 1 T:20C formula, that is, the dose of CBD and THC identified as most effective and best tolerated during the titration period ; 17 were female. Patients' epilepsy diagnoses included: 11 ‐ Lennox–Gastaut; 15 ‐ focal or multifocal; and three ‐generalized.The average dose per subject on the maximal dose phase was 5.9 mg THC and 117.2 mg CBD. The average dose per subject in mg/kg per day was 0.11 mg/kg/day for THC and 2.3 mg/kg/day CBD.Primary Outcome: Figure Median change in seizure frequency between baseline and maximal dose period −3 recorded seizures, change in IQR −11. There was no statistically significant difference found in frequency of seizures between the optimal and pre‐intervention phases using a Wilcoxon rank sum test Fig. .A. Median changes in average seizure duration between baseline and maximal dose periods was –2.5 seconds . B. Change in average duration of postictal state between pre‐intervention and maximal dose periods. Median change in postictal state duration was −50.7 seconds .Excluding the four seizure‐free patients, we found no differences in the average duration of seizures Fig.  or postiFigure Figure p = 0.66). Median change in percentage of required rescue medication between the baseline and maximal dose period was −10% (IQR ‐100%–300%).We found no difference in rescue medication use between baseline and maximal dose periods  . There were no significant differences in these groups, although there was a trend for reduced postictal state in the Focal/Multifocal and Generalized Epilepsy groups, as nine individuals had a > =50% decrease and one had a > =50% increase in postictal state.Thirteen anti‐seizure medication doses were reduced in seven patients, including benzodiazepines in six patients. Other reduced ASMs included phenobarbital, felbamate, levetiracetam, perampanel, lamotrigine, and zonisamide. For one patient, reductions occurred during the observation period and six afterwards. There were no increases of ASM during the observational period.Ten adverse events were reported in 6/29 (20.7%) patients, including emesis (n = 2 [6.9%]), gums/nose bleeding (n = 2 [6.9%]), upper respiratory infection (n = 1 [3.4%]), minor upper respiratory distress (n = 1 [3.4%]), fatigue (n = 1 [3.4%]), dizziness (n = 1 [3.4%]), and status epilepticus (n = 1 [3.4%]). Two adverse events caused Emergency Department evaluations, including respiratory distress and status epilepticus. There were 16 serious adverse events, all deemed unrelated to the study medication, in 7/29 (24.1%) patients. Serious adverse events included pneumonia (n = 5 [17.2%]) and ileus (n = 1 [3.4%]) and required hospitalization in two patients. Two patients were removed from the study due to serious adverse events: increased seizure frequency and increased maladaptive behaviors.No adverse or serious adverse events were deemed related to the study drug, and were consistent with patient's histories and diagnoses.p = 0.2). Nine had reduced target behaviors and three had increased target behaviors during maximal dose v. baseline periods.Twelve patients had behavioral disorders before study onset requiring behavioral tracking plans. Median percentage change in target behavioral episodes was −7.3% (IQR –16.1%–6.5%); there was no difference in average behavioral episodes between the maximal dose and pre‐intervention phases . No patient had a ≥50% change in average duration of sleep.There was no significant change in sleep duration between baseline and maximal dose periods using a Wilcoxon rank sum test with p = 0.064 a trend of more frequent bowel movements.There was a marginally significant difference in frequency of bowel movements between the maximal dose and pre‐intervention phases using a Wilcoxon rank sum test with There were no consistent or clinically significant abnormalities in blood cell counts or metabolic tests, including liver function studies, in our patients during the study period.This prospective observational study of a low‐THC/high‐CBD (1 T:20C or 1 T:50C) formulation in diverse TREs revealed that these MC formulations were well tolerated up to a predetermined maximum dose, but did not significantly reduce seizure frequency or duration, or postictal duration when analyzed at the group level. Similarly, at the group level comparing baseline and treatment periods, we found no significant differences in rescue medication use, maladaptive behavioral episodes, or sleep duration, although there was a trend for more frequent bowel movements during treatment. All patients achieved their predetermined maximal doses using a 1 T:20C formula: 27 patients achieved a 6‐mg THC with 120 mg CBD daily dose; two patients achieved a 4‐mg THC with 80 mg CBD daily dose. Although some patients achieved this dose of 1 T:50C beyond the 90‐day analysis period. For most patients on the 1 T:20C formulation, the daily doses of THC were higher than most previous studies, but the dose of CBD was lower, averaging 2.3 mg/kg/day of CBD. By contrast, Epidiolex is approved for Dravet and Lennox–Gastaut Syndromes with target doses of CBD at 10–20 mg/kg/day.There was considerable variability in convulsive seizure frequency, with some patients experiencing >50% increases or decreases during the treatment period. Similarly, seizure duration and postictal durations varied markedly between baseline and treatment periods. We cannot distinguish spontaneous variability from positive or negative effects of MC among individual patients. It is possible that both natural fluctuations as well as heterogeneous effects of MC contributed to the observed variation. Among those with increased seizure frequency or duration, or postictal dysfunction, all returned to baseline in these metrics after MC was reduced or eliminated, therefore, there were no lasting negative impacts from the trial. This observation can support either regression to the mean or reversible adverse effects of MC, or both.Of the 29 patients in the study, seven had their ASMs doses reduced during MC therapy, however, only one experience had ASMs decreased during the observational period while six had decreased after the observational period. No patients required increased ASMs while receiving the MC. The potential benefits of decreased ASMs in this population of highly medically complex patients deserves further study.5 this could reflect a milder effect, which was beneficial in those with constipation.Although there was no significant difference between rescue medication use between pre‐intervention and maximal dose phases at the group level, a third of patients had a ≥50% reduction in use of rescue medications with five of these 18 requiring no rescue medication during the optimal phase. Conversely, four patients who received no rescue medications during baseline required them during the optimal phase, although all had received rescue medications in past. We found no significant difference in sleep duration between the pre‐intervention and optimal phases. There was a trend for increased frequency of bowel movements during MC treatment. Since diarrhea is a potential adverse effect of higher dose CBDAdverse events among the patients on MC during the optimal dose phase was similar to baseline and patient's prior records. Overall, the study drug was well tolerated by most patients. One patient on 6 mg THC and 120 mg CBD daily had increased maladaptive behaviors necessitating removal from the study during the 90‐day observation period. The behaviors returned to baseline after the medication was stopped. These behaviors may result from THC since the CBD dose was relatively low and was rarely associated with adverse behavioral changes even at much higher doses. Another patient had increased seizure frequency on MC leading to discontinuation. Seizure frequency for this individual returned to baseline after MC was stopped. Unrelated to study drug toleration, four patients were removed before the completing the observation period because of unrelated hospitalizations. In summary, six patients were removed before completing the 90 days on the maximal dose.This study was limited by lack of blinding and small sample size. However, the study benefited from unusually structured and near‐continuous observation at TCFD before and during the study period, with detailed assessments of seizure activity as well as behavior, sleep, and other measures. Although this study was not powered to identify small but statistically significant reductions in seizure frequency or other measures, we found no trend for reduced seizure frequency or duration, suggesting that a larger sample may not yield a statistically significant difference. Two randomized controlled trials of CBD and the closely related CBDV for focal epilepsy reported was equivalent to placebo.Our data suggest that THC at doses of up to 6 mg daily did not reduce seizure frequency or severity in our patients. There are an infinite number of ratios and targeted doses for THC and CBD, but it may be worthwhile assessing higher ratios of CBD:THC  in populations with well‐defined epilepsy syndromes. The trend for reduced postictal duration deserves further study. Few investigations assess postictal duration, which may be a marker of seizure severity and long‐term cumulative effects of recurrent seizures on brain function.Overall, the MC formulations were well tolerated but had no significant reductions in seizure frequency, duration, postictal time, or recue medication use at the group level. Ten individuals continued on a‐1 T:50C formulation, with six experiencing a decrease in their ASMs after the study period, which supports potential benefits for a subset of our population. Given the widespread use of MC formulations to treat epilepsy patients with both CBD and THC in state dispensaries, our findings suggest that larger, randomized controlled studies should be conducted to more fully assess their safety and efficacy. Further, our study, one of the first to assess postictal state duration, rescue medication use, and behavioral changes should be replicated as these factors can significantly affect quality of life and be determining factors for use of MC or other therapies in TRE patients.This study was limited by lack of blinding, heterogeneous patient population and epilepsy syndromes, small sample size, and predetermined maximal dose ratios of THC:CBD.Persons responsible for the planning of the study, data collection and analysis, and manuscript preparation include Orrin Devinsky, MD; Theresa Hamlin, Ed.D; Philip Wilken, MD, MS; Angelica Marmanillo, BSN, RN; Daniel Ryan, M.Ed; and, Conor Anderson B.Eng. George J. Todd, MD, FACS served as the oversight medical advisor for TCFD. Daniel Friedman, MD provided supplemental analytics support and edited the manual.This study was funded in part by Columbia Care, Inc. who produced the study drug used in this observational study. They were not involved in any aspect of the treatment protocols, data collection, or interpretation of results. They were selected by a team of physicians and clinicians based on the consistency and efficacy of their product over time. None of the investigators received an honorarium from Columbia Care.Table S1 Percentage Change in Rescue Medication: Total and percentage change in rescue medication administered between pre‐intervention and optimal dose period, n = 18.Click here for additional data file.Table S2 ASM Reduction in Dosage: Of the 29 patients in the study, seven patients decreased their dosages of anti‐seizure medications while receiving MC: one decreased during the 6 months allotted for the analysis, and six decreased after.Click here for additional data file.


Since the human papillomavirus (HPV) vaccine was introduced in Norway in 2009, the vaccine uptake has increased. Whether this increase is similar regardless of the girls’ country background is unknown. We examined changes in HPV vaccine uptake from 2009 to 2014 and studied the impact of parental education and income on HPV vaccine uptake according to country background.Girls in the first six birth cohorts (1997–2002) eligible for HPV vaccination were identified through the National Registry. Information on HPV vaccination, country background and socioeconomic factors was extracted from the Norwegian Immunisation Registry and Statistics Norway. Risk differences (RDs) and confidence intervals (CIs) were estimated with linear binomial regression. A total of 177,387 girls were included in the study.The HPV vaccine uptake increased from 72.5% in 2009 to 87.3% in 2014. The uptake increased for girls in all country background categories. Highest vaccine uptake was observed in girls with East−/South-East Asian background, 88.9% versus 82.5% in the total population. Vaccine uptake decreased slightly with increasing parental education, RD = − 1.6% (95% CI: − 2.3% to − 0.8%) for highest compared with lowest education level. In contrast, the uptake increased with increasing household income, RD = 4.9%  for highest compared with lowest quintile. Parental education had largest impact in girls with Asian background, RD = − 8.1% (95% CI − 10.5% to − 5.6%) for higher vs lower education. The largest impact of household income was observed in girls with background from Middle East/Africa, RD for a 200,000 NOK increase in income was 2.1% (95% CI 1.2 to 3.0%).The HPV vaccine uptake differed with country background but increased over time in all country background categories. Moreover, the impact of education and income on vaccine uptake differed with country background.The online version contains supplementary material available at 10.1186/s12889-021-10877-8. Infection with human papillomavirus (HPV) is the most common sexually transmitted infection. HPV-infection is a necessary cause of cervical cancer. In addition, there is evidence linking HPV-infection with cancers of the anus, vulva, vagina, penis, and oropharynx . Since 2In a previous study, we reported socioeconomic differences in HPV vaccine uptake among girls in Norway . The proSince the HPV vaccine was introduced in the NCIP, the coverage has increased . HoweverThe study is based on national registries. Information on dates of birth, immigration, emigration, and death was obtained from the National Registry. Information on HPV vaccination was obtained from the Norwegian Immunisation Registry. Information on maternal and paternal education level, household income in 2011, country of birth of the study participants and their parents, number of siblings, and county of residence was extracted from Statistics Norway. Data from the different registries were linked using the unique identification number assigned to all residents of Norway. The current study was approved by The Norwegian Regional Committee for Medical and Health Research Ethics, “Ref 2012/1619/REK Sør-Øst”.n = 189,828  were identified through the National Registry, 997–2002 All girls were offered three doses of HPV vaccine. The outcome variable ‘HPV vaccine uptake’ was defined as receipt of at least one dose of the HPV vaccine, as of June 19, 2015.The main exposure was ‘Programme year’. Each programme year corresponds to an academic year running from 20 August to 20 June the following year . During ‘Country background’ was defined as “Norwegian” if at least one parent was born in Norway. Otherwise, country background was defined as the girl’s country of birth if she was foreign born, or as her mother’s country of birth if she was born in Norway. Country background was categorised as: ‘Norway’, ‘Western Europe’, ‘Central- and Eastern Europe (including previous Soviet republics)’, ‘Middle East and North Africa’, ‘South-Asia’, ‘East−/South-East Asia’, ‘Sub-Saharan Africa’, and ‘America and Oceania’ see  for coun‘Parental education’ was defined as maternal education level. If information on maternal education was missing, paternal education was used. Parental education was categorised into four categories: ‘Primary/ lower secondary school’ (≤ 10 years of schooling); ‘Upper secondary school’ (11–14 years of schooling); ‘Higher education, undergraduate level’ (14–17 years of schooling); and ‘Higher education, graduate level’ (≥ 18 years of schooling). We also used a two-category version of ‘Parental education’ with the categories lower education (≤ 14 years) and higher education (> 14 years).‘Household income’, the household total gross income, was divided into quintiles. For all birth cohorts, we used household income from 2011. This was the most recent information on household income that was available in our data. Thus, for the youngest birth cohorts, we did not have information on household income from the year before vaccination. Moreover, the proportion of girls with missing household income was substantially lower for 2011 than for household income from the previous years, due to possible non-residency prior to 2011.‘Number of siblings’ was categorised as 0, 1, 2, 3, and ≥ 4. ‘Maternal age at time of daughter’s birth’ was categorised into age categories ≤ 25, 26–30, 31–35, and > 35 years. ‘Region of residence’ was defined as Oslo, Eastern-Norway, Southern-Norway, Western-Norway, Mid-Norway, and Northern-Norway.We modelled the association between the main exposures  and the outcome variable (initiation of HPV vaccination), using linear binomial regression to estimate risk differences (RDs) and corresponding 95% confidence intervals (CIs). The multivariable model also included number of siblings, maternal age at daughter’s birth, and region of residence. These factors were considered potential confounders since they are associated with HPV vaccine uptake and with parental education and household income. The difference in earnings by educational level is smaller in Norway than in other high-income countries . This isChanges in vaccine uptake over time by country background (with four categories) was examined by including interaction terms between programme year (continuous) and country background. Programme year was treated as a continuous variable in this analysis in order to limit the number of parameters in the model. The impact of parental education and household income by country background was assessed by including interaction terms between country background and parental education (higher compared to lower), and between country background and household income (continuous). In these analyses, we used a Poisson model with robust standard deviation to estimate RDs because the linear binomial model failed to converge . In thesp <  0.05 was considered statistically significant. Statistical analyses were performed using STATA/SE 15.0 .All tests were two-sided, and Of the 177,387 girls included in the study, the majority had Norway as country background (89.5%)  girls initiated HPV vaccination  and 5.4% (95% CI: 4.3 to 6.6%), respectively Table . Girls wInitiation of HPV vaccination decreased slightly with increasing parental education. Compared to girls with parents in the category primary school/compulsory, girls with parents in the categories ‘higher education, undergraduate level’ and ‘higher education, graduate level’ were significantly less likely to initiate HPV vaccination, with multivariable RDs − 0.8% (95% CI: − 1.4% to − 0.3%) and − 1.6% (95% CI: − 2.3% to − 0.8%), respectively . The increase in HPV vaccine uptake was significantly higher for girls with Norwegian background than for girls with other country backgrounds , the multivariable RD corresponding to increase in HPV vaccine uptake per year was 2.7% (95% CI: 2.6 to 2.8%) for Norway, 1.0% (95% CI: 0.4 to 1.6%) for Europe/America/Oceania, 1.3% (95% CI: 0.7 to 1.8%) for Middle East/Africa, and 1.6% (95% CI: 1.2 to 2.1%) for Asia . Higher parental education was significantly associated with lower vaccine uptake among girls in all country background categories. However, the association was weak among girls with Norwegian background, RD was − 0.6% (95% CI: − 1.0% to − 0.2%). The association was stronger for girls with country background Asia or Europe/America/Oceania, RDs were − 8.1% (95% CI: − 10.5% to − 5.6%) and − 7.8% (95% CI: − 9.9% to − 5.6%), respectively. For girls with Middle Eastern and African background the RD was − 2.7% (95% CI: − 5.3 to − 0.02).Girls with Norwegian background were more likely to have parents with higher education 46.0%) than girls with background from Europe, America, and Oceania (39.3%), Middle East and Africa (17.2%), and Asia (19.5%) (Table 6.0% thanp <  0.001). There was a significant association between household income and initiation of HPV vaccination among girls with Norway and Middle East/Africa as country background. However, the association was weak among girls with Norwegian background, the RD for a 200,000 NOK increase in income was 0.2% (95% CI: 0.1 to 0.4%). The association was stronger for girls with Middle East/Africa as country background, RD = 2.1% (95% CI: 1.2 to 3.0%). Household income was not significantly associated with initiation of HPV vaccination for girls with Asia or Europe/America/Oceania as country background.Median household income (in NOK) was 930,136 for girls with Norwegian background, 685,123 for girls with European, American, and Oceanian background, 534,160 for girls with Middle Eastern and African background, and 698,839 for girls with Asian background , thereby limiting selection bias. Another strength of using national registries, is that our study does not rely on self-reported data, which contributes to high quality and less misclassifications of outcomes and exposures. Notification to the Norwegian Immunisation Registry is mandatory for all vaccinations provided within the childhood immunisation programme. Moreover, vaccinations received abroad are also notifiable to the immunisation registry . Thus, tStill, the present study also has limitations. With such a large dataset, we might detect significant results for clinically insignificant differences. Thus, results should be interpreted with caution. In order to detect a difference of 3 percentage points between the lowest income quintile and any other quintile with 90% power, we would need only 3519 individuals in each income quintile (10% of the number of girls in our study), if we assume that the vaccine uptake is 80% in the lowest income quintile and that the association between income and vaccine uptake is positive. Moreover, we would need 112,193 girls with Norwegian background and 1571 girls with East−/South-East Asian background (71% of the number of girls in our study) to detect a difference in vaccine uptake of 3 percentage points between these two groups with 90% power, if we assume that the vaccine uptake is 83% among girls with Norwegian background, that the ratio between the number of girls with Norwegian background and East/South-East Asian background is the same as in our study, and that the vaccine uptake is higher among girls with East−/South-East Asian background. For the last programme year (2014) there may be some delay in registration of vaccination in the immunisation registry  which maIn summary, the present study found inequalities in the uptake of the HPV vaccine related to both country background and socioeconomic factors in the publicly funded school-based programme in Norway.Our findings are encouraging; girls in all country background categories experienced a higher HPV vaccine uptake in 2014 than in 2009. Nevertheless, the vaccine uptake still differs with country background. Our observations indicate that education and income are important predictors of disparities in HPV vaccine uptake. Moreover, these factors differ with country backgrounds.Additional file 1: Supplementary table. Countries in each category of country background.


In this article, we examine and discuss the current situation for noncommercial clinical trials in Europe—two years after a new legal framework entered into force. The Clinical Trials Directive, issued in 2001 .,16.15,16


Previous studies have shown that ingroup/outgroup membership influences individual’s fairness considerations. However, it is not clear yet how group membership influences brain activity when a recipient evaluates the fairness of asset distribution. In this study, subjects participated as recipients in an Ultimatum Game with alleged members of both an experimentally induced ingroup and outgroup. They either received extremely unequal, moderately unequal, or equal offers from proposers while electroencephalogram was recorded. Behavioral results showed that the acceptance rates for unequal offers were higher when interacting with ingroup partners than with outgroup partners. Analyses of event related potentials revealed that proposers’ group membership modulated offer evaluation at earlier processing stages. Feedback-related negativity was more negative for extremely and moderately unequal offers compared to equal offers in the ingroup interaction whereas it did not show differential responses to different offers in the outgroup interaction. Analyses of event related oscillations revealed that the theta power (4–6 Hz) was larger for moderately unequal offers than equal offers in the ingroup interaction whereas it did not show differential responses to different offers in the outgroup interaction. Thus, early mechanisms of fairness evaluation are strongly modulated by the ingroup/outgroup membership of the interaction partner. Fairness considerations, i.e. comparisons of self-interest and other-interest, are a strong motivational force in social interactions223A social factor which seems to influence such fairness considerations is the group membership of the interaction partners. A growing body of studies have explored the group bias in fairness norm enforcement, but these results are inconsistent7891013We aimed to further address this issue by analyzing potential modulatory influences of experimentally induced group membership on recipient’s brain activity in the UG, as measured by event-related potentials (ERP) and event-related oscillations (ERO) in the electroencephalogram. We experimentally induced ingroup and outgroup status and compared subjects’ electrocortical responses to extremely unequal, moderately unequal and equal offers as they interacted with ingroup or outgroup proposers in the UG. Behaviorally, the acceptance rates should decrease with the fairness level of the offers. In addition, as prior research has been broadly consistent with the Social Identity Theory, we predicted that acceptances rates should be higher when interacting with ingroup than with outgroup members.In our ERP analyses, we focused on two components which reflect different stages of outcome-processing, the feedback related negativity (FRN) and the P3. The FRN is typically observed as a negative deflection in response to unfavorable compared to favorable action outcomes, peaking between 250–350 ms at frontocentral recording sites16151819202122212627The P3 is another ERP component that has frequently been found to be related to various aspects of outcome evaluation. It typically peaks about 250–500 ms post-stimulus onset and has a centro-parietal maximum. Some studies found that the P3 is sensitive to the magnitude of reward, with more positive amplitudes to a larger than to a smaller reward1629302223When analyzing ERP components of interest , their temporal overlap is often a troublesome problem. Principal components analysis (PCA) is a data reduction technique that can help to disentangle overlapping ERPs by providing a small set of statistically derived components which can explain the observed data323434In sum, the current study aims to examine the influence of proposers’ ingroup/outgroup membership on recipients’ fairness considerations in the UG as indexed by acceptance/rejection rates, ERP (FRN and P3) responses and ERO (theta band) responses.F = 122.11, p < 0.001, ηp2 = 0.89, with higher acceptance rates for offer 5:5  than for offer 3:7  and offer 1:9 , and for offer 3:7 than offer 1:9 (p < 0.001). The main effect of group membership was also significant, F = 13.34, p = 0.002, ηp2 = 0.47, with higher acceptance rates in intragroup interaction  relative to intergroup interaction . Importantly, the interaction between group membership and offer type was also significant, F = 6.21, p = 0.006, ηp2 = 0.29. Simple-effect tests showed that acceptance rates to offer 1:9 were significantly higher when interacting with ingroup proposers  compared to outgroup proposers , F = 5.22, p = 0.037. In addition, the acceptance rates for offer 3:7 were significantly higher when interacting with ingroup  than with outgroup proposers , F = 10.14, p = 0.006. For offer 5:5 there was no significant difference between proposer types, F = 0.04, p = 0.841.The acceptance rates for different offers are presented in F = 33.90, p < 0.001, ηp2 = 0.69, with longer reaction times to offer 3:7  than to offer 5:5  and offer 1:9 , and to offer 1:9 than offer 5:5 (p < 0.001). Moreover, the interaction between group membership and offer type was also significant, F = 7.60, p = 0.002, ηp2 = 0.37. Simple-effect tests showed that the reaction times to offer 3:7 was significantly higher when interacting with outgroup  compared to ingroup proposers , F = 7.26, p = 0.017. There was no significant difference between the two proposers for offer 5:5, F = 2.69, p = 0.122, nor for offer 1:9, F = 2.29, p = 0.151.The reaction times for different offers are presented in γ = 0.00, t = 0.72, p = 0.485, odds ratio = 1.00) or 3:7  did not change across time when interacting with ingroup members. When interacting with outgroup members, participants more often accepted offer 3:7  with increasing trial count, but their reaction to offer 1:9  did not change over time.Accordingly we analyzed changes in acceptance rates over time, we used the HLM6 software32F = 6.41, p = 0.005, ηp2 = 0.30, suggesting that the PCA-FRN was more negative for offer 1:9  and offer 3:7  than for offer 5:5 , ps < 0.05. PCA-FRN responses to offer 3:7 and offer 1:9 did not differ, p > 0.05. The interaction between group membership and offer type was significant, F = 5.48, p = 0.009, ηp2 = 0.27, Post-hoc analysis indicated that the different offers from outgroup proposers did not influence the amplitude of the PCA-FRN, F = 0.02, p = 0.978. The PCA-FRN did show differential responses to different offers from ingroup proposers, with offer 1:9  and offer 3:7  eliciting more negative amplitudes than offer 5:5 , ps < 0.01. Offer-wise contrasts between the ingroup and outgroup condition revealed no significant effects for offer 5:5, F = 3.63, p = 0.076, and offer 3:7, F = 2.66, p = 0.124. In contrast, for offer 1:9 we observed a significant difference between the ingroup and outgroup condition, F = 10.73, p = 0.005, with the PCA-FRN being more negative when interacting with ingroup  compared to outgroup proposers .The main effect of offer type was significant, t-tests. For the difference amplitudes for 1:9 minus 5:5, we found the difference amplitudes were more negative when interacting with ingroup proposers  than with outgroup proposers , t(15) = −3.15, p = 0.007. For the difference amplitudes for 3:7 minus 5:5, there was no significant difference between two interactive conditions, t(15) = −2.07, p = 0.056.In addition, we calculated the difference amplitudes between offer types (e.g. difference amplitudes for 1:9 minus 5:5 and for 3:7 minus 5:5), and then respectively performed a paired-samples F = 12.81, p < 0.001, ηp2 = 0.46, such that the PCA-P3 were more positive for offer 5:5  than for offer 3:7  and offer 1:9 , ps < 0.05. PCA-P3 responses to offer 3:7 and offer 1:9 did not differ, p = 0.609. The interaction between group membership and offer type was significant, F = 3.34, p = 0.049, ηp2 = 0.18. Post-hoc analysis showed that the PCA-P3 showed differential responses to different offers from ingroup proposers, F = 16.91, p < 0.001, with offer 5:5  eliciting more positive PCA-P3 amplitudes than offer 3:7  and offer 1:9 , ps < 0.01. Offer type also influenced the P3 responses when interacting with outgroup proposers, F = 4.54, p = 0.019, with offer 5:5  eliciting more positive PCA-P3 than offer 3:7 , p = 0.038. We did not observe any difference between the ingroup and outgroup condition when analyzing the offer types separately, all p > 0.154.The main effect of offer type was significant, t-tests. For the difference amplitudes for 1:9 minus 5:5, we found the difference amplitudes were more negative when interacting with ingroup proposers  than outgroup proposers , t(15) = −2.62, p = 0.019. For the difference amplitudes for 3:7 minus 5:5, there was no significant difference between two interactive conditions, t(15) = −1.85, p = 0.084.Moreover, we calculated the difference amplitudes between offer types (e.g. difference amplitudes for 1:9 minus 5:5 and for 3:7 minus 5:5), and then respectively performed a paired-samples F = 3.15, p = 0.027, ηp2 = 0.17. A further simple test found the theta power differentially responded to different offers from ingroup proposers over electrode Fz, F = 5.29, p = 0.011, with offer 3:7  eliciting larger theta power than offer 5:5 , p = 0.041. In contrast, we observed no significant theta power modulation by offer type in the outgroup condition over five electrodes, all p > 0.206. When directly comparing the ingroup and outgroup condition separately for each offer over five electrodes, group membership did affect theta power for 5:5 offers at electrode Fz, F = 4.91, p = 0.043, and FCz, F = 5.37, p = 0.035. At these two electrodes, theta power was larger when interacting with outgroup proposers  than with ingroup proposers . However, group membership did not have a significant effect for offer 1:9 and offer 3:7 over five electrodes, all p > 0.244.In the study presented, we aimed to investigate how this factor affects neurophysiological correlates of fairness considerations in the prominent UG. We found that participants were more likely to accept unequal offers from ingroup compared to outgroup members. In line with previous studies, this result supports the Social Identity Theory, suggesting that people are more tolerant to social norm violating behaviors executed by ingroup members. Moreover, group membership can modulate participants’ reaction to unequal offers over time. Participants’ reactions to unequal offers did not change across time in the ingroup interaction, while they do accept more unequal offer from outgroup members over time. In a repeated game, one would expect that reactions to unfairness change over time, based on partner’s behavior, and participants’ expectations would supersede the group conformity norm. However, the stable reaction to unequal offer from ingroup members indicates that participants might follow the group conformity norm in all interactions in order to maintaining and stabilizing the ingroup integrity. In contrast, the increasing tolerance to unequal offers from outgroup over time might support the expectation norm. As the interaction proceeds, participants might feel that the moderately unequal offers are exceeding their initial expectations about outgroup members, and consider these moderately unequal offers as acceptable during the context of group competition. So, these results indicates participants may adopt the group conformity norm towards ingroup members, and the expectations norm to outgroup members in our study.Our results indicate that ingroup/outgroup membership has a substantial influence on earlier and later processes of fairness evaluation as reflected by the well-known FRN and P3, respectively. Probably most strikingly, the differentiation between fair and unfair offers in the FRN amplitude was most pronounced when offers were made by an ingroup proposer. In contrast, this effect was much smaller and statistically insignificant when offers were made by an outgroup proposer. This modulatory effect is likely to be driven by group-based fairness expectations. Previous studies have repeatedly shown that the FRN is sensitive to expectancy violations, with FRN amplitude being especially high  for events that are worse than expected27317et al. reported a FRN by offer modulation for friends but not for strangerset al. found a more negative FRN for unfair compared to fair offers made by strangers, whereas this effect was reversed for offers made by friendset al. may be attributed to the participants’ more automatic responses to the violation of social norms associated with friendship. The third alternative possibility is related to the difference of anonymity. Wu et al. used two friends (and two strangers) to pair with one participant such that participants were uncertain about the identity of their partners. In contrast, Campanhã et al. used only one friend (and one stranger) to pair with one participant, making it clear with whom the participant was interacting. Although extensive research has demonstrated that anonymity is a significant factor influencing human behavior, a recent research found that degree of anonymity did not modulate the FRN patterns in response to unfairnessOur FRN results may also be interpreted in the light of two previous ERP studies which have shown that the FRN differentiation between fair and unfair proposals also differs, depending on whether proposer and receiver are friends or strangers21Besides disadvantageous unequal offers (i.e. offer 1:9 or 3:7) some researchers have also investigated the role of advantageous unequal offers (i.e. Offer 9:1 or 7:3). While chimpanzees show no evidence of sensitivity to advantageous inequalityIn addition, we also observed a P3 modulation by group membership. In detail and similar to our FRN results, the differentiation between offer types in the P3 amplitude was more pronounced when offers were made by ingroup compared to outgroup proposers. Thus, it may be inferred that also later mechanisms of fairness evaluation of others’ behavior are more strongly engaged when interacting with an ingroup member. In particular, previous research has indicated that P3 amplitude is closely linked to the motivational salience of a certain event27Moreover, we also observed a theta band power modulation by group membership. In detail and similar to our FRN results, the differentiation between offer types in the theta band power was more pronounced when offers were made by ingroup compared to outgroup proposers. Previous research has indicated that theta oscillations may  underlie the FRN effect in the EEG time-domain and reflect the activity of performance monitoring system in face of uncertainty36Altogether, our findings show that ingroup/outgroup membership strongly influences early as well as later neural mechanisms of fairness evaluation in the UG. In particular, these mechanisms appear to be much more engaged when interacting with a member of one’s own social group. Presumably, the observed effects, especially for the FRN and theta power, are mainly driven by norm-based expectations (and the violation thereof) with regard to social-rule conformity. Interestingly, when comparing behavioral and EEG results our study also points to a principle of “grin and bear it” in interpersonal bargaining. That is, while unfair offers might be quickly evaluated as negative when made by an ingroup compared to an outgroup member, individuals appear to overcome this negative evaluation, accepting unfair offers by ingroup members more often, maybe for the sake of group integrity. It is up to future research to uncover the cognitive-affective mechanisms involved in this group securing process.Sixteen undergraduate and graduate students  without prior history of neurological or psychiatric dysfunction completed the experiment. All of them were right-handed and had normal or corrected-to-normal vision. Informed written consent was obtained from all participants prior to the experiment. The experimental protocol was approved by the local Ethics Committee , and was in compliance with the ethical procedures of the American Psychological Association (APA).All the participants were paid 20 Chinese yuan (about $ 3) as basic payment and were informed that additional monetary rewards would be paid according to their performance in the task, although in the end all participants were paid extra 10 yuan on top of the basic payment. Four graduate students , who were strangers to the EEG participants, were recruited as confederates. To exclude possible influence of sex on fairness consideration, each participant was grouped with a pair of same sex strangers, who played the role of proposers in the UG.After arriving at the laboratory, each participant was asked to complete a picture selection task in which he/she took one of two photographs (upside down) of same sex confederates. Before taking the photograph, participants were informed that the two confederates had been assigned randomly either to the red group or the blue group, and that he/she and the selected confederate would belong to the same group. In other words, the selected confederate would be the ingroup member, while the unselected confederate would be the outgroup member for each participant. The in-group category (i.e. the red or blue group) was counterbalanced between subjects. After completing this task, the subject met the two same sex confederates and all of them were asked to stand against the wall and a picture of each person was taken using a digital camera. Finally, each of them was asked to wear a badge of the respective color.The participant and the two confederates were then told that they would sit in separate rooms to finish a task together through a computer network, and that the participant would play as a recipient and the others would be proposers. He/She was also informed about the rules of the UG and that he/she would interact respectively with the ingroup member and outgroup member in two separate UG blocks. Moreover, the subject was told that in order to reduce the difficulty and the range of offers, proposers have to make a choice from 1:9 offer , 3:7 offer or 5:5 offer. Participant was asked to press a button with the index finger of his/her left or right hand to accept or reject the offer, respectively. Finally, given that group affiliation is often strongest in contexts involving competition between groups, the participant was also informed that the experimenter would determine which group is victorious according to the average earnings of each group after all interactions. This manipulation was included as an attempt to further differentiate feelings toward ingroup and outgroup partners, and was just a verbal feedback that did not lead to any punishment or reward.At the beginning of each block, the subject was informed whether the proposer was an ingroup or outgroup member. Each trial started with the presentation of a fixation cross for 800 ms, followed by a black screen for 400–600 ms. see  AfterwarThe participant was seated comfortably about 1 m in front of a computer screen. The UG comprised two blocks (ingroup vs. outgroup) of 90 trials each. Block-order was counterbalanced between subjects. Unknown to the participants, each of the three offer types was presented 30 times per block in a pseudo-randomized order with the restriction that no more than 3 consecutive trials were of the same offer type. A practice block of 10 trials was administered before the formal test, in which the proposer was alternating between ingroup and outgroup member.We measured brain electric activity from 64 channels with the averaged bilateral mastoid reference and a forehead-ground, using a modified 10–20 system electrode cap (Neuroscan Inc.). The vertical EOG activity was recorded with electrodes placed above and below the left eye, and horizontal EOG activity was recorded with electrodes placed at the outer canthi of both eyes. All electrode sites were cleaned with alcohol and inter-electrode impedance was maintained below 10 kΩ. Bio-signals were amplified at a band-pass from 0.05 to 100 Hz and continuously sampled at 1000 Hz/channel. Eye blink artifacts were removed automatically using Scan software (Neruoscan Inc.). Data were segmented into offer-locked epochs of 1000 ms with a 200 ms pre-stimulus baseline window. All segments with EEG voltages exceeding ±100 μV were excluded from further analysis. Each offer condition contained at least 26 trials. After averaging, the mean amplitude of the baseline time window was subtracted from each data-point. The FRN and P3 were quantified using temporospatial PCA following the two-step procedure. PCA was conducted with the EP Toolkit (v2.45)33w has a Gaussian distribution in the time (σt) and frequency (σf) domains around the center frequency f0. All analyses were performed using custom in-house routines written using EEGLAB running under MATLAB. The epoching of continuous EEG files and artifact-related processing was performed with Scan software (Neruoscan Inc.). Single-trial epochs were extracted from −1000 to 2000 ms relative to stimulus, and the raw EEG was down-sampled to 500 Hz. A Morlet-based wavelet transform procedure as implemented in EEGLAB (v13.4.4b) was employed (3 through 35 Hz) in order to provide a continuous estimate of the power of a given frequency between −1000 and 2000 msTime-frequency analysis was performed using a complex Morlet wavelet transform as implemented by the EEGLAB toolbox (v13.4.4b) for MATLAB. The wavelet transform is a multiresolution analysis technique that provides a good compromise between time and frequency resolution2)]. Based on the previous studies on theta frequency and the results of permutation test implemented in the statcond function of EEGLAB toolbox, ERSPs in the range of 4–6 Hz during 200–350 ms were averaged for further statistical analysis. For statistical analyses, we focused on the midline electrode sites Fz, FCz, Cz, CPz, and Pz. The theta band power participated to repeated measures ANOVAs with three within-participant factors: group membership (ingroup vs. outgroup), offer type (offer 1:9 vs. offer 3:7 vs. offer 5:5), and electrode . Only significant effects were reported. P- values of all main and interaction effects were corrected using the Greenhouse-Geisser method for repeated-measures effects. The Bonferroni correction was used for multiple comparisons. Because the topographic distributions of power exhibited a frontal peak, the ERSP results at Fz was only illustrated.Event-related spectral perturbations (ERSP) were computed on the wavelet-transformed epochs for each condition at each time point and wavelet frequency to yield time-frequency mapsHow to cite this article: Wang, Y. et al. Ingroup/outgroup membership modulates fairness consideration: neural signatures from ERPs and EEG oscillations. Sci. Rep.7, 39827; doi: 10.1038/srep39827 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


BP) is an autoimmune disease characterized by tense blisters that are usually preceded by urticarial eruptions. Affected patients exhibit IgG and/or IgE autoantibodies against BP180 and/or BP230. Their relative importance in disease pathogenesis has not been fully elucidated.Bullous pemphigoid  and skin (n = 33) from patients with BP were analysed via enzyme‐linked immunosorbent assay (ELISA) and immunofluorescence, respectively.Sera  very often present with pruritus and hives.Circulating disease‐specific IgE is characteristic of patients with BP and correlates with urticarial lesions.What does this study add?This study demonstrates that IgE is found not only in the serum, but also in the perilesional skin of patients with BP, colocalizing with mast cells, eosinophils and BP180 fragments.Antigenic peptides of BP180 are capable of cross‐linking FcεRI‐bound IgE, ultimately resulting in degranulation.What is the translational message?IgE‐mediated events likely influence the onset and development of BP symptoms.Uncovering an IgE‐dependent disease pathway might impact therapeutic options and reduce the burden of steroids in this aged population.Br J Dermatol 2017; 177:1481–1482Ujiie. .Linked Comment: Plain language summary available onlineRespond to this article Bullous pemphigoid (BP) is a disease that typically occurs in elderly patients, clinically characterized by tense blisters that may follow an urticarial or erythematous phase. The detection of self‐reactive IgG in 1967Despite IgG and complement being hallmarks of BP, their sole action does not easily explain all the characteristics of the disease. For example, animal models using BP180‐specific IgG are characterized by a neutrophilic infiltrate,in vivo models of BP supposedly mimic the human disease more closely – with both blister formation and eosinophilic infiltrates – than those dependent on IgG and complement.In 1974, it was first reported that patients with BP display elevated IgE in the serum.The location of IgE in BP skin has been a matter of controversy, with positive staining for IgE deposits being described along the BMZIn this study, we wanted to address this issue by taking a closer look at these BP‐specific IgE autoantibodies, investigating their antigenic specificity, cell and tissue distribution, cellular and molecular partners, as well as their functionality.n = 19) and healthy donors (n = 18) were collected from subjects who voluntarily signed an informed consent form. Skin biopsies from patients with BP (n = 33) and those with non‐BP skin conditions (n = 8) were collected for routine diagnostic purposes. Non‐BP controls comprised patients diagnosed with linear IgA disease (n = 1), vasculitis (n = 1), urticaria (n = 1), epidermolysis bullosa acquisita (n = 2), pemphigus vulgaris (n = 2) and dermatitis herpetiformis (n = 1). Normal human skin (n = 5) was obtained from patients undergoing abdominoplastic surgery.Sera from patients with BP  and conducted in accordance with the Declaration of Helsinki.NC16A and BP230 peptide‐covered enzyme‐linked immunosorbent assay (ELISA) plates were obtained commercially  and adapted for IgE detection. Details of the detection antibodies used can be found in Data Overlapping peptides spanning the NC16A and C‐terminal domains of BP180 t‐tests were employed for comparing means between two groups at a time. A P‐value of < 0·05 was considered statistically significant.Graphics and statistical analyses were performed using Prism 5 . Student's In line with previous literature, we found that patients with BP have significantly higher levels of circulating anti‐BP180 and anti‐BP230 IgE than healthy controls (HCs) , which are likewise known to express FcεRI.By DIF, a large percentage of patients with BP 22 of 33) presented with IgE+ cells in perilesional skin .Click here for additional data file.Fig S1. Verification of the specificity of the α‐IgE antibody.Click here for additional data file.Fig S2. Patients with bullous pemphigoid (BP) have elevated levels of total circulating IgE.Click here for additional data file.Fig S3. No significant competition, between IgG and IgE, for BP180‐NC16A can be detected by enzyme‐linked immunosorbent assay.Click here for additional data file.Fig S4. Tissue IgE does not appear to correlate with total or specific circulating IgE.Click here for additional data file.Fig S5. Rare case of a patient with bullous pemphigoid with linear deposits of IgE in direct immunofluorescence and indirect immunofluorescence.Click here for additional data file.Fig S6. Eosinophils from patients with bullous pemphigoid express FcεRI.Click here for additional data file.Fig S7. Bullous pemphigoid (BP) perilesional and non‐BP disease skin have significantly more eosinophils than healthy skin; in contrast, all skin samples exhibit similar amounts of mast cells and BP180 dermal fragments.Click here for additional data file.Fig S8. IgG autoantibodies shift from a balanced distribution in serum to an IgG4 dominant profile in skin.Click here for additional data file.Fig S9. BP180 is found along the basement membrane zone and in the dermis.Click here for additional data file.Fig S10. No significant differences are found between cohorts of healthy controls and patients with bullous pemphigoid for the degranulation of basophils.Click here for additional data file.Video S1 Author video.Click here for additional data file.


One of the most reported neural features of autism spectrum disorder (ASD) is the alteration of multiple long-range white matter fiber tracts, as assessed by diffusion-weighted imaging and indexed by reduced fractional anisotropy (FA). Recent methodological advances, however, have shown that this same pattern of reduced FA may be an artifact resulting from excessive head motion and poorer data quality and that aberrant structural connectivity in children with ASD is confined to the right inferior longitudinal fasciculus (ILF). This study aimed at replicating the observation of reduced FA along the right ILF in ASD, while controlling for group differences in head motion and data quality. In addition, we explored associations between reduced FA in the right ILF and quantitative ASD characteristics, and the involvement of the right ILF in visual processing, which is known to be altered in ASD.Global probabilistic tractography was performed on diffusion-weighted imaging data of 17 adolescent boys with ASD and 17 typically developing boys, matched for age, performance IQ, handedness, and data quality. Four tasks were administered to measure various aspects of visual information processing, together with questionnaires assessing ASD characteristics. Group differences were examined and the neural data were integrated with previously published findings using Bayesian statistics to quantify evidence for replication and to pool data and thus increase statistical power.  correlations were calculated to investigate associations between measures.The ASD group showed consistently reduced FA only in the right ILF and slower performance on the visual search task. Bayesian statistics pooling data across studies confirmed that group differences in FA were confined to the right ILF only, with the evidence for altered FA in the left ILF being indecisive. Lower FA in the right ILF tended to covary with slower visual search and a more fragmented part-oriented processing style. Individual differences in FA of the right ILF were not reliably associated with the severity of ASD traits after controlling for clinical status.Our findings support the growing evidence for reduced FA along a specific fiber tract in ASD, the right ILF.The online version of this article (10.1186/s13229-018-0188-6) contains supplementary material, which is available to authorized users. Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairments in social reciprocity and communication, combined with restricted, repetitive and stereotyped patterns of behavior, interests or activities (RRBIs) . AtypicaHere, we investigated structural brain connectivity in ASD using diffusion-weighted imaging (DWI). This non-invasive magnetic resonance imaging (MRI) technique indirectly assesses the structural properties and orientation of white matter tracts based on the diffusion of water molecules . TypicalPrevious studies have observed widespread reductions of FA in individuals with ASD and have interpreted these findings as indicative of generally reduced white matter connectivity in ASD , 9. A meRecent findings, however, revealed that differences in diffusion properties may be an artifact resulting from excessive head motion and poorer DWI data quality , 15. IndThe ILF is a white matter association tract, extending from the occipital cortex into the anterior temporal lobe . The rigThe overall aim of this study was threefold. First of all, we aimed to replicate the findings of Koldewyn and colleagues , obtaineT-scores above 65 on the Social Responsiveness Scale (SRS) ), because our samples have equal size. An effect size ranging from 0.2 to 0.3 is considered small, values around 0.5 are medium, and values of 0.8 or above are considered large effects [All reported  effects .equality-of-effect-size Bayes factor quantifies the evidence for the null hypothesis that the effect sizes in our study equal the effect sizes in the study of Koldewyn and colleagues [replication Bayes factor quantifies the evidence that the data provide for the hypothesis that the effect that we found in our replication attempt is consistent with the effect found in the original study of Koldewyn and colleagues [fixed-effect meta-analysis Bayes factor, in which we pooled the data from our study and the study of Koldewyn and colleagues [p < 0.05). We therefore lack power to detect more subtle group differences with an effect size smaller than 0.69. However, by pooling the data from both studies and by calculating the fixed-effect meta-analysis Bayes factor, we can quantify the combined evidence for the presence or absence of an effect for each of the 18 fiber tracts. For all Bayes factors (BFs), values in between 3 and 1/3 indicate that the data are ambiguous, making the outcome indecisive.Finally, to quantify the success or failure of our attempt to replicate the findings of Koldewyn and colleagues  concernilleagues , versus lleagues , versus lleagues . This islleagues . Thirdlylleagues . This faStatistics for the DWI data quality measures average volume-by-volume translation, average volume-by-volume rotation, percentage of slices with signal drop-out, and average signal drop-out severity are displayed in Table Given our aim to replicate the findings of Koldewyn and colleagues , we primp > .10), with the exception of significantly increased RD in ASD in the right ILF  = 4.54, p = .041, d = .73).Concerning AD, RD, and MD, a one-way ANOVA comparing ASD vs. TD revealed similar values for both groups on each of the 18 tracts  = 5.09, p = .031, d = − 0.77), marginally significantly reduced FA in ASD in left ILF  = 4.07, p = .052, d = − 0.69), and increased RD in ASD in right ILF  = 6.61, p = .015, d = 0.88), with all other measures similar for both groups (p > .10). Second, we calculated weighted DWI averages by weighting each DWI measure at each voxel in the tract by the pathway probability at that voxel. This analysis revealed reduced FA in ASD in right ILF  =.4.58, p = .040, d = − 0.73) and increased RD in ASD in right ILF  = 4.49, p = .042, d = 0.72), with all other measures similar for both groups (p > .098). Third, we reanalyzed the data of FA of right ILF after excluding two outlying TD subjects  = 12.40, p = .0014, d = − 1.25), which survives Bonferroni multiple comparison correction for the 18 assessed tracts (α = .05/18). Fourth, we reanalyzed the data of FA of the right ILF after excluding the four left-handed subjects, as handedness may reflect lateralization of language, which in turn may impact upon lateralization of face-sensitive areas [F = 6.11, p = .019, d = − 0.90).Four supplementary analyses were performed on the DWI statistics. First, we calculated the DWI statistics only for the center of each tract, revealing reduced FA in ASD in right ILF .As expected, individuals with ASD scored significantly higher on each of the questionnaires assessing ASD characteristics, both for impairments in social communication and interaction and for RRBIs , although the effect size was medium to large (d = 0.72). No significant group differences were found on the other visual processing measures   = − 0.27, p = .15; SRS Social Communication and Interaction scale: partial r(29) = − 0.26, p = .17; SRS RRBI scale: partial r(29) = − 0.18, p = .34), none of these correlations were significant, and the association only approached significance for the RBS-R questionnaire  = − 0.37, p = .05). Pertaining to the visual measures, lower FA in the right ILF showed a marginally significant association with slower visual search (r(29) = − 0.34, p = .059) and a more part-oriented processing style as indexed by the fragmentation score on the ROCF (r(30) = − 0.34, p = .05). However, these results did not survive Bonferroni correction (α = 0.006). No association was observed with coherent motion sensitivity (r(30) = − 0.23, p = .22) or performance on the Fragmented Object Outlines task (r(30) = − 0.23, p = .21). Due to the small sample size, none of these correlations sustained in the separate participant groups, except for associations in the ASD sample between lower FA in right ILF and a more fragmented processing style (ROCF: r(15) = − 0.50, p = .04) and a trend towards more RRBIs on the RBS-R questionnaire (r(14) = − 0.46, p = .07). In the TD group, the association with ASD characteristics was substantial but not significant (SRS: r(13) = −.42, p = .12). None of the 17 other white matter tracts showed an association with quantitative ASD characteristics or visual processing measures, except for two tracts: lower FA in the left and right cingulate-cingulum gyrus (CCG) bundle was associated with slower performance on the Fragmented Objects Outline task (r(30) = − 0.43, p = .01 and r(30) = − 0.35, p = .04, respectively) and with slower visual search (r(29) = − 0.45, p = .009 and r(29) = − 0.34, p = .049, respectively) . Furthermore, the Bayes factor tests provided strong evidence that we replicated the findings of Koldewyn and colleagues [Similar to the findings of Koldewyn and colleagues , we founlleagues  and thatlleagues –52. Togelleagues , this sud = − 0.72) in the ASD group. However, this group difference was less consistently observed across different analysis approaches, and—as it was not a priori predicted—it did not survive correction for multiple comparisons. A closer look at the data of Koldewyn and colleagues [d = − 0.34). The replication Bayes factor also indicated that the observed lower FA value in the left ILF for the ASD group corresponds to the effect reported by Koldewyn and colleagues [Contrary to previous studies and reviews , 9, 16, selective alterations in one particular white matter tract in ASD, it should be noted that our sample was too small to reliably demonstrate the absence of more subtle group differences in white matter properties. To overcome this limitation, we calculated the fixed-effect meta-analysis Bayes factor on the pooled data from our study and the study of Koldewyn and colleagues [Concerning the issue of lleagues . This BaAdditionally, our findings also illustrate the heterogeneity in white matter properties within the ASD population see Fig. b, indicaThe observed alterations in white matter organization can potentially underlie some of the cognitive characteristics of ASD. In this study, we focused on the role of the right ILF in visual processing. According to one of the dominant theories on visual processing in ASD , individuals with ASD show relatively impaired global integrative processing and are more inclined to process and attend to parts or details . NeverthReduced brain connectivity has been suggested as the biological mechanism underlying atypical visual processing in individuals with ASD . Here, wOne cognitive process in which the right ILF is particularly implicated is face processing, by interconnecting several key brain regions  , 54–57. only the right ILF and no other tracts may display reduced FA in ASD. Likewise, the power of our study was limited to demonstrate reliable associations with visual processing and ASD characteristics, especially at the subgroup level. Therefore, larger studies are needed to examine these associations more thoroughly, including the association with face processing abilities. Second, although both our study and the one of Koldewyn and colleagues [Although this study replicated the previously observed reduction in FA in the right ILF in individuals with ASD , some lilleagues  demonstrlleagues , 59 and lleagues . Third, lleagues ) includeIn this regard, future research should also investigate how heterogeneity at the behavioral level in the ASD population  may relate to heterogeneity at the neural level . Future To conclude, our results replicate the findings of Koldewyn and colleagues  and suppAdditional file 1:Supplementary R-script. The applied R-script to calculated the three Bayes factor tests, to quantify the result of our replication attempt .  and individual differences in quantitative ASD characteristics and visual processing measures (depicted on the Y axis). First row: Associations with (square root transformed) scores on the SRS questionnaire, the SRS Social and Communication subscale, the SRS RRBI subscale, and the RBS-R questionnaire. Second row: Associations with reaction time on the Fragmented Object Outlines task, (log-transformed) percentage coherence threshold on the Coherent Motion Task, (log-transformed) reaction time on the visual search task, and (square root transformed) fragmentation score on the Rey-Osterrieth Complex Figure task. ASD subjects are depicted by empty squares, TD subjects by filled diamonds. (PPTX 130 kb)Scatter plots displaying the association between individual differences in FA in right ILF  correlations between ASD characteristics, visual processing measures and fractional anisotropy (FA) in the white matter tracts. (DOCX 19 kb)


To compare the sodium‐glucose‐cotransporter‐2 (SGLT‐2) inhibitor dapagliflozin with dipeptidyl peptidase‐4 (DPP‐4) inhibitors with regard to risk associations with major adverse cardiovascular (CV) events , hospitalization for heart failure (HHF), atrial fibrillation and severe hypoglycaemia in patients with type 2 diabetes (T2D) in a real‐world setting.All patients with T2D prescribed glucose‐lowering drugs (GLDs) during 2012 to 2015 were identified in nationwide registries in Denmark, Norway and Sweden. Patients were divided into two groups: new users of dapagliflozin and new users of DPP‐4 inhibitors, matched 1:3 by propensity score, calculated by patient characteristics, comorbidities and drug treatment. Cox survival models were used to estimate hazard ratio (HR) per country separately, and a weighted average was calculated.After matching, a total of 40 908 patients with T2D were identified as new users of dapagliflozin (n = 10 227) or a DPP‐4 inhibitor (n = 30 681). The groups were well balanced at baseline; their mean age was 61 years and 23% had CV disease. The mean follow‐up time was 0.95 years, with a total of 38 760 patient‐years. Dapagliflozin was associated with a lower risk of MACE, HHF and all‐cause mortality compared with DPP‐4 inhibitors: HRs 0.79 , 0.62 (95% CI 0.50‐0.77), and 0.59 (95% CI 0.49‐0.72), respectively. Numerically lower, but non‐significant HRs were observed for myocardial infarction (0.91 [95% CI 0.72‐1.16]), stroke (0.79 [95% CI 0.61‐1.03]) and CV mortality (0.76 [95% CI 0.53‐1.08]) Neutral associations with atrial fibrillation and severe hypoglycaemia were observed.Dapagliflozin was associated with lower risks of CV events and all‐cause mortality compared with DPP‐4 inhibitors in a real‐world clinical setting and a broad T2D population. All citizens have a unique personal identification number (person‐ID), which is mandatory for all administrative purposes , thus providing a complete full population medical history. In the present study, we used data from Prescribed Drug Registers, the Cause of Death Registers, and the National Patient Registers covering all hospitalizations with discharge diagnoses and all outpatient hospital visits. Individual patient‐level data from the national registers were linked using the person‐ID. The linked databases were managed separately by the Steno Diabetes Centre Copenhagen, Gentofte, Denmark (Danish database) and Statisticon AB, Uppsala, Sweden (Swedish and Norwegian databases). Anonymized data were used and analyses were performed within each separate country database .2.4The primary analyses used an on‐treatment approach. Patients were observed from index date until index drug discontinuation (defined as the first gap of 6 months between filled prescriptions), death, or December 31, 2015. In addition, intention‐to‐treat analyses were performed also including the follow‐up time after index treatment discontinuation.2.5Outcomes were: MACE, defined by main diagnosis of non‐fatal myocardial infarction or non‐fatal ischaemic/haemorrhagic stroke or CV mortality; HHF, defined by in‐ or outpatient visits with a main diagnosis of heart failure; all‐cause mortality, defined as death from any cause; MACE+, MACE with the addition of unstable angina; and MACE++, MACE with the addition of unstable angina and HHF. Other predefined outcomes were atrial fibrillation and severe hypoglycaemia. Detailed outcome definitions are given in Supporting Information Table 2.6Propensity scores were used to match each patient who initiated dapagliflozin with patients who initiated a DPP‐4 inhibitor . The probability of having a new drug initiation of dapagliflozin was estimated using a logistic regression model with patient characteristics, age, time since first GLD initiation, comorbidity, coronary revascularization, frailty, all separate classes of GLDs, CV disease preventive drugs, drugs associated with treatment of heart failure, and date of both index drug and first‐line initiation as independent variables. Detailed information on variables included in the propensity score are given in Supporting Information Tables 2.7P value was <.05 and the hazard ratio (HR) was <1. Proportional assumptions were tested. Pooled Kaplan–Meier plots from all 3 countries were used for descriptive purposes only.Standardized differences of >10% were used to detect significant group imbalance between baseline variables.33.1During the observation period years 2012 to 2015, 94 064 patients with T2D initiated new therapy with dapagliflozin or a DPP‐4 inhibitor Figure . The dap3.2After matching, a total of 40 908 patients with T2D could be included as new users of either dapagliflozin n = 10 227) or a DPP‐4 inhibitor (n = 30 681). The groups were well balanced at baseline: the mean age was 61 years, 40% were women, 23% had CV disease, 15% microvascular disease and 84% had been prescribed CV disease preventive drugs  and 0.62 (0.50‐0.77), respectively  in the dapagliflozin group compared with the DPP‐4 inhibitor group . The dapagliflozin group showed neutral associations for atrial fibrillation and severe hypoglycaemia compared with DPP‐4 inhibitor: HRs 0.92 (95% CI 0.76‐1.12) and 0.94 (95% CI 0.74‐1.19), respectively.3.5When including time after treatment discontinuation , the analysis showed similar risk associations between the dapagliflozin group and the DPP‐4 inhibitor group  associated lower risk of MACE, a recent meta‐analysis of dapagliflozin treatment in patients with 30% established CV disease at baseline showed a numerically 23% (HR 0.77 [95% CI 0.54‐1.10]) lower risk of MACE compared with placebo.The EMPA‐REG OUTCOME trial showed that empagliflozin reduced the risk of MACE, HHF, and all‐cause mortality compared with placebo by 14%, 35% and 32%, respectively, also similar to canagliflozin in the CANVAS trial.Clinicaltrials.gov: NCT03030235, NCT02653482 and NCT03036124).Heart failure is a both common and frequently underdiagnosed complication in T2D,Interestingly, both atrial fibrillation and severe hypoglycaemia showed no associations between the two treatment groups in the present work. The risk of atrial fibrillation has not, to our knowledge, been reported to show associations with either SGLT‐2 or DPP‐4 inhibitors, and should thus be expected to be neutral. In addition, it is known that both dapagliflozin and DPP‐4 inhibitors have low and similar risks of hypoglycaemia.In contrast to the EMPA‐REG OUTCOME trial, reporting non‐significant higher stroke risk (HR 1.24 [95% CI 0.92‐1.67]), we report a lower risk association for stroke (HR 0.79 [95% CI 0.61‐1.03]) with dapagliflozin compared with DPP‐4 inhibitor treatment. This finding is of particular interest because it is more in line with the CANVAS trial results (HR 0.90 [05% CI 0.711‐1.15]) and considers benefit in stroke rates that could be expected by a mild reduction in blood pressure. The seemingly discrepant findings between our results and those of the EMPA‐REG OUTCOME trial could be attributable to chance and/or presence of a potential unknown confounding factor at baseline; however, the virtually identical baseline variables, similar risk estimates for other outcomes compared with the EMPA‐REG OUTCOME and CANVAS trialsclinicaltrials.gov, NCT01730534), including >17 000 patients with both low and high CV risk, will further elucidate dapagliflozin‐specific findings.Previous observational multi‐country studies have recently shown that parts of the EMPA‐REG OUTCOME and CANVAS trial results do translate to the SGLT‐2 inhibitor class, and into a real‐world setting with patients with T2D having broader CV risk profilesThe strengths of the present study include its population‐based, nationwide and unselected real‐world design, which provides high external validity and a large enough population to allow country‐wise propensity score‐matched analyses to be performed. The results were consistent across all 3 countries and across several subgroup analyses. In addition, national registers with full coverage for hospitalizations, filled drug prescriptions and cause of death were used in 3 countries with established and complete public healthcare systems. Because diagnostic accuracy can be challenging for HHF in registries, it is reassuring that CV diagnoses in Denmark, Norway and Sweden have high validity.Limitations of the study include the fact that the results are only representative of patients who have been initiated on SGLT‐2 inhibitor treatment or who are similar with regard to available clinical variables and, therefore, cannot be extended to all patients with T2D. The present work has no information on laboratory measurements, lifestyle variables, primary healthcare data, or socio‐economic data, and consequently there may be remaining confounding factors. The close matching on a large number of essential variables ensures that some confounding factors are controlled for, but even propensity score matching does not remedy all confounding; for example, residual confounding by indication. Further, there was no information on diabetes duration, therefore, we used a proxy for time since diagnosis by matching for age at index date, time since first registered GLD treatment and classes of GLD at baseline. Because dapagliflozin has only been on the market since 2012 the mean follow‐up was short, ~1 year; however, in the EMPA‐REG OUTCOME study, similar early effects to those of the present study have been observed, suggesting that the 1‐year mean follow‐up time might be sufficient. We did not examine safety, but recent reports have not identified any new safety signals with dapagliflozin.In conclusion, dapagliflozin, when compared with DPP‐4 inhibitor treatment, was associated with a lower risk of MACE, heart failure and all‐cause mortality in a real‐world T2D population, where 23% had previously established CV disease. A large ongoing prospective dapagliflozin trial will further elucidate these findings.Jan W. Erikssonhttp://orcid.org/0000-0002-2639-9481Johan Bodegardhttp://orcid.org/0000-0001-5423-3967Appendix S1 Data sources.Click here for additional data file.Table S1A. Definitions of glucose lowering drugs.Table S1B. Definitions of patient characteristics.Table S1C. International Classification of Diseases [ICD] code 8/9/10 diagnoses and Classification of Surgical Procedures NOMESCO  codes used to define comorbidities and treatments.Table S1D. Prior medications using the ATC  codes.Table S2. Baseline patient characteristics of unmatched type 2 diabetes patients being new users of dapagliflozin versus dipeptidyl peptidase‐4 inhibitor (DPP‐4i).Table S3. Baseline of propensity matched 1:3 type 2 diabetes patients being new users of dapagliflozin versus dipeptidyl peptidase‐4 inhibitor (DPP‐4i).Table S4. Weighted means of hazard ratios (HRs) in Denmark, Norway and Sweden for new users of dapagliflozin versus dipeptidyl peptidase‐4 inhibitor (DPP‐4i) including the follow‐up time after index treatment discontinuation (intention to treat) and separate analysis on inpatient hospitalization for heart failure. The groups were matched 1:3 using propensity scores based on age, sex, frailty  comorbidity and treatmentClick here for additional data file.


Sarcoptes scabiei.The development of alternative approaches in ectoparasite management is currently required. Essential oils have been demonstrated to exhibit fumigant and topical toxicity to a number of arthropods. The aim of the present study was to assess the potential efficacy of ten essential oils against Sarcoptes mites collected from experimentally infected pigs. For contact bioassays, essential oils were diluted with paraffin to get concentrations at 10, 5, and even 1% for the most efficient ones. The mites were inspected under a stereomicroscope 10, 20, 30, 40, 50, 60, 90, 120, 150, and 180min after contact. For fumigation bioassay, a filter paper was treated with 100 μL of the pure essential oil. The mites were inspected under a stereomicroscope for the first 5min, and then every 5min until 1h.The major chemical components of the oils were identified by GC-MS analysis. Contact and fumigation bioassays were performed on Using contact bioassays, 1% clove and palmarosa oil killed all the mites within 20 and 50min, respectively. The oils efficacy order was: clove > palmarosa > geranium > tea tree > lavender > manuka > bitter orange > eucalyptus > Japanese cedar. In fumigation bioassays, the efficacy order was: tea tree > clove > eucalyptus > lavender > palmarosa > geranium > Japanese cedar > bitter orange > manuka. In both bioassays, cade oil showed no activity.S. scabiei infections in humans or animals, as well as to control the mites in the environment.Essential oils, especially tea tree, clove, palmarosa, and eucalyptus oils, are potential complementary or alternative products to treat The online version of this article (doi:10.1186/s13071-016-1889-3) contains supplementary material, which is available to authorized users. Sarcoptes scabiei, a mite of the family Sarcoptidae, causes a contagious pruritic skin disease in humans and in animals. The human infection is called scabies and is estimated to affect more than 130 million people worldwide at any time [S. scabiei is devastating and causes significant morbidity and mortality in wild and domestic mammals. It affects more than 100 species worldwide including companion, livestock, and wild animals and is considered as an emerging problem in many countries [ountries , 3.S. scabiei infection is hindered by the suboptimal efficacy of the few available therapies [S. scabiei) infections is progressively undermined by the development of resistance to acaricides [In humans, the treatment of herapies . In vetearicides . In addiaricides –8. For aaricides  describearicides . They haaricides , 8. In Aaricides .S. scabiei var. suis.The aim of this study was to assess the potential efficacy of 10 chemically-characterized essential oils against The acaricide activity was evaluated for 10 essential oils  with an Agilent 5977A Series GC/MSD System. The ability of the columns to separate compounds depends on their stationary phases. According to this principle and considering the potential composition of the oils, a DB-1 MS column (30 m × 0.25 mm × 0.25 μm) was used to analyze lavender, tea tree, geranium, manuka, and bitter orange oils, whereas an HP-INNOWax column (30 m × 0.25 mm × 0.25 μm) was used to analyze clove, eucalyptus, cade, palmarosa, and Japanese cedar oils. Helium was the carrier gas. The chemical structure of each component was identified by comparing its fragmentation patterns seen in mass spectra with standard library data. The relative percentage of the oil components was calculated from the GC peak areas.Sarcoptes mites were collected from pigs maintained at CRBM , Maisons-Alfort, France. Pigs were experimentally infected as described by Mounsey et al. [y et al. .Inoculation was performed by directly inserting mite-infected skin crusts deep into the ear canals of five-week old female piglets. The cutaneous lesions developed first in the ear then spread to the entire body of the pigs. In the present study, mites were collected 9 and 10 weeks post-experimental infection. Skin crusts of the external ear canal were gently removed and placed in a Petri dish. Mites were picked up with a needle one h after crust collection; giving time to the mites to climb out of the crusts  were placed in a plastic Petri dish (3 cm in diameter). In the first experiment, all the pure essential oils included in the present study were successively tested against mites. The following bioassays were conducted using the essential oils that killed all the mites within 1 h of direct contact. The selected essential oils were diluted with paraffin to get concentrations at 10, 5 and even 1% (for the most efficient ones). In each Petri dish, 1 ml of the diluted solution was added in direct contact with the mites. A control Petri dish was inoculated with 1 ml of paraffin oil. The mites were inspected under a stereomicroscope  10, 20, 30, 40, 50, 60, 90, 120, 150 and 180 min after inoculation. Mites were considered dead when no movement was seen even after touching it with a needle, and no gut movement was observed over 2 min [For each experiment, alive mites of all motile stages . A covering filter paper was put on the lid of the Petri dish and treated with 100 μl of the pure essential oil. A control Petri dish was treated with paraffin oil. All the Petri dishes (tested oils and control) were closed and turned over. The mites stayed firmly attached to the bottom of the Petri dish  of scabies mites were calculated using JMP 12.0 software. The statistical differences between data obtained with each essential oil and the control for each experiment  were measured by Log-rank test expressed by χ2 results and P-values (degree of freedom (df) = 1). P-value of ≤ 0.05 was considered significant.The data were analyzed by Kaplan Meier survival curves and the median lethal times (LTP < 0.0001) except for the contact test of Japanese cedar oil at 5% concentration   and tea tree  oils were specifically examined for the purpose [Syzygium aromaticum), nugmeg (Myristica fragrans), and ylangylang (Cananga odorata) oils, as well as plant extracts from Eupatorium adenophorum, Ailanthus altissima and Ligularia virgaurea [Sarcoptes mites have already been reported, the current study allowed us, for the first time, to compare these two oils together and with other oils. Pasay et al. reported that 1.56% clove oil killed all S. scabiei mites after 15 min in contact bioassay [Dermatophagoides pteronyssinus [To our knowledge, the research work covering the efficacy of essential oils against  purpose –23. Otheirgaurea , 24–26. bioassay , which inyssinus . The letnyssinus , whereasnyssinus , 28. Thinyssinus .Psoroptes mites [S. scabiei. A notable finding to report here is the strong fumigant efficacy the tested oils have on scabies mites. However, the fumigant efficacy order of the oils did not exactly match their contact efficacy order. Similar discrepancies were reported in studies on contact and fumigant effects of essential oils against agricultural pests [50 = 4 ± 0.4) was the most effective, followed by clove (LT50 = 5) and eucalyptus oils (LT50 = 5 ± 0.3)  [Sitophilus oryzae and Tribolium castaneum insects, but was much less effective in contact bioassays. Exactly opposite geraniol which had strong contact toxicity but low fumigant activity [Psoroptes cuniculi, whereas linalyl acetate and estragole were almost ineffective against this parasite [S. scabiei, as demonstrated in the present study, could be attributed to the variation in their percentages in these oils. On the other hand, the discrepancies between contact and fumigation bioassays can also be explained by the mode of absorption of the active ingredient by the mites. In contact bioassays, the toxic compounds directly penetrate the cuticle layer of the arthropod. In fumigation bioassays, the toxic compounds are inhaled via the respiratory system. Therefore, compounds which are lipophilic and viscous might have better performance in contact bioassays, whereas in fumigation bioassays, the vapor pressure may determine the compound efficacy [The fumigant effect of essential oils was studied on stored products pests, head lice, ticks, and es mites , 30–32, al pests , 34. Fores (C10) , and depparasite . Since lefficacy .In this study, the observation time was relatively short (180 min for contact bioassays and 60 min for fumigation bioassays). This is the reason why we believe that using a mixture of instars had a limited effect on our results, although females and nymphs were found surviving longer than males and larvae in the environment . Twenty Sarcoptes mites. For instance, Hu et al. [S. scabiei mites. The same compound was also shown to affect the nervous system of the mite by increasing the activity of monoamine oxidase and inhibiting acetylcholinesterase.New research work has been directed to study the essential oils mechanism of action on u et al.  have recEssential oils have extensively been used as fragrances, aromatherapy and in pharmaceutical industry for centuries . Given tS. scabiei var. suis. Essential oils, especially tea tree, clove (for both fumigant and contact use), eucalyptus (for fumigant use), and palmarosa (for contact use), should be considered as complementary or alternative therapies to chemical acaricides in the treatment of S. scabiei infections, as well as in the environmental control of the mites.The present study demonstrated that several essential oils (except cade oil) have both contact and fumigant effects against