Background: Tuberculosis (TB) surveillance data are crucial to the effectiveness of National TB Control Programs. In South Africa, few surveillance system evaluations have been undertaken to provide a rigorous assessment of the platform from which the national and district health systems draws data to inform programs and policies.Objective: Evaluate the attributes of Eden District’s TB surveillance system, Western Cape Province, South Africa.Methods: Data quality, sensitivity and positive predictive value were assessed using secondary data from 40,033 TB cases entered in Eden District’s ETR.Net from 2007 to 2013, and 79 purposively selected TB Blue Cards (TBCs), a medical patient file and source document for data entered into ETR.Net. Simplicity, flexibility, acceptability, stability and usefulness of the ETR.Net were assessed qualitatively through interviews with TB nurses, information health officers, sub-district and district coordinators involved in the TB surveillance.Results: TB surveillance system stakeholders report that Eden District’s ETR.Net system was simple, acceptable, flexible and stable, and achieves its objective of informing TB control program, policies and activities. Data were less complete in the ETR.Net (66–100%) than in the TBCs (76–100%), and concordant for most variables except pre-treatment smear results, antiretroviral therapy (ART) and treatment outcome. The sensitivity of recorded variables in ETR.Net was 98% for gender, 97% for patient category, 93% for ART, 92% for treatment outcome and 90% for pre-treatment smear grading.Conclusions: Our results reveal that the system provides useful information to guide TB control program activities in Eden District. However, urgent attention is needed to address gaps in clinical recording on the TBC and data capturing into the ETR.Net system. We recommend continuous training and support of TB personnel involved with TB care, management and surveillance on TB data recording into the TBCs and ETR.Net as well as the implementation of a well-structured quality control and assurance system. South Africa has the third highest tuberculosis (TB) incidence rate  in the world . Given tIn South Africa, the Electronic TB register (ETR.Net) surveillance system has been operational since 1995 and provides the National TB Control Program (NTCP) with information on key indicators essential to guide and inform public health action and TB programmatic activities . PrimaryIn line with World Health Organization (WHO) recommendations, it is critical to periodically evaluate TB surveillance systems in order to identify challenges and barriers to complete and accurate data essential to TB management and control activities. To ensure the NTCP’s objectives are being met, we evaluated Eden District’s TB surveillance system using the US Centers for Disease Control and Prevention’s (CDC’s) guidelines for evaluating public health surveillance systems .A quantitative and qualitative evaluation of the Eden District, Western Cape Province, ETR.Net surveillance system was conducted from May to June 2014 and March 2015 using CDC guidelines for evaluating public health surveillance systems as described in Supplementary Table 1 . We perf2 . Key. Key8]. e system .http://www.cdc.gov/epiinfo/) and analyzed using STATA 13.0 . Data quality was evaluated by assessing the completeness, concordance, sensitivity and PPV of reported values in the ETR.Net. Completeness of data was calculated as the total number of values available for each variable divided by the total number of records. Data concordance, a measurement of agreement, between TBCs and ETR.Net was calculated using Cohen’s Kappa (κ) coefficient for categorical variables and intraclass correlation coefficient (ICC) for continuous variables. We interpreted the κ values as follows: 0.81–1 as near perfect agreement, 0.61–0.80 as substantial agreement, 0.41–0.60 as moderate agreement, 0.21–0.40 as fair agreement, <0.21 as poor agreement and −1 as perfect disagreement. An ICC value of >0.75 represents excellent reliability, 0.40–0.75 represents fair to good reliability and <0.40 represents poor reliability. Sensitivity and PPV of variables in the ETR.Net were computed using a diagt command in STATA. Sensitivity was defined as the probability of having a positive value in the ETR.Net given a positive value in the TBC. PPV was defined as the probability of a documented positive value in the TBC given a positive value in the ETR.Net.Data were captured in Epi-Info7  TBC variables had a 95–100% completion rate compared with 9/14 (64%) of ETR.Net variables . Gender,The estimated sensitivity of reported values in the ETR.Net ranged from 90% to 98% for gender, patient category, on ART, treatment outcome and pre-treatment smear grading, and 75–79% for HIV status, pre-treatment smear results and CPT . All varSub-district coordinators noted that they used ETR.Net to generate reports on TB cases, sputum smear conversion and TB treatment outcomes. They also acknowledged other benefits of ETR.Net: ‘As part of HIV and TB service integration, we use the ETR.Net to evaluate and report on basic data relating to HIV status of TB patients and ART initiation of those with co-infection’ (sub-district coordinator).Sub-district and district HIV/AIDS, STIs and TB (HAST) coordinators spoke of how useful the ETR.Net was:Interviewer: ‘Do you think the information contained in the ETR.Net is adequate to help the sub-district and district to manage the performance of TB control program at the health facilities?’District coordinator: ‘Yes, reports generated from the ETR.Net are useful to inform and guide resource allocation at the facilities.’Sub-district coordinator: ‘We can also track the burden of TB at the sub-districts, monitor clinic’s catchment areas with high burden and TB case trends by age, sex HIV status, patient category, health facilities and sub-districts.’Overall, eight of nine (88%) respondents  reportedInterviewer: ‘Do you find it easy to transfer information from a TBCs into a TB register by yourself?’TB nurse: ‘I think it is a lot easier for us to transfer information and in fact information on the TBCs are similar to those contained on the TB register except that there are no detail on treatment doses, clinics note and household contacts on TB register.’At the sub-district level, health information officers and HAST coordinators also spoke of the willingness to use the ETR.Net:Interviewer: ‘Do you find it easy to enter data into the ETR.Net?’Health information officer: ‘When I started using the ETR.Net it wasn’t easy at all but with training and working on the system on a daily basis, it is much easier to enter information on the system and to generate reports for the health facilities.’The TB surveillance system has a simple data flow structure and reporting scheme as illustrated in Health information officers and sub-district coordinators spoke of how they experience interruption and downtimes on the ETR.Net:Interviewer: ‘Were there any unscheduled outage and downtimes in the ETR.Net, where data was not captured, analyzed and submitted to the facilities?’Sub-district coordinator: ‘Yes, at times there were periodic downtimes of 2–6 hours in a week in 2014 where we couldn’t use the system and this was due to the installation of ETR.Net v2.’Health information officer: ‘We had some problems with login and random freeze of the system due updates and software changes.’Prior to the introduction of GeneXpert testing and results capture on ETR.Net, it was not possible to distinguish whether TB patients tested positive using GeneXpert or culture, as during this time GeneXpert results were entered in the culture results section. Since this was not optimal for flexibility, the system was later adapted to accommodate the changing information needs for the revised WHO algorithms for TB diagnosis to include GeneXpert testing. Subsequent to this, in 2014, more improvements were made to ETR.Net to include enhanced security features, locking of records, data migration tool and data exchange with other electronic health information systems . However, despite these improvements, sub-district HAST coordinators expressed concerns that ETR.Net was not networked and lacked unique TB patient IDs.One of the key findings in this evaluation was the perceived usefulness of the TB surveillance system and the high level of acceptability among its stakeholders. Stakeholders were very satisfied with information gathered through the surveillance system and its use for public health program planning, implementation and resource allocation. Given the importance of surveillance data to the monitoring of TB programmatic indicators, it is critical that health facility and TB program staff ensure quality and completeness of surveillance system data. Our findings of usefulness and acceptability of the system in Eden District is encouraging, and demonstrate a direct engagement of stakeholders with TB surveillance system to inform TB programmatic activities and policies. Continuous engagement and support of TB staff is essential to improve the understanding and objectives of TB surveillance, and to identify challenges and barriers critical in the management and control of TB in the district.Our evaluation also showed that the system was simple, stable and flexible. Most TB clinical staff found the surveillance data collection tools easy to use, while TB program managers felt that data collection tools collected appropriate programmatic indicators to inform programs and resource allocation. While there was periodic downtime of the system due to regular updates and random system freezes, stakeholders believed the system was stable and adaptable to changing information needs; changes were made to enhance security features and accommodate the revised WHO algorithms for GeneXpert testing. However, stakeholders also suggested that additional efforts are needed to network the system and to include a unique ID for TB patients. Without a unique patient ID, it is difficult to update patient information of those that move between or transfer clinics, thus impacting patient tracking, planning and allocation of resources in the sub-districts.Data discrepancies were found within the TB surveillance tools. Specifically, date of birth, age, treatment outcomes, baseline CD4 counts and on ART were not adequately completed in the ETR.Net; data completeness for these key variables ranged from 66% to 89%. Sub-optimal data quality can undermine the ability of the NTCP to properly evaluate and plan for TB control program activities in the district, and the utility of the TB surveillance system to inform public health actions and policies. Sound, reliable data quality is also critical to instill confidence of stakeholders in the performance of the surveillance system. We attribute the findings of sub-optimal data quality in Eden to the lack of dedicated sub-district health information officers, inadequate computers, staff training, and standard operating procedures for tracking updates and changes to patient information in the ETR.Net.Furthermore, it is evident that there is a need for TB program managers at the sub-district and district levels to implement structured quality control and assurance processes to improve the integrity of data collected in the TB surveillance system. Continuous training of personnel involved with data quality at different levels of the TB surveillance system is also an important mechanism to engage them on the purpose and uses of the TB surveillance data to achieve the objective of the South African NTCP.Despite this, our findings demonstrated a good sensitivity of reported values in the ETR.Net for gender, patient category, on ART, pre-treatment smear grading and treatment outcomes. In addition, we report good PPV for gender, patient category, treatment outcome, HIV status, pre-treatment smear results and CPT variables ranging from 92% to 99%, indicating that less than 10% of these variables were missing in the ETR.Net. Such findings demonstrate the robustness of the TB surveillance system to capture these key variables which are critical to the performance of the TB surveillance system and control of TB in the district. To our knowledge, this first study to report on the sensitivity and PPV of recorded variables on the district’s South African TB surveillance system. Additional data might be needed to establish whether this finding is generalizable to other districts in the Western Cape Province or in South Africa as whole.This evaluation has several limitations. The scope of the evaluation was limited to Eden District and is not generalizable to other districts in Western Cape Province. Our data review was restricted to specifically to TBCs and ETR.Net, not the processes or procedures associated with the flow and transfer of data from TBCs to the TB register, and finally to ETR.Net. Recent evaluation of TB data sources in South Africa suggest that information is often lost when transferred from TBCs to TB register and from TB register to ETR.Net .Despite its limitations, ETR.Net provides valuable surveillance data and information for Eden District. ERT.Net is simple, acceptable, flexible, stable and useful in achieving the objectives of guiding the district’s TB control program and activities. To strengthen the ETR.Net surveillance system we recommend the following: (i) implementation of a quality assurance mechanism to improve data quality, (ii) continuous training of TB surveillance staff on proper data collection and entry, (iii) ensure that there are dedicate health information officers at sub-district and district levels, (iv) institute a web-based ETR.Net data collection portal to allow direct input at facility-level, and (v) implement a unique TB patient identifier to link information of TB patients who move between or transfer to other health facilities and/or sub-districts.


Objective: To investigate parent reports of childhood symptoms of inattention as a predictor of adolescent academic achievement, taking into account the impact of the child’s intellectual functioning, in two diagnostically and culturally diverse samples.Method: Samples: (a) an all-female sample in the U.S. predominated by youth with ADHD , and (b) a mixed-sex sample recruited from a Norwegian population-based sample . Inattention and intellectual function were assessed via the same measures in the two samples; academic achievement scores during and beyond high school and demographic covariates were country-specific.Results: Childhood inattention predicted subsequent academic achievement in both samples, with a somewhat stronger effect in the BGALS sample, which included a large subgroup of children with ADHD. Intellectual function was another strong predictor, but the effect of early inattention remained statistically significant in both samples when intellectual function was covaried.Conclusion: The effect of early indicators of inattention on future academic success was robust across the two samples. These results support the use of remediation procedures broadly applied. Future longitudinal multicenter studies with pre-planned common inclusion criteria should be performed to increase our understanding of the importance of inattention in primary school children for concurrent and prospective functioning. Academic achievement is an important predictor of later vocational career success and adult financial stability . IdentifSymptoms of inattention, however, are not restricted to a specific diagnostic group. Most patients with neuropsychiatric disorders e.g., , as wellWe note that most previous studies in this area feature teacher ratings of inattention, often to the exclusion of parent ratings of this variable e.g., . In addiItems used to assess inattention are closely related to cognitive/executive functions like flexibility, inhibition, and working memory, and are thus related to broader issues of executive function, as articulated in the classic review of Selection of cognitive measures is, however, challenging. Cognitive function may be defined from a single test or clusters of tests designed to assess the parallel cognitive domains or subfunctions  the Berkeley Girls with ADHD Longitudinal Study (BGALS), comprising girls with an ADHD diagnosis and a matched comparison group who attend or have attended high school see ; and (b)In both samples, parents rated their children’s inattentive behavior according to the nine inattention items from the Swanson, Nolan, and Pelham Questionnaire SNAP-IV, . IntelleSD = 1.7) if they met full ADHD diagnostic criteria for the combined or inattentive types on the parent-administered Diagnostic Interview Schedule for Children  were recruited from schools, mental health centers, pediatric practices, and direct advertisements to participate in summer research programs in the San Francisco Bay Area, California, United States in 1997, 1998, and 1999. After initial screening, participants with ADHD were included at Wave 1 , results on a test of intellectual function performed about 1 year after the parent reports , and an academic achievement score from the School registry when the participants attended high school . To make the sample similar to the BGALS, 11 participants with an IQ score below 70 were excluded, together with one child defined as an outlier (>3 SD) according to casewise statistics. In total, 92 participants are included in the final BCS sample for this report. According to the Kiddie-SADS interview in the clinical phase of the first study wave, diagnosis was confirmed in 27 of the children .The sample for the present study includes participants with parent reports on inattentive behavior from the initial study wave , Western Norway. Parents gave written consent for participation when the children were below 16 years, and the adolescents consented themselves to participate when they were above 16 in accordance with Norwegian regulations.Parents completed the nine relevant items from SNAP-IV  in both Full-scale IQ (FSIQ) was calculated from the WISC-III, scored according to American norms in the BGALS study  and accoThe Wechsler Individual Achievement Test WIAT;  was inclFor the BCS, academic achievement scores were provided by official registers from the Hordaland County for those who consented to the school registry linkage. In Norway, grades are assigned on a scale ranging from 1 to 6, with 6 being the highest grade (outstanding competence). A score of 2 (low level of competence) is the lowest passing grade, and 1 signifies a fail. The scores represent the average of all grades except for gym class. The achievement scores from both BGALS and BCS are calculated as continuous measures.In the BGALS sample, several important background variables were included as covariates: (a) mother’s highest-level education, (b) total household income, (c) ethnicity, and (d) age at baseline, all of which were ascertained via a demographic questionnaire at baseline. In the BCS, covariates were restricted to age at WISC-III testing and biological sex. Among families who answered the question about mother’s education (73.1%), all but two reported at least 12 years of education. Furthermore, all but one of the participants had at least one parent born in Norway, confirming the homogeneous ethnicity of the Norwegian population. Maternal education and ethnicity were therefore not included as covariates when analyzing the BCS data.Data from both studies were analyzed (SPSS version 23/24) according to the same analytic plan, including covariates that were specific to characteristics of the two countries. Group differences  were initially calculated. Hierarchical linear regression analyses with academic achievement as the late-adolescent/early adult outcome measure were computed to investigate the effect of parent-reported inattention during childhood. In all analyses, relevant demographic variables were included in the first step. We then added inattention in the next step to investigate its direct effect on academic achievement. Then, to probe whether an effect of inattention was retained when intellectual function was covaried, intellectual function was included in a second step after demographics, followed by parent reports of inattention in a final step. Standardized change scores were used to reflect the unique contribution from the variables included in the regression models.Table 1. At inclusion, the ADHD group obtained significantly higher parent rated inattention scores than the comparison group , as well as significantly lower scores for FSIQ . The WIAT composite of reading and math achievement obtained during Wave 3 was far lower in the ADHD than in the comparison group . There were no significant group differences for the variables of age, household income, and mother’s education, but the % Caucasian was significantly higher in the ADHD group (p < 0.05).Results are presented in r = -0.353, p < 0.001), (b) parent reports of childhood inattention and academic achievement at Wave 3 , and (c) childhood FSIQ and Wave 3 achievement . When considering the two subsamples separately, only the correlations between FSIQ and academic achievement were statistically significant, in both the ADHD  and comparison subsamples . The entire sample was therefore included in the regression analysis.Bivariate correlation analyses including the full sample showed statistically significant negative correlations between (a) parent reports of childhood inattention and concurrent FSIQ (Table 2) revealed that parent-reported inattention at Wave 1 significantly predicted academic achievement scores in high school (p < 0.001), explaining 10.4% of the variance beyond the covariates . Academic achievement was also significantly predicted by FSIQ (p < 0.001), explaining 36.9% of the variance beyond the covariates. When inattention was included in the final stage after entry of FSIQ, the contribution from inattention remained statistically significant (p < 0.05), bringing the overall explained variance of the model to 52.9%.The regression analyses (see n = 66) than girls (n = 26). Boys and girls obtained similar results on all measures except for parent-reported inattention, which was significantly higher in boys than in girls  (see Table 1). There were 14 boys and 5 girls who reported at least one psychiatric disorder at the late adolescent data collection point. ADHD was reported in only boys (n = 11), all of whom had a comorbid disorder . None of the participants had psychosis, intellectual disability, or known neurological disorders.The BCS sample included a higher number of boys , childhood inattention and the high-school academic achievement score , and childhood FSIQ and the high-school achievement score . In a sex-specific correlation analysis, all correlations retained their statistical significance for boys , but for girls, only the correlation between inattention and academic achievement, r = -0.517, p = 0.007 remained significant. Sex was therefore kept as a covariate in the following regression analyses.The correlations in the full sample were statistically significant for the relation between the childhood inattention and FSIQ scores (Table 2) revealed that parent reports of inattention significantly predicted academic achievement in high school (p = 0.001), explaining 13.8% of the variance beyond the two covariates (age and sex). Introducing intellectual function as a predictor explained 23.8% of academic achievement (p < 0.001), with 18.2% incremental variance explained by FSIQ. Inclusion of parent reports of inattention in the final step, following inclusion of the covariates and intellectual function, increased the explained variance to 30.9%, leaving the contribution from inattention statistically significant (p < 0.05).The linear regression analyses (see p = 0.032) and girls (p = 0.008), while the effect of intellectual function was statistically significant only for boys (p < 0.001).Because the correlational pattern was different for boys and girls, the regression analysis was re-conducted separately for boys and girls with age as the only covariate. Inattention was still statistically significant for boys  and the measures of cognitive function  and inattention were motivated largely by our objective of including parallel measures in both studies. Still, we believe that these selections have given us results of clinical importance. Parent reports are commonly the most available information about a child’s behavior when assessed in a clinical situation; the total score of the included questionnaire on inattention is well-validated; and the full-scale IQ score is commonly available as a general measure of cognitive function. Still, questions may be raised about the specificity of the parent reports, in that parents may find it difficult to contrast inattention with characteristics of other aspects of problematic behavior. Other limitations are related to critical differences in study designs, leading to somewhat different age groups and not quite overlapping time-lines in our longitudinal designs.Inattention is one of the hallmarks of ADHD. The present study shows that features of this behavior, in particular from a parent’s perspective, are of crucial importance in predicting high-school achievement about a decade later—an effect that is not restricted to children within the diagnostic category of ADHD. Furthermore, the time span between parent reports of inattention and assessment of academic achievement was longer than in most previous studies (∼10 years), suggesting that the effects of inattention in primary school may yield consequences into adult life. Inattention tends to lead to a cascade of other problems , includiAL: data collection, design, statistical analysis and main responsibility for the manuscript; JM: data collection, design, statistical analysis and comments on the manuscript; MH: data collection, statistical analysis, comments on the manuscript; SH: data collection, design and main comments on the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cross-sex hormone treatment (CSHT) is an important option for gender dysphoria (GD) individuals to improve the quality of life. However, in Japan, sex reassignment surgery (SRS) and CSHT for GD had been discontinued until 1998 (over 30 years). After resumption, the number of GD individuals wishing treatment rapidly increased. On the other hand, the number of medical institutions available for evaluation was limited. For this reason, hormonal treatment has been administered to GD individuals requiring the prompt start of CSHT in the absence of mental health assessment by specialists. In this study, we examined the efficacy of CSHT and psychotherapy.n = 53, Group no-CSHT: n = 102). In all participants, Minnesota Multiphasic Personality Inventory (MMPI) and blood hormone tests were performed on the initial consultation. In addition, CSHT was combined with psychotherapy for a specific period in Group no-CSHT, and FtM individuals in whom an additional MMPI test could be conducted (Group combined treatment (CT), n = 14) were enrolled in a longitudinal study.The participants were 155 female-to-male (FtM) individuals who consulted our gender identity clinic, and were definitively diagnosed. A cross-sectional study was conducted by dividing them into two groups: groups with and without CSHT on the initial consultation , and was approved at  In Japan, gender dysphoria (GD) refers to a condition in which biological sex is not consistent with self-consciousness or self-recognition of gender . In the On the other hand, formal epidemiologic studies on the incidence and prevalence of transsexualism specifically or transgender and gender nonconforming identities in general have not been conducted, and efforts to achieve realistic estimates are fraught with enormous difficulties , 5. And,In Japan, a survey was performed by the Special Committee for Gender Dysphoria of the Japanese Society of Psychiatry and Neurology in 2013, and the number of GD individuals who consulted a medical institution, complaining of GD, was estimated to be approximately 17,000. This number corresponds to 1/7000 in Japan. It is higher than the prevalence rate described by De Cuyperera . HoweverConcerning medical practice for GD, there are specific circumstances in Japan. In 1969, sex reassignment surgery (SRS) for MtF individuals was evaluated as guilty for the following reasons: evaluation for diagnosis was not performed, and informed consent regarding SRS was not obtained. However, SRS was misunderstood as violating the laws. As a result, not only SRS but also physical treatment, including CSHT, was officially avoided. In 1997, the “Guidelines for the Diagnosis and Treatment of GD” were published by the Japanese Society of Psychiatry and Neurology . CurrentAlthough medical practice for GD, which had been discontinued over approximately 30 years, was resumed, there are few physicians who can evaluate GD and make a definitive diagnosis or such medical institutions. GD individuals had to wait for 6 to 12 months until consultation at a gender identity clinic. To GD individuals requiring the prompt start of CSHT, hormonal treatment has been administered based on gynecologists’ or urologists’ evaluation at local clinics in the absence of assessment by mental health specialists. As a result, GD individuals who consulted our gender identity clinic consisted of those who had not been evaluated and those in whom hormonal treatment was started without evaluation.Recently, several studies in other countries evaluated the effects of hormonal treatment on FtM. The efficacy of CSHT was reviewed by Colizzi and Costa . ConcernThe Minnesota Multiphasic Personality Inventory (MMPI) involves many clinical scales, facilitating many-sided personality assessment. Therefore, it has been used for the psychological assessment of GD –26. SeveIn this study, the effects of assessment-free CSHT were initially examined. Subsequently, the effects of CSHT with evaluation and psychotherapy were investigated. As the number of MtF individuals was small, FtM individuals were adopted as participants. The participants were divided into two groups based on the presence or absence of CSHT, and its effects were evaluated based on the MMPI test results and blood hormone levels.The protocol of this study was approved by the Ethics Review Board of Kansai Medical University. Written informed consent was obtained from all FtM individuals on the initial consultation after explaining that information on medical and psychological examinations may be published in academic journals.Since 1999, Kansai Medical University Medical Center has been responsible for GD treatment. According to the guidelines in Japan, comprehensive treatment has been performed by a medical team consisting of psychiatrists, urologists, gynecologists, plastic surgeons, psychiatric social workers, clinical psychologists, and clerical employees .The participants of this study were 183 FtM individuals who were definitively diagnosed at our gender identity clinic before the end of December 2008. The participants comprised our series. Informed consent regarding study participation was obtained from all participants. For diagnosis, it was confirmed that the participants met the diagnostic criteria for GID presented in the DSM-IV-TR , and thaOf the 183 FtM individuals, we analyzed 155 (mean age: 25.6 ± 5.9 years [15–43 years]), excluding 28 in whom hormone levels were measured before July 1, 2004 because hormone-measuring methods were changed on July 1, 2004. The 155 participants had not undergone assessment or psychotherapy in other hospitals.They were divided into two groups: FtM individuals who had received CSHT before the initial consultation at the gender identity clinic (Group CSHT) and those who had not received it (Group no-CSHT). In addition, in the latter, FtM individuals in whom CSHT was started in combination with psychotherapy (combined treatment) based on the results of evaluation after the start of outpatient care were assigned to Group CT.Of the 155 FtM individuals, 53 comprised Group CSHT (mean age: 26.8 ± 6.0 years [17–43 years]), 102 comprised Group no-CSHT (mean age: 24.9 ± 5.8 years [15–41 years]) , depression (D), hysteria (Hy), psychiatric disease qualitative deviation (Pd), masculinity/femininity (Mf), paranoia (Pa), psychasthenia (Pt), schizophrenia (Sc), hypomania (Ma), and social introversion (Si) scales). Therefore, this test may also be important to obtain suggestions regarding the indication of treatment. Furthermore, the clinical scales include a scale regarding male and female sex , which is characteristic. Since the 1970’s, this scale has been used to evaluate the psychological properties of GD .In addition to the MMPI test, the blood levels of testosterone and estradiol were measured as blood hormone levels on the initial consultation. In addition, in Group CT, the second MMPI test was conducted after performing CSHT for a specific period (519 ± 365 days) in parallel with psychotherapy.To confirm the effects of previous CSHT, the blood levels of testosterone and estradiol on the initial consultation in Group CSHT were compared with those in Group no-CSHT. In addition, to evaluate the relationship between the hormone levels and mental state, we analyzed the relationship between the clinical scales of the MMPI and blood testosterone and estradiol levels in the same groups using Pearson’s test.To evaluate the effects of CSHT, the MMPI clinical scale scores on the initial consultation were compared between Groups CSHT and no-CSHT using the Mann-Whitney U-test.To evaluate the effects of starting CSHT during psychotherapy following assessment on the mental state, the MMPI clinical scale scores after a specific period of CSHT in Group CT were compared with those on the initial consultation using Wilcoxon’s coded rank test.To examine the effects of CSHT with respect to the presence or absence of psychotherapy, the MMPI clinical scale scores on the initial consultation in Group CT were compared with those in Group CSHT using the Mann-Whitney U-test. In addition, the scores after the start of CSHT in Group CT (on the second MMPI test) were compared with those in Group CSHT.Essentially, participants in whom hormonal treatment was started after the initial consultation should be randomly divided into two groups: CT and non-psychotherapy-combined groups, and the results should be compared. However, according to the guidelines in Japan, CSHT cannot be started in the absence of psychotherapy. Therefore, this study involved Group CSHT, in which CSHT had been performed before the initial consultation in the absence of psychotherapy. , and assistance to improve the QOL in a new life).With respect to hormone levels, the blood testosterone level in Group CSHT was significantly higher Fig. , and theThere were no significant differences in any MMPI clinical scale between Groups CSHT and no-CSHT Table . After tWhen comparing the results on the initial consultation in Group CT with those in Group CSHT Table , the D, CSHT is technically easier than surgery, and its cost is relatively inexpensive. For this reason, CSHT for FtM individuals is routinely performed to relieve psychiatric symptoms associated with gender dysphoria-related mental distress and acquire a better mental well-being. Therefore, its effects on the mental state should be particularly evaluated in detail.We initially investigated the blood levels of hormones to evaluate the presence or absence of CSHT, and confirmed that the influence of hormone agents was strongly reflected in Group CSHT. The blood testosterone level was significantly higher Fig. , and thePrevious cross-sectional , 34–37 aSeveral longitudinal studies demonstrated the efficacy of CSHT using the SAS, SDS, SCL-90-R, Structured Clinical Interview for Diagnostic and Statistical Manual of Mental Disorders I (SCID-I) , SCL-90-In this study, there was no significant difference in any MMPI clinical scale score between Groups CSHT and no-CSHT. The results of previous cross-sectional studies suggest the positive effects of CSHT in FtM individuals , 34, 36.In Group CT, improvements in the clinical scale scores other than the Mf scale score were achieved after CSHT. In particular, there were significant improvements in the D, Hy, Pd, and Pt scale scores. In addition, the D, Sc, and Si scale scores on the initial consultation in Group CT were significantly higher than in Group CSHT, but the Pd scale score was significantly lower after treatment. This suggests that CSHT combined with psychotherapy improves the mental health. Although the efficacy of CSHT was confirmed, CSHT-related apparent masculinization may affect the FtM individuals’ relationship with others. Furthermore, the effects of CSHT are marked in FtM individuals, but it may stimulate aggression . The comConcerning the Mf scale, Bonierbale et al. reported that the scale score in the CSHT group was significantly lower than in the no-CSHT group . They inA mental health screening and/or assessment is needed for referral to hormonal treatment for GD , 10. In However, our study has a limitation: when performing CSHT, it was impossible to assign the participants to psychotherapy and psychotherapy-free groups and evaluate the results, because CSHT cannot be started in the absence of psychotherapy in accordance with the guidelines in Japan.We examined the effects of CSHT and psychotherapy in FtM individuals using the MMPI through cross-sectional and longitudinal studies. In the cross-sectional study, we evaluated the results with respect to the presence or absence of CSHT. However, there were no significant differences in the MMPI clinical scale scores. In the longitudinal study, the combination of CSHT and psychotherapy significantly improved the MMPI clinical scale scores. CSHT improve mental health, and, when combined with psychotherapy, further improvements may be achieved.


Rhizobium sullae type strain IS123T nodulating the legume Hedysarum coronarium, was sequenced and resulted in 317 scaffolds for a total assembled size of 7,889,576 bp. Its features were compared with those of genomes from rhizobia representing an increasing gradient of taxonomical distance, from a conspecific isolate (Rhizobium sullae WSM1592), to two congeneric cases (Rhizobium leguminosarum bv. viciae and Rhizobium etli) and up to different genera within the legume-nodulating taxa. The host plant is of agricultural importance, but, unlike the majority of other domesticated plant species, it is able to survive quite well in the wild. Data showed that that the type strain of R. sullae, isolated from a wild host specimen, is endowed with a richer array of symbiotic genes in comparison to other strains, species or genera of rhizobia that were rescued from domesticated plant ecotypes. The analysis revealed that the bacterium by itself is incapable of surviving in the extreme conditions that its host plant can tolerate. When exposed to drought or alkaline condition, the bacterium depends on its host to survive. Data are consistent with the view of the plant phenotype as the primary factor enabling symbiotic nitrogen fixing bacteria to survive in otherwise limiting environments.The prominent feature of rhizobia is their molecular dialogue with plant hosts. Such interaction is enabled by the presence of a series of symbiotic genes encoding for the synthesis and export of signals triggering organogenetic and physiological responses in the plant. The genome of the  We had previously provided the first description of the infection process of this legume by its bacterial symbiont and their morphological peculiarities  as type strain. Phylogenetic analyses  and Rhizobium etli (symbiont of beans).The interaction between rhizobia and legumes in root nodules is an essential element in sustainable agriculture, as this symbiotic association is able to enhance biological fixation of atmospheric nitrogen (NR. sullae type strain IS123T in order to: (1) compare this genome with other members of the order Rhizobiales, including a conspecific isolate , and various genera within the legume-nodulating taxa; (2) determine whether or not the type strain of R. sullae, which comes from a plant that still grows in the wild, carries a richer array of genes for the symbiotic interaction with its host; (3) assess whether or not the traits allowing the host plant to endure extreme soil conditions  are also mirrored by appropriate determinants in the bacterial genome.Focusing, in particular, on the genes ruling the symbiotic association with the host plant, we sequenced the genome of the R. sullae strain IS123T has been previously described as a new species by Squartini et al. , as previously described by Goris et al.   was used to estimate function abundance and evaluate metabolic and physiological potential. The reference database was KEGG (Kyoto Encyclopedia of Genes and Genomes), and R. sullae proteins were mapped and normalized on the ribosomal proteins counts in its pathway database. KAAS (KEGG Automatic Annotation Server) was used for ortholog assignment  and pathway mapping. The PHASTER tool was used to search for sequences of phages and prophages  reads (2 × 100 bp) were obtained, accounting for a total of 9.1 Gb sequence information.R. sullae, R. leguminosarum, and R. etli genomes reported so far. A total of 51 tRNA genes and 6 rRNA (rrn) operons were identified.The draft genome assembly contains 447 contigs with a total assembly size of 7,889,725 bp and an N50 of 72.64 kb, and 317 scaffolds with a total assembly size of 7,889,576 bp and an N50 of 118.24 kb. The longest contig was 296,399 bp, the longest scaffold 488 bp. The whole genome GC content was 59.88% (Table R. sullae IS123T genome are available in the European Bioinformatics Institute (EBI) database under project number PRJEB9435. The genome assembly can be accessed under the code ERZ403196 .The raw reads of the The gene predictions, CDS FASTA files, and the corresponding proteins are all provided as Supplementary Datasets http://enve-omics.ce.gatech.edu/ani/) was calculated to assess the similarity between the assembled genome and the genomes of R. sullae WSM1592 and R. etli CFN42. The analysis revealed a high average ANI (97–98%) between the sequenced genome and the genomes of the two afore-mentioned related species  and symbiotic N2 fixation (nif and fix). The R. sullae type strain genome displayed all the essential nodulation genes, which are required for early symbiotic nodulation via nod factor production  in the nodABC operon on the complementary strand, there is a copy of nodD interrupted by the insertion element ISRh1 , O-acetylation (nodL and nodX), sulfation , and a putative O-carbamoylation (nolO). These features, encoding for precise nod factor decorations, may account for the very tight host specificity of this rhizobium for its symbiont plant H. coronarium.Sequence annotation confirmed the presence of genes involved in nodulation . Among the fix genes, we detected fixL, fixS, and fixC, which potentially encode a symbiotically essential cbb3 high-affinity terminal oxidase, a rate-limiting component for symbiotic N2 fixation : R. sullae WSM1592 (6985), R. leguminosarum bv. viciae 3841 (7281), R. etli CFN42 (5963), Sinorhizobium meliloti 1021 (6217), Mesorhizobium loti MAFF303099 (7283), and Bradyrhizobium japonicum USDA110 (8317).In order to assess the uniqueness of the genes and the level of conservation of those shared across related taxa, a whole genome orthology search was run on six additional rhizobial genomes. Besides Occurrences of the corresponding orthologous genes are shown in the “Supplementary Dataset R. sullae IS123T, its conspecific R. sullae WSM1592 lacks nifQ (the molybdenum donor for nitrogenase synthesis), the second copy of the structural nifH gene for nitrogenase, a copy of the fixN Cytochrome c oxidase subunit, a copy of the fixL oxygen sensor protein, a copy of the fixK nitrogen fixation regulation protein, and the gene for ferredoxin I, an enzyme linked to the electron flux for N2 reduction to nitrogenase.On inspecting all the genes known to correspond to symbiotic traits, , we found that, compared with the type strain R. sullae IS123T but have no orthologs in either of the two species of the same genus and closest to R. sullae according to 16S rRNA taxonomy, namely R. leguminosarum bv. viciae and R. etli, include, in addition to the above: the nodG (fabG) 3-oxoacyl acyl-carrier protein reductase; nodM, encoding functions for efficient Nod signal production; fixJ, the response regulator inducing nif operons in response to microaerobiosis within the nodule tissue; the putative nitrogen fixation protein gene fixT; a copy of the nod factor synthase nodA; a copy of the nodD2 and syrM genes encoding for flavonoid-responsive transcriptional activators; and a copy of the nodU putative carbamoyl transferase.The symbiotic genes that are present in Bradyrhizobium, Mesorhizobium, and Sinorhizobium (Ensifer), revealed the R. sullae IS123T genome to be the one displaying the highest number of genes related to symbiosis, which was 59.Interestingly, the ortholog search across the seven taxa, including the three different genera H. coronarium, as mentioned in the introduction, has remarkable properties of tolerance to several environmental stress factors, such as salinity, drought, and alkaline soil pH, and displays regular root nodulation under such conditions, we inspected the genome of its R. sullae microsymbiont to search for genes which could account for corresponding genotypes on the bacterial side.Since the host plant We identified genes important in maintaining osmoregularity, which included the osmotically inducible protein C. However, in bacteria this gene is reported to be related not to saline conditions, but rather to responses to organic hydroperoxides and reactive oxygen species in general  glucan export ATP-binding/permease protein NdvA, which is responsible for the synthesis of osmoregulated periplasmic glucans. These oligosaccharides are common not only to most rhizobia but to the whole Proteobacteria phylum, in which glucan concentration in the periplasm increases in response to a decrease in environmental osmolarity  is present, but this gene appears to be widespread among bacteria, including y Bohin, . This fiR. sullae, including the present type strain IS123T, compared with other rhizobia  and R. leguminosarum bv. viciae (pea symbiont). The limit was 1% in only two strains, but this was still lower than in S. meliloti , which tolerated values up to 3%. As for alkali tolerance, R. sullae did not perform very differently from the others, and R. sullae IS123T, in particular, was able resist up to pH 8.5, the same level attained by the pea and clover symbionts, and lower than that recorded for the alfalfa symbiont , the other from an agricultural context (WSM1592), and to investigate the subtle ways in which they differ in spite of an overall conserved genome. The host plant is considered to be native to Algeria, Tunisia and Morocco, as well as Spain (http://www.ildis.org/), where it is found essentially in the southernmost region. The presumed origin of the plant species in the north-western African belt above the Saharan Atlas range is supported by the high frequency of sites in which H. coronarium is encountered in natural populations in that area compared with European countries, and by the fact that the related species Hedysarum flexuosum is the only legume nodulated by rhizobia, whose 16S rRNA sequence displays a >99% similarity with the R. sullae type strain IS123T (Aliliche et al., R. sullae is, as far as is currently known, fully symbiotic only with H. coronarium.The genome of R. sullae IS123T was isolated and the transcontinental crossing point.In conclusion, the present study enabled us to verify ecological correspondences between host–plant lifestyles and bacterial symbiont genotypes. In addition to the interest in comparing genomic features of isolates from cultivated vs. wild Sulla, the present genome, a strain collected from a wild specimen thriving in an arid region of the Mediterranean, provided us with the possibility to investigate, and to rule out, the presence of genes related to drought and salt tolerance, which are two major characteristics of the naturally-occurring ecotypes of its plant host. Its range extends throughout the near-desert belt in Northern Africa, and its invasion of Mediterranean Europe has apparently followed a route via Gibraltar, the site where AS and RM: conceived the project. Rv, RG, AG, and EP: performed the sequencing GS, TS, NL, and RR: were responsible of bioinformatics analysis. AS: wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The Department of Health and Human Services’ 2010 Strategic Framework on Multiple Chronic Conditions called for the identification of common constellations of conditions in older adults.To analyze patterns of conditions constituting multimorbidity (CCMM) and expenditures in a US representative sample of midlife and older adults .n=17,912). The following measures were used: (1) count and combinations of CCMM, including (i) chronic conditions , (ii) functional limitations , and (iii) geriatric syndromes ; and (2) annualized 2011 Medicare expenditures for HRS participants who were Medicare fee-for-service beneficiaries . Medicaid beneficiaries were also identified based on their self-reported insurance status.A cross-sectional study of the 2010 Health and Retirement Study (HRS; No large representations of participants within specific CCMM categories were observed; however, functional limitations and geriatric syndromes were prominently present with higher CCMM counts. Among fee-for-service Medicare beneficiaries aged 50–64 years, 26.7% of the participants presented with ≥10 CCMM, but incurred 48% of the expenditure. In those aged ≥65 years, these percentages were 16.9% and 34.4%, respectively.Functional limitations and geriatric syndromes considerably add to the MM burden in midlife and older adults. This burden is much higher than previously reported. In 2011, nearly two thirds of Medicare fee-for-service (FFS) beneficiaries had two or more chronic conditions, and 15% presented with six or more chronic conditions ); depressive symptoms ; visual impairment; hearing impairment; often experience severe pain; incontinence; and dizziness.Thus, the total count of conditions that an individual may potentially present with is 20.The MBSF includes the total amount of Medicare expenditure incurred by a beneficiary in a given year. To account for partial-year enrollment caused by entering mid-year or exiting the program (mainly through death), we calculated the per-person per month (PPPM) expenditures by aggregating the Medicare expenditures in 2010 and 2011, and dividing the total amount by the total number of FFS months during the 2-year period. To obtain more robust estimates, we calculated the PPPM based on 2-year rather than 1-year data. To annualize, we then multiplied the PPPM expenditures by 12, and afterwards by 0.01 to account for inflation from 2010 to 2011. Data are presented as median to address the skewedness of expenditures.Other data retrieved from the HRS included age ; sex; race ; marital status (married and non-married); years of education ; income ; and self-reported Medicaid receipt (yes/no).Using survey weights to account for the complex survey design of the HRS, we conducted a descriptive analysis to identify patterns of CCMM count in the sociodemographic strata of our study population and to report the median annualized expenditures for CCMM. To analyze the combinations of CCMM, we used two different approaches. First, we used association rule mining (ARM) to identify the most common monads, dyads, triads, quadriads, and pentads, and to determine whether or not they co-occur with any other conditions. ARM was developed in the realm of marketing research to identify items that are commonly purchased together and is now applied in a variety of settings, including medicine and bioinformatics –24. SecoOf the 17,912 HRS participants included in our study, about half (50.9%) were younger than 65 years of age, 42.7% were men, 31.3% were non-Hispanic Black or Hispanic, 21% had less than 12 years of education, 32.3% had an income of less than 200% of the federal poverty level, and 6.2% were on Medicaid or dually eligible Medicare–Medicaid beneficiaries .Only 15.6% of study participants presented with no or only one condition, and 22.3% presented with two or three conditions. The remaining 62% of the population presented with at least four conditions, including 11.4% with 10 or more conditions.A higher count of conditions was observed among older than younger individuals. For example, 7.4% of individuals aged 50–64 years presented with 10 or more conditions, compared with 31.8% of those aged ≥85 years. Similarly, higher counts were observed among women than among men; among those with lower income and/or educational attainment than among those with higher income and/or educational attainment; and among those on Medicaid or dually eligible Medicare–Medicaid beneficiaries .the most common combinations of conditions  that appear regardless of whether or not they co-occur with other conditions ; next, we show the number of individuals with specific combinationsof conditions, without the co-occurrence of other conditions; and lastly, we present results showing the association between the number of conditions with which individuals present and the Medicare expenditures.Our analysis of the combinations of conditions is described below, and is as follows: first, we show monads, dyads, triads, quadriads, and pentads of combinations within CCMMs are shown in The most common Among individuals aged 50–64 years, the most common condition was hypertension (50.0%), whether presenting with or without the co-occurrence of any other condition . The mosAmong individuals aged ≥65 years, the most frequent monads, dyads, triads, quadriads, and pentads were identical to those observed in the younger age group (50–64 years), but the percent of individuals presenting with these combinations of conditions differed markedly  and 2B. individuals presenting with the top five most frequent combinations of CCMM is shown in The percent of In both age groups of the population, our analysis did not show any large representations of HRS participants with specific CCMM  and 3B. The distribution of individuals and expenditures by CCMM, and stratified by age, is shown in Using a unique resource combining survey and administrative data for a representative sample of US midlife and older adults, we evaluated the MM burden by examining the count of CCMM across sociodemographic strata, as well as in terms of Medicare expenditures. We conducted this analysis using data for all HRS participants, as well as on the subset of HRS participants who are also Medicare FFS beneficiaries. Our findings indicate that functional limitations and geriatric syndromes considerably add to the MM burden in midlife and older adults, and that expenditures increase sharply with higher counts of CCMM.The disproportionate consumption of Medicare expenditure by individuals with multiple chronic conditions has been reported previously, though the extent of the disproportionality varies across different sources –27. In aone that lasted or was expected to last twelve or more months and resulted in functional limitations, and/or the need for ongoing medical care” [Despite the difference in the study measures, the high burden of MM shown in our current analysis is somewhat consistent with a report by the Agency for Healthcare Research and Quality (AHRQ), indicating that 80% of individuals aged ≥65 years have multiple chronic conditions , using dal care” . Data fral care” .et al. [Our results are also comparable to that of a recent study by McClintock et al.  who asseet al. [Regarding CCMM, our findings failed to identify large groups of individuals with specific combinations of CCMMs  and 3B, et al. . A notabIndependence at Home [These results have important implications both in research and in clinical practice, warranting a shift of paradigm to account for functional limitations and geriatric syndromes when studying MM and not simply focusing on chronic conditions. In addition to being closely inter-related –35, the  at Home , authori at Home . HoweverFindings from this study also have implications regarding MM burden in midlife adults. Indeed, as shown in In conclusion, the prominence of co-occurring functional limitations and geriatric syndromes in CCMMs provides a basis for developing clinical systems that consider the interaction among co-existing conditions and treatments, and the needed resource allocation. Studies on MM bear increasingly greater significance, especially given the aging of the population, as their findings will guide future developments in clinical care for older adults, research methodology, and policy analysis.


Transcranial direct current stimulation (tDCS) is increasingly used in both research and therapeutic settings, but its precise mechanisms remain largely unknown. At a neuronal level, tDCS modulates cortical excitability by shifting the resting membrane potential in a polarity-dependent way: anodal stimulation increases the spontaneous firing rate, while cathodal decreases it. However, the neurophysiological underpinnings of anodal/cathodal tDCS seem to be different, as well as their behavioral effect, in particular when high order areas are involved, compared to when motor or sensory brain areas are targeted. Previously, we investigated the effect of anodal tDCS on cortical excitability, by means of a combination of Transcranial Magnetic Stimulation (TMS) and Electroencephalography (EEG). Results showed a diffuse rise of cortical excitability in a bilateral fronto-parietal network. In the present study, we tested, with the same paradigm, the effect of cathodal tDCS. Single pulse TMS was delivered over the left posterior parietal cortex (PPC), before, during, and after 10 min of cathodal or sham tDCS over the right PPC, while recording HD-EEG. Indexes of global and local cortical excitability were obtained both at sensors and cortical sources level. At sensors, global and local mean field power (GMFP and LMFP) were computed for three temporal windows , on all channels (GMFP), and in four different clusters of electrodes . After source reconstruction, Significant Current Density was computed at the global level, and for four Broadmann's areas (left/right BA 6 and 7). Both sensors and cortical sources results converge in showing no differences during and after cathodal tDCS compared to pre-stimulation sessions, both at global and local level. The same holds for sham tDCS. These data highlight an asymmetric impact of anodal and cathodal stimulation on cortical excitability, with a diffuse effect of anodal and no effect of cathodal tDCS over the parietal cortex. These results are consistent with the current literature: while anodal-excitatory and cathodal-inhibitory effects are well-established in the sensory and motor domains, both at physiological and behavioral levels, results for cathodal stimulation are more controversial for modulation of exitability of higher order areas. Although tDCS is one of the most used non-invasive brain stimulation techniques, there are relatively few studies addressing the mechanisms underlying its action  modulation, a certain amount of evidence suggests that the after-effects of both anodal and cathodal tDCS share a mechanism involving glutamatergic synapses  recorded with this technique reflect cortical excitability and connectivity of the targeted area, representing a direct measure of the neural state. TMS-EEG studies on tDCS effects on cortical excitability showed an increase of TEPs during and 10 min after the end of anodal tDCS, which were not restricted to the stimulated area, but rather affected different cortical networks according to the brain activation state. Effects spread following structural connections at resting state  participated in the study. To define the sample size, we run a-priori computations in GPower to determine the number of participants needed to highlight a potential effect of stimulation with a power of 0.90 and an alpha level of p = 0.05. In detail, we ran two a-priori sample size computations, one with the smallest significant effect found in Romero Lauro et al. , during (during-tDCS) and 10 min after cathodal tDCS (post-tDCS) applied over the right PPC. Each recording lasted about 7 min during which participants were in a resting condition, fixating a white cross in a black screen (17′′). The second group of 15 participants took part in a control Sham session. Six participants of this sample were taken from a previous study  Y = −70 (SD = 6.6) Z = 54 (SD = 3.7). The NBS system allowed to continuously monitoring the position and orientation of the coil, thus assuring precision and reproducibility of the stimulation across sessions. Moreover, the NBS system estimated on-line the distribution and intensity (V/m) of the intracranial electric field induced by TMS. It uses a locally best-fitting spherical model, accounting for the head and brain shape of each participant, and taking into consideration the distance from scalp, coil position, and orientation. Mean stimulation intensity, expressed as a percentage of the maximal output of the stimulator, was 58% (range = 50–63%), corresponding to an electric field of 100 ± 14 V/m. The coil was placed tangentially to the scalp, and adjusted for each participant in order to direct the electric field perpendicularly to the shape of the cortical gyrus, following the same procedure of previous studies  using a focal figure-of-eight bi-pulse 70 mm-coil. As in Romero Lauro et al. , stimula2; current density = 0.08 mA/cm2) was placed over the right PPC under the EEG cap, in a site corresponding to P2 electrode, which was previously removed from the cap together with the CP2 electrode, as in Romero Lauro et al.  was positioned over the left supraorbital area. A constant current of 0.75 mA was applied for 15 min, with 8 s of fade-in/fade-out period. Different sized electrodes were used to increase the focality of stimulation  using a pair of rubber electrodes and a conductive paste  to attach them to participants' head and reduce impedance. An intracephalic montage was used. The cathode , which gates the TMS artifact and prevents saturation by means of a proprietary sample-and-hold circuit  for the pre-tDCS, 123 (SD ± 5) for the during tDCS block, and 122 (SD ± 3) for the post-tDCS condition. TEPs were computed by averaging selected artifact-free single trials and by filtering them between 2 and 40 Hz. Bad or missing channels, as P2 and CP2 for each session, were interpolated using the spherical interpolation function of EEGLAB . First, recordings were down-sampled to 725 Hz. Continuous signal was then split in single trials, from 800 ms before to 800 ms after the TMS pulse. Trials with artifacts due to eye blinks/movements, or spontaneous muscle activity were removed following a semi-automatic procedure . This method includes 3 concentric spheres with different homogeneous conductivities, representing the best-fitting sphere of inner and outer skull and scalp. The model was constrained to the meshes of these tissues obtained from the individual MRIs of the experimental subjects in the Statistical Parametric Mapping software package : for each participant, binary masks of skull and scalp obtained from individual MRIs were warped to the canonical meshes of the Montreal Neurological Institute (MNI) atlas. Then, the inverse transformation was applied to the MNI mesh of the cortex for approximating to real anatomy. The cortex, in particular, was reconstructed as a 3D grid of 3004 fixed dipoles, normally oriented with respect to the cortical surface. For each participant, EEG sensor position was aligned to the canonical anatomical markers (pre-auricular points and nasion), and the forward model was computed. The inverse solution was computed on the average of all artifact-free TMS-EEG trials using the weighted minimum norm estimate with smoothness prior, following the same procedures as in Casali et al. . These BAs approximately corresponded to the original four clusters of LMFP (left/right BA 6 and 7).Source modeling was performed in order to assess the impact of tDCS on cortical excitability, avoiding the potential confound of volume conduction and allowing a better definition of the spatial distribution of the tDCS effects  and Time Window  were tested as fixed factors. The by-subject intercept was included as random factor. The inclusion of a main effect or interaction in the final model was assessed by means of Likelihood Ratio Test , the second (BF01 = 4.6), and the third (BF01 = 5.3) time window.The final model on GMFP values did not include the main effect of Analyses run on LMFP values did not support any effect of cathodal tDCS in any of the considered clusters of electrodes , the second (BF01 = 3.9) and the third (BF01 = 5.7) time window.In particular, concerning C1, the cluster under the tDCS cathode, LRT indicated not to include Condition in models for any time window . Crucially, Bayesian analyses moderately supported the null hypothesis, indicating no effect of tDCS on LMFP computed in this cluster in any time window .Analyzing C2, the cluster under the TMS coil, LRT indicated to not include Condition in models for any time window . Yet, Bayesian analyses moderately supported the null hypothesis, indicating no effect of tDCS on LMFP computed in this cluster in any time window .Similarly, in C3 LRT indicated to not include Condition in the 0–50 ms [p = 0.49] in the 50–100 ms [p = 0.30] or in the 100–150 ms [p = 0.22] time windows. Even in this case, Bayesian analysis supported moderately the null hypothesis, indicating no effect of tDCS on LMFP computed in this cluster in any time window .The final model on C4 values did not include the main effect of Source modeling analyses confirmed results from the sensor analyses. A full report of statistical results of analyses run on Global and Local SCD is reported in Table Condition in any time window, indicating no effect of cathodal tDCS on cortical activation induced by TMS .As expected, sham stimulation did not modulate global indices of cortical excitability. In particular, the final model run on GMFP did not include the main effect of Analyses run on LMFP confirm the lack of any effect of sham tDCS in any of the considered clusters of electrodes in modulating local cortical excitability .In C1, LRT values were non-significant for the first . Similarly, Bayesian analyses moderately supported the data under the null hypothesis for the first (BF01 = 3.4), the second (BF01 = 5.4), and the third (BF01 = 5) time window.The same holds for C2. LRT values were not significant for the factor Condition for any time window  in the final model. Yet, Bayesian analyses moderately supported the data under the null hypothesis for the first (BF01 = 4.7), the second (BF01 = 5), and the third (BF01 = 5.4) time window.Similarly, in C3 LRT indicated not to include factor p = 0.95], the second [p = 0.86] and the third [p = 0.89] time window. Even in this case, Bayesian analyses moderately supported the null hypothesis, indicating no effect of sham tDCS on LMFP computed in this cluster .Finally, also for C4 LRT values were non-significant for the first [Source modeling analyses on sham recordings mirrored results from the sensor analyses. A full report of statistical results of analyses run on Global and Local SCD is reported in Table Condition in any time window, indicating no effect of sham stimulation on cortical excitability  nor at a local level (LMFP for 4 clusters of electrodes). Furthermore, no significant results were found for any of the considered TEPs' temporal windows, namely an early (0–50 ms), a middle (50–100 ms), and a late (100–150 ms) one, chosen to assess different TEP's components. Source modeling confirmed the results observed at the sensor level, since SCD did not change during or after stimulation with respect to the pre-tDCS condition, both when computed at a global level or in BAs, matching the clusters of the LMFP analysis. A similar pattern of results without any significant changes in cortical excitability among the three pre-, during- and post-tDCS conditions was found when sham tDCS was delivered. This result confirms the reliability of the TMS-EEG approach and no effect of test-retest of TEP-derived indices of brain activation  affected the neuron's input/output function, namely the synaptic efficacy  and the likelihood of eliciting an action potential , by modeling a 2-compartment neuron, including the soma and the dendrites, and by recording in vitro from hippocampal pyramidal cells.In this study, a combination of computational modeling and Results showed that opposing polarization of soma and dendrite may account for the asymmetry in the strength of the effects of stimulation for opposite polarities. In particular, anodal tDCS modulates I/O functions by increasing the likelihood that neurons elicit an action potential in response to a fixed input and, in addition, increasing the synaptic current entering the cell. The opposite occurs for cathodal stimulation, i.e., a decrease in output likelihood by soma hyperpolarization. However, in cathodal tDCS this effect is canceled by the depolarization of the dendrites, hence increasing the probability of spike initiation at this location. Therefore, cathodal, compared to anodal, tDCS creates an opposite shift in the threshold of I/O function, but with a weaker impact because of these two counterbalancing opposite effects on the two neuron's compartments.By applying this model to the conventional M1-SO electrodes' montage, Lafon et al.  analyzedA polarity asymmetry has also been shown in the induction of LTP effects. For instance, in M1 mice slices, only anodal, but not cathodal, DCS coupled with simultaneous synaptic activation was able to induce LTP (Fritsch et al., Another important point is that in our study TMS-EEG recordings were collected while our participants were in a resting state. We have recently shown how cortical excitability, as measured by means of TEPs, is modulated by the activation state of the target area (Pisoni et al., Possible limitations could derive from the dimensions of the electrodes and the montage used here. In a recent study, indeed, Roy et al.  used a cIn conclusion, the results of the present study show no significant modulation of cortical excitability as measured by TEPs, when the cathodal tDCS is applied over the right parietal cortex. In contrast, previous studies showed a significant rise of cortical excitability both stimulating the same area at resting state (Romero Lauro et al., EV, AP, GM, AV, and LR: designed the study; EV, AP, GM, AV, AG, and LM: performed the experiments; EV, AP, and LR: analyzed the data; EV, AP, and LR: mainly contributed to the manuscript; GM, AV, MR, NB, and GV: contributed to the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In contrast, meta-analysis could not demonstrate a significant effect of probiotics on BPD, defined either as oxygen dependency at 28 days of life  or at 36 weeks of postmenstrual age . Meta-regression did not show any significant association between the RR for NEC or LOS and the RR for BPD. In conclusion, our results suggest that NEC and LOS prevention by probiotics does not affect the risk of developing BPD in preterm infants.Probiotic supplementation reduces the risk of necrotizing enterocolitis (NEC) and late-onset sepsis (LOS) in preterm infants, but it remains to be determined whether this reduction translates into a reduction of other complications. We conducted a systematic review and meta-analysis to evaluate the possible role of probiotics in altering the risk of bronchopulmonary dysplasia (BPD). Fifteen randomized controlled trials  were included. None of the included studies assessed BPD as the primary outcome. Meta-analysis confirmed a significant reduction of NEC (risk ratio (RR) 0.52, 95% confidence interval (CI) 0.33 to 0.81,  Bronchopulmonary dysplasia (BPD), a chronic lung disease of prematurity, is considered one of the major complications of premature birth ,2,3,4. TThe pathogenesis of BPD is initiated by the arrest in alveolar and lung vascular development, due to premature birth, and sustained by inflammatory events that play a paramount role in the progression of BPD ,4,8,9. TSeveral treatments, most of which focused on anti-inflammatory or homeostasis-restoring properties, have been attempted in order to prevent or treat BPD . HoweverProbiotics are defined as live micro-organisms which, when administered in adequate amounts, confer a health benefit on the host ,22. ProbA protocol was developed prospectively that detailed the specific objectives, criteria for study selection, the approach to assessing study quality, clinical outcomes, and statistical methodology. The study is reported according to the PRISMA checklist .A comprehensive literature search was undertaken using PubMed, EMBASE and CENTRAL  from their inception to 1 July 2017. Combinations of the following terms (including MeSH terms) were used to search for relevant publications: (probiotic(s) OR lactobacillus OR saccharomyces OR bifidobacterium OR streptococcus) AND (“preterm infant” OR “premature infant” OR “extremely low birth weight infant” OR “very low birth weight infant”). Language was not restricted. Additional strategies to identify studies included manual review of reference lists of key articles that fulfilled our eligibility criteria, use of the “related articles” feature in PubMed, use of the “cited by” tool in Web of Science and Google Scholar, and manual review of reference lists of meta-analyses on probiotics in preterm infants ,42,43,44The initial search was performed by two reviewers (EV-M and EV), who eliminated clearly irrelevant articles based on the title and abstract as defined by the pre-set selection criteria. The final selection of articles was made by mutual consideration of both authors. Studies were included if they were RCTs involving the use of probiotics in preterm infants  and reported results on BPD. BPD was defined as dependence on supplementary oxygen either at 28 days of life (BPD28) or at a postmenstrual age (PMA) of 36 weeks (BPD36) [Studies were reviewed to ensure that study populations did not overlap by checking subject sources and studying time-frame. Where two or more studies reported on the same population, the most recent study was preferentially used (provided it reported data on BPD) to avoid duplicate data.Two groups of investigators (EV-M/EV and MP/GC) extracted the data independently by using a data collection form designed for this review. Data extracted included: gestational age (GA) and birth weight (BW) of participants, patient inclusion criteria, study design , and outcomes of interest .Two reviewers (EV-M and EV) independently assessed risk of bias in each trial by using the Cochrane “Risk of Bias Assessment Tool” . For eac2I statistic, which is derived from Q and describes the proportion of total variation that is due to heterogeneity beyond chance [I2 value of 0% indicates no observed between-study heterogeneity, and large values show increasing between-study heterogeneity. The risk of publication bias was assessed by visual inspection of the funnel plot and using an Egger test. To identify any study that may have exerted a disproportionate influence on the summary effect, we calculated the summary effect excluding studies one at a time. To explore differences between studies that might be expected to influence the effect size, we performed subgroup sensitivity analysis and univariate random-effects meta-regression (method of moments) [Studies were combined and analyzed using comprehensive meta-analysis V3.0 software . We used a random-effects model to account for anticipated heterogeneity, resulting from the differences in methodology between studies. However, analysis using a fixed-effect model was also carried out to ensure that the model used for the meta-analysis would not affect the results. Effect size was expressed as Mantel–Haenszel risk ratio (RR) and 95% confidence interval (CI). Statistical heterogeneity was assessed with the Cochran’s Q statistic and by the d chance . An I2 vmoments) ,47. A poThere was no substantial disagreement between reviewers on articles for inclusion, data extraction, and risk of bias assessment. Based on the titles and abstracts of 1456 citations, we identified 63 potentially relevant studies, of which 15 met the inclusion criteria ,60,61,62p = 0.900, p = 0.999). In sensitivity analyses, excluding one study at a time, the summary RR ranged from 0.99 , when the study of Totsu et al. [p = 0.703), when the study of Jacobs et al. [p = 0.983). Exclusion of the study by Stratiki et al. [p = 0.829). Further sensitivity analysis and assessment of publication bias were not performed for BPD28 due to the low number of studies.BPD was not the primary outcome in any of the included studies. Six studies ,60,61,62s et al.  was excls et al.  includedi et al. , in whicp = 0.043). In contrast, neither the other individual studies nor the meta-analysis could detect a significant effect of probiotic supplementation on BPD36 . In sensitivity analyses, excluding one study at a time, the summary RR ranged from 1.04 , when the study of Lin et al. [p = 0.138), when the study of Al Hosni et al. [Data on BPD36 were available from 11 studies ,60,61,62n et al.  was exclp = 0.200).One study  also incBifidobacterium was part of the supplementation, studies where Lactobacillus was part of the supplementation, studies where multiple-strain supplements were used, studies where single-strain supplements were used, and studies where infants had a mean BW < 1250 g. No subgroup analysis could demonstrate a significant effect of probiotics on BPD36 , and when we pooled the studies that reported BPD36 , and the meta-analysis of studies that reported BPD36 found a close to significant reduction in LOS , and neither could the meta-analysis of studies that reported BPD36 . We performed meta-regression analyses (methods of moments) to investigate the possible correlation between the effect size for mortality and the effect size for BPD36. This meta-regression could not detect a statistically significant correlation between the changes in mortality produced by the probiotics and the effect size for BPD36 .All the included studies reported data on mortality , but metInflammatory events, such as NEC and LOS, are not only life-threatening for (very) preterm infants but also may mediate major short- and long-term adverse outcomes ,64. Currr2) of 0.08, suggesting that only the 8% of the reduction of BPD is attributable to the reduction in nosocomial infection rates [Inflammatory processes such as NEC and LOS may increase the risk of developing BPD through direct and indirect mechanisms. Proinflammatory cytokines may exert a direct effect on lung development or sensitize the lung to the effects of oxygen, mechanical ventilation, or other stressors ,9,15,65.on rates .The 15 studies included in our meta-analysis represent a subset of the larger number of RCTs included in the meta-analyses on probiotics for NEC and LOS prevention. Therefore, we analysed whether the protective effects of probiotics on NEC and/or LOS were also present in the RCTs included in our study. Pooling the 15 studies showed a significant reduction of NEC  and a close to significant reduction of LOS (RR 0.82 95% CI 0.65 to 1.03) in the probiotics group. We speculated that studies with higher protective effects against NEC and/or LOS would show more effect on the development of BPD. However, meta-regression did not show a significant correlation between the RR for NEC and LOS and the RR for BPD. This suggests that the reduction in postnatal inflammatory events did not translate into a reduction of BPD.Several meta-analyses showed that probiotics reduce mortality among VLBW infants ,25,38. IOne important limitation inherent to any meta-analysis on BPD is the heterogeneity of the definition of the condition ,69,70. ILactobacillus or Bifidobacterium species showed a different effect on BPD. We also performed a separate analysis for multi-strain probiotics because recent meta-analyses suggest that the use of more than one strain has a stronger effect in the prevention of NEC [As mentioned above, the RCTs included in our analysis had important differences in the type, amount, and timing of probiotic supplementation. The choice of probiotic strain(s) is crucial and meta-analyses on probiotics have been criticized because, in most of them, probiotics administered for treatment/prevention of a specific disease or condition were all evaluated together ,71,72,73n of NEC . None ofBesides their effect on NEC and LOS prevention, there are some other mechanisms of action ascribed to probiotics which may directly counteract the disruption of lung development prompting to BPD ,75. In tIn conclusion, our study could not demonstrate any significant effect of probiotic supplementation on the risk of developing of BPD. Given the remarkable theoretical benefits of probiotics supplementation in ameliorating several aspects of BPD pathogenesis and the limitations of the analysis, our data should be seen as a starting point rather than definitive results. The main merit of our study was to collect, for the first time, the available data on the role of probiotic supplementation in the prevention of BPD, and to revise the possible specific mechanisms of action. Nevertheless, further experimental and clinical data are needed to draw more solid conclusions. Particularly, more studies designed to select the optimal probiotic preparation, dosing, and duration of therapy are still needed . These s