The Enquiring About Tolerance (EAT) study examined whether the early introduction of 6 allergenic foods from 3 months of age in exclusively breastfed infants prevented the development of food allergy. The intervention was effective in the per-protocol analysis for allergy to 1 or more foods and for egg and peanut individually, but only 42% of early introduction group (EIG) children met the per-protocol criteria.We sought to identify which factors were responsible for nonadherence in the EAT study.Factors influencing adherence within the key early introduction period in the EIG (up to 6 months of age) were divided into enrollment and postenrollment factors, and their association with nonadherence was explored.In an adjusted analysis, at enrollment, increased maternal age, nonwhite ethnicity, and lower maternal quality of life were independently and significantly associated with overall nonadherence in the EIG. Enrollment eczema and enrollment serum allergen-specific IgE sensitization to 1 or more foods (≥0.1 kU/L) were not related to overall nonadherence. After enrollment, 2 factors were significantly related to EIG overall nonadherence: parent-reported IgE-type symptoms with infant allergenic food consumption by 6 months of age and reported feeding difficulties by 4 months of age.If early introduction of allergenic foods were to be considered a strategy to prevent food allergy, families of nonwhite ethnicity, those with older mothers, and those with infants with reported feeding difficulties or early-onset eczema would benefit from support to promote early and sustained consumption.  The Enquiring About Tolerance (EAT) study was a large randomized food allergy prevention trial of the early introduction of 6 allergenic foods from 3 months of age in exclusively breast-fed infants recruited from the general population.There are 2 explanations for this discrepancy. First, the per-protocol analysis was effective because the population adhered to the intervention. Second, the intention-to-treat analysis was not effective because among the large group of children who did not adhere to treatment, those with nascent allergy with food sensitization had subclinical symptoms, food aversion, or both and were therefore unable to adhere. Further analyses suggested that food allergy prevention through early introduction of multiple allergenic foods in normal breastfed infants might depend on adherence and dosage.The EAT study per-protocol criteria for the EIG were stringent. By 6 months of age, EIG infants were expected to have achieved sustained high-dose consumption of 5 or more of the 6 early introduction foods. Achieving such per-protocol adherence in the EIG proved difficult. Only 42% (223/529) of adherence-evaluable EIG children complied entirely with the protocol (34% (223/652) of the whole EIG group).After the completion of the study, we measured serum allergen-specific IgE sensitization in the EAT participants and demonstrated in an intention-to-treat analysis that the intervention successfully reduced the development of food allergy among EIG infants who were sensitized on specific IgE testing at enrollment. Infants with the early emergence of food-specific IgE sensitization to foods are known to be at high risk of developing a food allergy.4In this article we explore how these and other data collected in the EAT study influenced the issue of nonadherence to the recommended level of consumption of the 6 early introduction foods , as well as nonadherence to consumption of individual foods (food-specific nonadherence). We determined whether occult sensitization and early symptoms were responsible for the high nonadherence rate.The UK Scientific Advisory Committee on Nutrition has issued the following statement after its review of the evidence on feeding in the first year of life: “The available evidence indicates that the deliberate exclusion or delayed introduction of peanut or hen's egg beyond 6 to 12 months of age may increase the risk of allergy to the same foods.”One thousand three hundred three 3-month-old infants were recruited from the general population in England and Wales through direct advertising and were enrolled between November 2009 and July 2012. Details of the EAT study methodology have been published elsewhere.Participants were randomized to the SIG or the EIG. The SIG was asked to exclusively breastfeed to around 6 months of age in accordance with the recommendation in place in the United Kingdom since 2003.www.jacionline.org).The EIG parents were asked to introduce 6 allergenic foods to their infants: cow's milk (yogurt), peanut, cooked (boiled) hen's egg, sesame, white fish, and wheat.An online questionnaire was sent to each family monthly to 1 year of age and then every 3 months until the child reached 3 years of age. Each questionnaire allowed families to report any suspected symptoms with food ingestion and to list suspected foods. Clinical judgement was used to divide these into IgE-type and non–IgE-type symptoms. The online questionnaires also ascertained maternal quality of life using the World Health Organization Quality of Life BREF questionnaire,Food-specific IgE levels to each of the 6 foods were measured at enrollment and at 1 and 3 years of age in both groups using ImmunoCAP  assays. The primary outcome of the EAT study was the proportion of participants with challenge-proved food allergy to 1 or more of the 6 early introduction foods between 1 and 3 years of age.www.jacionline.org. The key criterion for overall adherence in the EIG was consumption of at least 5 of the allergenic foods in at least 75% of the recommended amount  for at least 5 weeks between 3 and 6 months of age. Food-specific per-protocol adherence was based on the same criteria . The window in which 5 or more weeks of consumption could be achieved was narrow, as explained in more detail in the “Restricted window to achieve per-protocol status” section in the The definitions of overall per-protocol adherence are presented in www.itntrialshare.org).Univariate and multivariable logistic regression models were used to identify factors associated with nonadherence. Penalized logistic regression was used where appropriate for situations with very sparse data. Eczema was analyzed as the presence of visible eczema at enrollment by using the UK diagnostic criteria (“visible eczema”).www.jacionline.org.Overall per-protocol adherence status could only be determined in 81% (529/652) of the EIG participants see . An explwww.jacionline.org). Although randomization had been effective in ensuring the median age of introduction of peanut, egg, sesame, and fish was the same at 19.6 weeks, there remained the possibility that the position in the order in which a specific food was introduced might be associated with overall or food-specific adherence. Specific foods introduced later in the sequence reduced the likelihood of being per-protocol adherent to that food (see www.jacionline.org). However, the order of introduction of each of the foods was unrelated to overall per-protocol adherence.Although overall adherence to the EAT early introduction regimen was low at 42%, families found it easier to introduce some foods than others , with thThe key period for defining per-protocol adherence was consumption through 6 months of age. However, www.jacionline.org for univariate analysis). Nonwhite ethnicity and increased maternal age were associated with delaying the introduction of solids in both the SIG and EIG infants (see www.jacionline.org). Differences were observed in other enrollment demographic characteristics between ethnic groups: visible eczema and enrollment sensitization were both more common in nonwhite ethnic groups, and a history of a sibling having a parent-reported food allergy was more common in infants of Asian, black, or Chinese ethnicity (see www.jacionline.org) and are reviewed in the “Association between ethnicity and baseline demographic characteristics” in the Nonwhite ethnicity, increased maternal age, and lower enrollment maternal quality-of-life scores were significantly associated with not being per-protocol adherent in the EIG  was significantly associated with overall nonadherence, whereas any visible eczema at enrollment was of borderline significance  to be able to determine reliably the effect on subsequent food-specific adherence . More EIwww.jacionline.org). Sensitization to 1 food also predicted food allergy developing to other foods. The group of infants with early food-specific IgE sensitization accounted for 69% of the food allergy cases that developed in the EAT study. We have shown that the EAT study early introduction intervention was effective in an intention-to-treat analysis of this high-risk population.Food-specific IgE sensitization at enrollment strongly predicted the development of food allergy to the same food, and sensitization to 1 or more foods strongly predicted overall food allergy (see www.jacionline.org), both of which are likely to be a consequence of the study design and discussed in more detail in the “Consumption of each allergenic food by enrollment-specific IgE sensitization status” section in the The great majority of EIG infants sensitized to a specific food at enrollment did not report any symptoms when that food was introduced into their diets . Similarly, the great majority of infants whose families reported symptoms with a specific food were not sensitized to that food at enrollment . Both are reviewed in detail in the “IgE-type symptom reporting and enrollment sensitization” section in the A multivariable analysis was undertaken to determine whether overall nonadherence in the EIG could have been predicted from certain enrollment characteristics  all remained significantly related to increased EIG overall nonadherence. There was no statistically significant relationship with any measure of eczema at enrollment, be this visible eczema, eczema severity (SCORAD severity group), or SCORAD itself, although all odds ratios were greater than 1.0.P = .77] and egg odds ratio of 2.32 ) and not statistically significant. Among the other variables, nighttime waking frequency was significantly associated with egg nonadherence.Models were created for each individual food (www.jacionline.org). These were new onset of parent-reported eczema after enrollment but before 6 months, maternal reporting of feeding difficulties at the very beginning of solid food introduction , and reporting of IgE-type and non–IgE-type symptoms with consumption of early introduction foods before 6 months of age (see www.jacionline.org). Although new-onset eczema showed no association with overall or food-specific adherence, there was a strong association between adherence and early reported feeding difficulties and between adherence and the reporting of IgE-type symptoms to the early introduction foods in the key early introduction period .The EAT study SIG participants can be used to explore the natural history of the development of food allergy independent of the early food introduction intervention. Children with visible eczema or food sensitization at enrollment or those of nonwhite ethnicity, although representing a small subgroup of the overall population, contributed disproportionately to the burden of overall food allergy (A).SIG participants with eczema at enrollment made up 24.3% of the study population but were responsible for 61.9% of the food allergy cases. Likewise, 15.6% of the SIG were sensitized on IgE testing at baseline to 1 or more foods but were responsible for 69.2% of food allergy cases in the SIG. Of the SIG, 15.3% were of nonwhite ethnicity but accounted for 28.6% of food allergy cases in the SIG, and a significantly greater proportion of nonwhite participants had visible eczema and/or IgE food-specific sensitization at enrollment see . FurtherB). Certain assumptions were made: an 80% intervention effect was assumed given the high efficacy observed in the EAT per-protocol analysis and the efficacy seen in the Learning Early About Peanut Allergy (LEAP) study intention-to-treat analysis.B, displays the percentage reduction in total allergy burden and the number needed to treat within the different subgroups.We modelled the effects of improved adherence in infants at high risk of developing food allergy  and how this might affect the prevalence of food allergy , B. CertTargeting the intervention to the group of nonwhite and/or eczematous infants would comprise a high-risk population of 71.4% of the food allergy burden. If 50%, 75%, or 85% adherence rates can be achieved in this high-risk group, reduction in the overall burden of food allergy in the whole population would be approximately 29%, 43%, and 49%, respectively.Whilst 5.6% of EIG participants had a food allergy in the EAT study, 58% were nonadherent with the early introduction protocol. Hence the nonadherence rate was 10-fold greater than the food allergy rate. Notably, the nonadherence rate also significantly exceeded the prevalence rate of risk factors associated with developing a food allergy: visible eczema at enrollment (25%), enrollment sensitization to 1 or more foods on specific IgE testing (16%), and nonwhite ethnicity (15%).www.jacionline.org. However, unexpectedly, this delay was present in the EIG as well, despite all EIG families having being asked to introduce the allergenic foods as rapidly as possible.In univariate analysis nonwhite ethnicity, older maternal age, lower enrollment maternal quality-of-life scores, and increasing SCORAD were associated with nonadherence to the EIG protocol. The enrollment factors that were found to remain significantly associated with nonadherence in the adjusted analysis were nonwhite ethnicity, older maternal age, and lower enrollment maternal psychological quality of life. Nonwhite ethnicity and older maternal age were associated in both study groups with postponing the introduction of allergenic foods, compromising the ability to be per-protocol adherent because the window for per-protocol–defined adherence was so short. This delay being present in the SIG could have been anticipated, having been reported in the Infant Feeding Survey undertaken in 2010 (Infant Feeding Survey 2010),After enrollment, although the reporting of IgE-type symptoms in EIG participants in the key early introduction period was common (16%), this too fell far short of the 58% nonadherence rate. Furthermore, the great majority of EIG participants reporting such symptoms were neither sensitized to any food at enrollment (76%) nor had a food allergy (82%). The strongest association with nonadherence was the early emergence of feeding difficulties, with 40% of non per-protocol EIG families reporting some or great difficulty feeding their infant at 4 months of age compared with 20% of the per-protocol EIG families.Enrollment eczema was not associated with overall adherence in the adjusted analyses . This is likely to reflect the mild phenotype of infants with eczema in the EAT study. Of the 160 EIG infants with enrollment eczema, in 123 it was mild (SCORAD <15), in 31 moderate (SCORAD 15 to <40), and in only 6 was it severe (SCORAD ≥40). Having eczema in childhood is strongly associated with dietary restriction and a reluctance to include allergenic foods in the diet. In a study of 100 children attending a pediatric dermatology clinic, 75% were having some form of parent-instigated dietary exclusion, and allergenic foods, including dairy products, eggs, and cow's milk, were being omitted by 48%, 27%, and 25%, respectively.It might have been anticipated that enrollment sensitization could lead to subclinical symptoms, resulting in a subclinical form of pre-existing food allergy that prevented adherence. This was not the case. Enrollment sensitization was not associated with overall or food-specific adherence. Furthermore, the EAT intervention was effective in an intention-to-treat analysis in the enrollment-sensitized EIG infants.The early emergence of parentally perceived feeding difficulties and aversive feeding behavior by 4 months of age, when families had only just started introducing solids to their EIG infants, had the strongest associations with nonadherence in the EIG and were also associated with later introduction of allergenic foods. This finding suggests that families were trying to feed their infants the allergenic food, but if the family perceived that the infant was not ready or mature enough and they were struggling to achieve the study's stipulated consumption levels, then, particularly for certain foods and having reached a specific level of consumption in the key early introduction period, they did not attempt to escalate this level further beyond 6 months.That infant maturity might be a key factor was demonstrated in our recent analysis of sleep data within the cohort. In this study we showed in an intention-to-treat analysis that early introduction in the EIG was associated with greater duration of sleep and fewer night wakings, as well as significantly less parent-reported very serious sleep problems.Feeding difficulties were also shown in the Infant Feeding Survey 2010 to have a strong association with ethnicity (considered in more detail in the “Comparison with the Infant Feeding Survey 2010” section findings in the The primary strength of this analysis is that the EAT study cohort was recruited from the general population. The children were meticulously studied and have been shown to have demographic characteristics broadly similar to those of the population of England and Wales.It has been recognized by others that when clinical efficacy has been demonstrated in trials, such as EAT and particularly LEAP, translating this into a public health intervention is complex, and the results are likely to be subject to effect modification in different populations.Our findings suggest that there are certain groups who could benefit from directed support should the recommendation of early introduction of allergenic foods be adopted as a way to prevent food allergy. Specifically, the important factors were found to be nonwhite ethnicity, older mothers, and mothers with poorer quality of life. This also applies to infants with early-onset eczema in that although their adherence to per-protocol consumption of specific foods was not compromised compared with children without eczema, it is within this group that the majority of food allergy develops. Although these factors explain only a small part of the 58% of EIG families who were nonadherent, these groups of infants contributed disproportionately to the overall prevalence of food allergy in the EAT population. Indeed, our modelling shows that improved adherence in infants manifesting early eczema and from ethnic minorities raises the possibility of a substantial reduction in the burden of food allergy, with a 49% reduction if 85% adherence were achievable. Sufficiently high adherence rates with an early introduction regimen in these populations will be more challenging yet of great value.The issue of ethnic differences in adherence to public health recommendations is well recognized.A recent study of 1000 expecting and 1000 new caregivers of infants less than 1 year of age reported questionable support for early allergenic solid food recommendations.Nonwhite families, those with older mothers, and those with infants with early reported feeding difficulties or early-onset eczema would benefit from support to achieve early and sustained allergenic food consumption.A number of countries, including the United States,


The current prospective study aimed at determining the educational characteristics of MCQresident and at comparing them with those of MCQteacher.We hypothesized that multiple-choice questions written by radiology residents resident and 63 of MCQteacher. The difficulty index, the discrimination power, and the distractor’s quality were calculated in the two series of MCQs and were compared by using Student t test. Two radiologists classified each MCQ according to Bloom’s taxonomy and frequencies of required skills of both MCQ series were compared.Fifty-one radiology residents of the first to the fifth year of training took the 2017 exam that contained 58 MCQresident was statistically significantly higher than that of MCQteacher . The mean ± SD discrimination index of MCQresident was statistically significantly higher than that of MCQteacher . The mean number of non-functional distractors per MCQresident was statistically significantly higher than that per MCQteacher . MCQresident required recalling skills more frequently than MCQteacher which required more advanced skills to obtain a correct answer.The mean ± SD difficulty index of MCQresident differ from those of MCQteacher. This study highlights the characteristics to optimize the writing of MCQs by radiology residents.Educational characteristics of MCQ Scores obtained by PGY1-5 at their annual evaluation increase with education year whoever wrote the MCQs (radiology residents or teachers).MCQs written by radiology residents are easier and contain more nonfunctional distractors than MCQs written by radiology teachers, but their discriminant power is higher.Memory skills play a more important role in answering MCQs written by residents than by teachers.Training of radiology residents is generally based on supervised daily clinical practice during scheduled core rotations, autonomous activity during night calls, attendance to multidisciplinary consultations and dedicated radiology-centered lectures, and presentations of clinical cases. Evaluation of their knowledge, skills, and attitudes is an important part of their training and depends on opinions of faculty staff and written or oral exams. In our institution, opinions of faculty staff and MCQ exams are used. Each year, all PG1-5 residents perform a written examination containing 125 single best-option multiple-choice questions addressing knowledge in all fields of medical imaging as outlined in the ESR training curriculum [Multiple-choice question (MCQ) is a validated educational tool for both formative and summative assessment because of its objective output, simplicity of utilization, and informative feedback to exam-takers and teachers –6. For fresident) and those written by teachers (MCQteacher), combined in a single computer-supported test.Writing of MCQs is a difficult task, and teaching staff rarely have the time or incentive to develop high-quality formative questions, focusing instead on material for high-stakes assessments , 10. In resident was prospectively classified by its author according to eight organs or anatomic systems, three estimated degrees of frequency , and of clinical relevance . The residents were aware that some of their MCQs would be used for their annual evaluation (PGY1-5). In November 2016, 221 resident-authored MCQresident were available.Since October 2015, radiology residents from the third to the fifth year of residency (PGY3-5) rotating in our academic hospital are asked to integrate two single best-option multiple-choice questions (MCQs) into each of their presentations of clinical cases. MCQ templates including a stem and four-answer options consisting of one correct answer and three distractors were proposed . Each MCresident, addressing items from all organ systems, with a frequent or an occasional occurrence and with a high or an intermediate degree of clinical relevance. Sixty-five MCQteacher written by nine radiologists after their lectures were also included in the annual evaluation. The writing of all MCQs was checked for spelling and format (four-option format with only one correct answer) [In January 2017, a first-year EDIR-certified fellow in radiology with interest in education and the staff member radiologist responsible for the resident’s training  have in consensus selected 65 MCQ answer) . In Febrrpbis) [The internal consistency reliability was verified by the Kuder-Richardson formula 20 (KR-20) , 25. Thrrpbis) . A high rpbis) . Usual wrpbis) . Finallyrpbis) . IdeallyThe two radiologists in charge of the evaluation separately classified each MCQ according to Bloom’s cognitive taxonomy that indicates the most probable cognition process needed to correctly answer the item . This hit test  for independent samples was performed to assess statistical differences between scores from two groups: the MCQresident and MCQteacher. Due to the multiple comparisons that were performed, a Bonferroni correction of type p < 0.05/ncomparisons was applied to the tests cited above, and the significance levels were adjusted accordingly.The exam scores of residents were expressed in mean value ± standard deviation (SD) for both questionnaires and were plotted against year of training. Distribution of scores was found to be normal according to the Kolmogorov-Smirnov test. Therefore, a two-sided resident and MCQteacher were determined. Difficulty and Discrimination Index from residents’ and teachers’ MCQs were compared using the t test . The number of non-functional distractors of the two series was compared using the non-parametric Mann-Whitney test (U test) and the two-sided Fisher’s exact test with the mid-P approach at p < 0.05. The p value less than 0.05 was considered to indicate statistically significant difference. The frequency of the highest levels of cognitive process involvement reached by MCQresident was compared with those of MCQteacher according to Bloom’s taxonomy [The mean values ± SD for Difficulty Index, Discrimination Index, and Distractor Functionality of MCQtaxonomy .The authorization of our ethical committee was not asked because our study did not involve patients. The project had been validated by resident representatives and faculty staff. All residents were aware of the projects, and they had signed a form for the use of their presentations.resident and 63 MCQteacher were validated and 7 MCQresident and 2 MCQteacher were excluded due to technical difficulties during the examination (failure of video on some PCs) (Table teacher and 35 MCQresident). The mean scores (± SD) obtained at the MCQresident were statistically significantly higher than those at the MCQteacher for the residents of each year of residency (p < 0.01 for each year)  from the first to fifth year of training took the exam that initially contained 130 MCQs. Fifty-eight MCQresident than MCQteacher with an ideal difficulty index without statistically significant difference (p = 0.94), and their mean Difficulty Index (0.81 ± 0.1) was statistically significantly higher than that of MCQteacher (0.64 ± 0.2) (p < 0.0001) , and the mean Discrimination Index ± SD of MCQresident (0.34 ± 0.2) was statistically significantly higher than that of MCQteacher (0.23 ± 0.2) (p = 0.0007) , and the mean number of NFD per MCQresident (1.36 ± 0.9) was statistically significantly higher than that per MCQteacher (0.86 ± 0.9) (p = 0.0031). Application-type (for reviewer 1) and understanding-type (for reviewer 2) cognitive processes were less frequently needed for MCQresident than for MCQteacher.The frequency of each cognitive process required for a correct answer is given in Table In our institution, the annual evaluation task of the residents consists of three parts: a self-fulfilled logbook ; a summary of the evaluation by the supervising faculty staff of their knowledge, skills, and attitudes; and our MCQ test addressing all radiology subspecialties. The MCQ test is performed on a yearly basis to provide the resident an insight on his/her learning curve throughout the 5 years of residency. The results of the PGY4 and PGY5 MCQs are validated by the National Accreditation Board and integrated in the qualification process, in the absence of a national board examination.The current study demonstrated that the scores obtained by the residents varied according to their level of training in radiology with a non-exponential improvement throughout the 5 years of residency. Our learning gain curve of radiology residents that seems to decelerate over time was similar to that observed by Ravesloot .resident were statistically significantly higher than in the MCQteacher for all residents, independently of their post-graduate year. The most likely explanation was that the degree of difficulty of the MCQresident was lower than that of the MCQteacher and that the number of NFD was higher in MCQresident than in MCQteacher. We cannot exclude the hypothesis that the residents deliberately lowered the difficulty level and included NFD because they knew that their MCQs would be used for their annual evaluation. It is most likely that these characteristics are inherent to the degree of qualification of the MCQ writer [Second, the scores obtained in the MCQQ writer .resident was higher than that of MCQteacher warrants further assessment as this feature is important when composing high-quality MCQs. A likely explanation was that MCQresident were written by PGY3–5 and not by PGY1–2 residents. Therefore, the PGY3–5 residents who, in overall, should obtain the highest scores obtain much better scores than the PGY1–2 residents in the MCQs of their peers than in the MCQteacher, thus artificially increasing the discriminating index of the MCQresident.Third, the observation that the discrimination index of MCQresident focused more on recalling skills than the MCQteacher that required the capacity to analyze and apply knowledge. This feature indicates the difficulty in writing high-quality MCQs that require more experience in solving problems [Finally, the analysis of the MCQs according to Bloom’s taxonomy demonstrated that the MCQproblems –31. Howeproblems . Actuallproblems .The effects of this collaborative approach for MCQ writing are controversial although it at least contributes to create questions that can support formal or summative evaluations . Aflalo The effect of MCQ format on the resident’s scores was not assessed as questions with videos were eliminated because of technical problems on certain personal computers. While taking the exam, residents were not able to scroll into images. The Clinically Orientated Reasoning Evaluation (CORE) computer-based format that replaced the oral examination in EDIR using DICOM viewer simulating the daily work of radiologists is most likely a better way to evaluate radiology residents .resident and MCQteacher that will be explained to current and future radiology residents in order to increase the quality of their MCQs. In addition, we plan to share this collaborative approach with other training centers to provide a broader supply of MCQ that would decrease the influence on the exam takers.The current study highlighted differences between MCQOur study had several limitations. First, it was a monocenter study with a limited number of MCQs, from residents and from teachers. Second, both MCQs were selected by two radiologists to create a series of MCQs that would cover all fields of diagnostic and interventional radiology. To minimize selection bias, items were selected based on their characteristics indicated by the residents and the teachers and not by reading the MCQs. In addition, questions with a high degree of importance and a frequent occurrence in clinical practice have been privileged. Finally, our results were influenced by the facts that PGY3–5 residents composed the MCQs and that PGY1–5 residents took the examination. Residents were also aware of the fact that their MCQs would be used in the annual evaluation.resident differ from those of the MCQteacher in many ways. The clear identification of these differences enabled us to indicate points of attention to address in MCQ writing guidance in order to achieve higher quality examinations with the collaboration of the teaching staff.In conclusion, the current study demonstrated that the educational characteristics of MCQ


The ability of HIV-1 to integrate into the genomes of quiescent host immune cells, establishing a long-lived latent viral reservoir (LVR), is the primary obstacle to curing these infections. Quantitative viral outgrowth assays (QVOAs) are the gold standard for estimating the size of the replication-competent HIV-1 LVR, measured by the number of infectious units per million (IUPM) cells. QVOAs are time-consuming because they rely on culturing replicate wells to amplify the production of virus antigen or nucleic acid to reproducibly detectable levels. Sequence analysis can reduce the required number of culture wells because the virus genetic diversity within the LVR provides an internal replication and dilution series. Here we develop a Bayesian method to jointly estimate the IUPM and variant frequencies  from the sequence diversity of QVOAs.Using simulation experiments, we find our Bayesian approach confers significantly greater accuracy over current methods to estimate the IUPM, particularly for reduced numbers of QVOA replicates and/or increasing actual IUPM. Furthermore, we determine that the improvement in accuracy is greater with increasing genetic diversity in the sample population. We contrast results of these different methods applied to new HIV-1 sequence data derived from QVOAs from two individuals with suppressed viral loads from the Rakai Health Sciences Program in Uganda.Utilizing sequence variation has the additional benefit of providing information on the contribution of clonality of the LVR, where high clonality (the predominance of a single genetic variant) suggests a role for cell division in the long-term persistence of the reservoir. In addition, our Bayesian approach can be adapted to other limiting dilution assays where positive outcomes can be partitioned by their genetic heterogeneity, such as immune cell populations and other viruses.The online version of this article (10.1186/s12977-018-0426-1) contains supplementary material, which is available to authorized users. HIV-1 persists in individuals, despite fully suppressive anti-retroviral therapy (ART), due to the presence of resting CD4+ (rCD4) T cells that are latently infected with replication competent, integrated copies of the virus . The sizAdditionally, sequencing of outgrowth virus can provide information on the contribution of rCD4 T cell division to persistence of the LVR. The long half-life of the HIV-1 LVR that averages several years in duration , 9, in cN is the number of wells in the QVOA; Y is the number of p24-positive wells; and n cells. The rate parameter n (the number of cells per well) is known without error, so that the maximum likelihood estimate (MLE) of n across sets of wells, which reduces the chance that the wells are either all positive or all negative; neither of these extreme outcomes can be used effectively to estimate D is the number of dilution factors, i-th dilution factor, The current standard approach for estimating the IUPM from QVOA results is based on the single-hit Poisson model, which has long been used for the analysis of limiting dilution assays . The expper well .A through E) with a uniform frequency of 0.2 in the sample population of viruses, and the true IUPM is 5. Then we can simulate a random outcome represented by the following binary (presence/absence) matrix:The defining feature of the single-hit Poisson (SHP) model is that each well has a binary all-or-nothing outcome, in which the well either tests positive or negative for viral outgrowth Fig. . EstimatK wells containing the i-th variant. This estimator stipulates that every well contains the same number of cells, such that the estimate Recently, Lee et al.  describeonality) .pol and gp41) of viral outgrowth populations from positive wells, derived from peripheral blood samples from two ART-suppressed individuals with markedly different levels of HIV sequence diversity in the LVR.In this study, we developed a Bayesian method  used in the IUPMStats can only assume a finite number of values because the outcome space is discrete. When there are a large number of wells in which a positive outcome is likely to occur, then there is a high probability that the true value is close to the MLE. As the expected number of positive wells declines, however, the distribution of potential MLEs becomes increasingly sparse. For example, the most frequent IUPMStats estimates when the true IUPM was set to 1 were 0.51 and 1.61 Fig. , which cSecond, the wells with high cell counts become less informative with increasing IUPM, since they will nearly always be positive when the IUPM is sufficiently high. For example, a well with mentclass2pt{minimn is the total number of replicates. This lack of information in turn results in greater uncertainty in estimating IUPM. For this situation, IUPMStats uses the posterior median estimate assuming a uniform prior on IUPM from 0 to 1 cells per million, whereas we used the same prior distribution across the same set of simulations irrespective of outcomes.We conducted an additional set of simulation experiments in which the same number of cells . On a single core of an Intel E5-1620v2 processor, it required an average of 358.8 seconds (roughly six minutes) to run a chain sample for one million steps, which we have found to be adequate for convergence under a variety of simulated conditions. In contrast, our implementation of IUPMStats in We applied both the IUPMStats and IUPMBayes methods to actual experimental results derived from two patients (106 and 111). These patients had been selected from a larger study in progress because the overall sequence diversity in viral outgrowth wells derived from the latent reservoirs of the respective individuals were characterized by either very high (106) or very low (111) levels of clonality. We use the term ‘clonality’ to refer to the scenario where the virus population is dominated by a variant or variants that are repeated, such that most sequences in a random sample will be identical or have another well containing that same variant. Based on this prior information, we adjusted the hyperparameters of the Dirichlet prior distribution to entclass1pt{minimaR and the online calculator at http://silicianolab.johnshopkins.edu. These experimental data and results are summarized in Table R produced numerically identical estimates to the online calculator for both patients. The IUPMStats estimate was significantly higher for patient 106 (8.2 per million) than 111 (1.6 per million). Additionally, we observed fewer distinct sequence variants in well samples from patient 106 than 111 for both the gp41 and pol regions.We generated ML estimates under the SHP model using both our implementation of this method in gp41 and 3.03  for pol. This difference was driven in part by the occurrence of the predominant gp41 variant in all eight wells carrying pol variant occurred in only seven of these eight. Combining these estimates, we predict that the IUPM in patient 106 was about 3.8 infected cells per million—substantially lower than the IUPMStats estimate, but within its 95% confidence interval  for pol, respectively.Results from our Bayesian analysis of the same data sets under the MTP model are summarized in Fig. entclass1pt{minimaMaximum likelihood (ML) is a powerful technique for estimating the rate parameter of the single-hit Poisson model , 20. HowOur results support the concept of applying Bayesian methods to sequence diversity to overcome the limitations of maximum likelihood for small samples (fewer wells). Although previous studies have implemented Bayesian methods to parameterize the single-hit Poisson model , we are A core assumption of our method is that detecting more than one sequence variant in a culture well indicates the presence of at least that number of latently infected cells. It is possible that a cell is multiply infected with two or more integrated HIV DNA variants . This scP(f|M), becomes directly influenced by the number of observed variants in the data. Strict adherence to Bayesian principles discourages this repeated use of the data, but there is growing recognition that such ‘data-dependent priors’ are routinely used for empirical Bayesian inference and can retain statistically desirable properties, e.g., [The considerable depth at which next-generation sequencing samples templates from the outgrowth population makes it unlikely that variants present in the well are unobserved. However, the rapid evolution of HIV-1 means there may be an unknown number of rare genetic variants in the LVR that fail to become sampled from the source population into any replicate wells of the QVOA experiment, immediately precluding these variants from outgrowth and sequencing. In fitting the multi-hit Poisson model, we have out of necessity restricted the model to the observed variants in the experiment by marginalizing out the number of unobserved variants, s, e.g., . For exainfinite . FurtherAlthough our method was developed for the specific purpose of measuring the latent HIV reservoir, it can be adapted to other applications of limiting dilution assays where the target has genetic variation. For example, limiting dilution has been used to measure the frequency of reactivation in cells latently infected with human cytomegalovirus . It coulThe existence of the latent viral reservoir is a key barrier to curing HIV-1 despite highly effective drug treatments. In this study, we demonstrate that sequencing individual viruses from the positive wells of a viral outgrowth assay can provide a more robust and accurate measure of the latent reservoir. Furthermore, this approach can facilitate scaling up the assay to large numbers of samples by reducing the number of wells necessary to measure the latent viral reservoir.R programming language. For brevity, we will use the notation http://silicianolab.johnshopkins.edu/, version 1.0). We estimated the 95% confidence interval from the likelihood function using numerical root-finding [To facilitate batch processing on simulated and experimental data sets, we re-implemented the SHP model in the entclass1pt{minima-finding  to solveλm= .K wells and M sequence variants. Let i-th sequence variant in well k. Let i-th variant in the population of infected cells in the undiluted QVOA wells. The likelihood that at least one cell in a particular well was infected with the i-th variant is described by combining the Bernoulli and Poisson distributions:i in well k. Following standard practice such as the single-hit Poisson model [K wells and M variants:Suppose that an experiment has on model , we assuentclass1pt{minimaquencies —accordinM now refers to the observed number of variants instead of the total number. Our objective is use this likelihood to estimate f, M and f, which become obscured by the highly stochastic nature of viral amplification. Thus, we used Bayesian inference to estimate the posterior distribution of f given i to obtain a flat uninformative prior distribution over all possible values of f. We evaluated different prior distributions for f is renormalized so that P(f|M) are assumed to be independent of M is set to the observed number of variants in the data  samples were propagated over the model parameters R by first sampling the number of infected cells per well, N sequence variants by drawing from the multinomial distribution defined by the frequency vector M and To evaluate the performance of different methods to estimate IUPM, we simulated virus outgrowth data sets in hood Eq. . Put anoR module parallel. The concordance of model estimates and actual values was visually assessed with density plots and quantified by the relative error, which was calculated as the absolute difference between the actual and true values, normalized by the true value.To systematically evaluate the impact of different simulation conditions, we conducted simulations for all combinations of rate parameters Actual HIV-1 sequence data was obtained from QVOA previously performed on a population of HIV-individuals recruited through the Rakai Health Science Program in Uganda, originally enrolled to examine the size of the HIV-1 LVR . Particigp41 (pol (cutadapt (version 1.8) [AdapterRemoval [p24 positive viral outgrowth wells derived from QVOA of rCD4 T cells from these Ugandan subjects were collected and stored at escribed  for sequion 1.8)  and materRemoval . The remMAFFT ver. 7) was generated from the resulting set of sequence variants and used to reconstruct a phylogenetic tree using the implementation of the neighbor-joining method [R for visual inspection. Each unique sequence variant was assigned a unique number and the presence/absence of the variant was recorded for each well.Wells containing three or more sequence variants  were manually examined for evidence of recombination as a result of ex vivo recombination in the well or crossover events during library preparation and sequencing, by visually inspecting a Highlighter plot  generateg method  in R forpol and gp41 amplicons, for a total of four data sets. The clonal prediction scores, which quantify the proportions (maximum 100) of unique sequence variants that would be correctly identified by a particular subsequence [R package coda.We used the SHP (IUPMStats) and Bayesian MTP (IUPMBayes) methods to analyze sequence data from viral outgrowth assays for samples derived from two individuals (denoted 106 and 111) from the Ugandan study cohort. There were two sequence data sets per patient corresponding to the sequence , were 90Additional file 1. Fig. S1. Relative errors in IUPM estimates for simulated data with highly skewed variant frequencies. The composition of this figure is similar to Fig. Additional file 2. Fig. S2. Effect of misspecified prior distributions on posterior estimates of IUPM from real data sets. The filled curves represent the same posterior distributions as in Fig. α = {10, 1,… , 1} for patient 106 and α = {1,…, 1} for patient 111. The dashed curves represent the posterior distributions obtained when these priors were swapped between the patient data sets. These results illustrate that misspecification of the prior distribution on variant frequencies can have a measurable effect on posterior estimates of IUPM where the underlying variant frequencies are skewed toward a single common variant (patient 106). However when the virus population has low clonality and there is a mixture of positive and negative wells at the lowest dilution of the QVOA  on the same simulated data set, where there were eight wells with 106 cells, four wells with 4 × 104 cells, and four wells with 320 cells; the true IUPM was set to λ = 1; and the variant frequencies were set to f = {0.5, 0.25, 0.125, 0.0625, 0.0625}. (left) Decay of autocorrelation with increasing lag between samples in the Markov chain. Throughout the study, we thinned chain samples at a lag of 1000 steps. (right) Traces of posterior probability for the first 10,000 steps of the three replicate Markov chains, which corresponds to the length of the burn-in period used in this study. Based on these results, the rate of approach to the posterior distribution was fairly rapid: on the order of 1000 steps.Additional file 4. Fig. S4. Prominent species identification and elimination of potential outgrowth derived recombinant sequences. a All Pol derived consensus sequences with amplicon totals greater than 0.02% of the total amplicon number for well 5M4  from patient 111 were aligned and viewed in a Neighbor-Joining (NJ) phylogenetic tree. Prominent species were defined as those with amplicon totals > 2.5% of the total amplicon read number for well 5M4 and are indicated with red arrows. b Since the number of prominent species in well 5M4 were ≥ 3 the prominent sequences were aligned and viewed in a highlighter plot. The probable outgrowth derived recombinant sequence is indicated with a red X and was removed from the analysis (probable recombination area highlighted in red box). c All Pol prominent outgrowth sequences for patient 111 were viewed in a NJ tree and variants assigned based on clonality (well 5M4 indicated with red arrows).


Autosomal dominant polycystic kidney disease (ADPKD) is a genetic systemic disorder causing the development of renal and hepatic cysts and decline in renal function. It affects around 1 in 1,000 live births. Early hypertension and progressive renal failure due to massive enlargement of cysts and fibrosis are hallmarks of the disease. This article reviews recent advances in ADPKD and focuses mainly on diagnosis, management, and prediction of the course of the disease. ADPKD arises owing to mutations in thePKD1 gene (about 85% of cases) or thePKD2 gene (about 15% of cases), which encode the proteins polycystin-1 (PC1) and -2 (PC2), respectively3. Compared with patients who havePKD1 mutations, those withPKD2 mutations have a milder phenotype and reach end-stage renal disease (ESRD) about 20 years later6. ADPKD is characterized by progressive development and enlargement of cysts in all nephron segments but preferentially in the collecting duct, and ESRD occurs in 50% of patients by the age of 60 years1. It is associated with cyst formation in other organs, primarily the liver and pancreas, and with cardiovascular anomalies such as intracranial arterial aneurysms and vascular dissections. Huge variation is observed in disease phenotype and progression, even within families; the reason for this is poorly understood, but genetic modifiers, including hypomorphic alleles, probably play a significant role.Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic cause of kidney failure, occurring in more than 1 in 400 to 1,000 live births and accordingly representing a major socioeconomic medical problem globally8. This article guides the reader through recent achievements, and the clinical consequences, within the field of ADPKD.Although a definitive curative treatment is not currently available, there is a disease-modifying therapy using a V2 receptor antagonist (tolvaptan)GANAB andDNAJB11, have recently been identified in a small subgroup of patients10, but the phenotypes related to these mutations are not exactly the same as those caused by mutations inPKD1 orPKD2. This may suggest that, instead of using the name “ADPKD”, it would be appropriate to name these diseases after the mutated gene, as has been done, for example, for ADTKD11. Whereas mutations inGANAB give rise to a mild form of cystic disease that does not progress to ESRD9, mutations inDNAJB11 cause cystic disease in the context of which ESRD may develop in the absence of marked kidney enlargement10.Mutations in other genes, such asPKD1 andPKD2 genes were first identified, genetic testing was performed by means of linkage analysis5. Not only was this time consuming but also, more importantly, several affected and non-affected family members were needed in order to identify the risk haplotype. As soon as thePKD1 gene was identified, it became evident that genetic testing for the gene would be burdensome owing to the large transcript size (>14 kb) and reiteration of the genomic area encoding about 75% of the protein on the same chromosome12. The group of Harris then developed a technique that used long-range polymerase chain reaction and benefited from the few non-repeated nucleotides13. Although this allowed diagnosis of a single individual, it remained time consuming and was also expensive. Over the past 3 years, next-generation sequencing (NGS) has changed the scenario16. The use of NGS for genetic diseases such as ADPKD has reduced costs, improved sensitivity, and reduced turnaround time18. A further advantage of this relatively new technique is that gene panels or whole exome sequencing can be used, allowing the detection of mutations in ultrarare genes that account for a very small number of cases of renal cystic disease17. The drawback of testingPKD1 is the huge number of variants of unknown significance (VUSs) that can be identified, around 10 per patient18. Some of these VUSs have previously been published as mutations, but for many pathogenicity is still to be elucidated19. This is of the utmost relevance when a patient seeks preimplantation genetic diagnosis (PGD) or when a disease-modifying treatment for ADPKD can be offered but there is uncertainty over the diagnosis. PGD has evolved over recent years to become a valuable technique to offer patients with severe genetic diseases, although there are ethical issues regarding its use for non-severe genetic diseases. ADPKD is halfway between these categories, and genetic counseling is highly recommended to ensure sound understanding of the pros and cons of such a procedure20. Probably, each case or family should be counseled differently according to the severity of the disease, but patient attitudes to PGD vary considerably depending on how well the family is dealing with the disease, religious aspects, age, disease severity, and so on.Genetic testing has undergone remarkable progress, as is very evident if one considers the case of ADPKD. When the21, and whereas some are modifiable, many are not22. Among the latter are male sex,PKD1 mutations (worse if protein-truncating (PT)), early development of renal symptoms, early detection of high blood pressure23, and large kidney volumes in relation to age and height25. Modifiable factors that should be highlighted in patients with ADPKD include smoking; blood pressure; lipid levels; water, protein, and calorie intake; and body mass index (BMI)31. Also, some biomarkers, such as MCP-1, FGF23, and copeptin, have been found to be predictive factors of ADPKD progression34.A large number of factors have been identified as influencing disease progression35. Although in the past few years many attempts have been made to provide tools predictive of rapid disease progression, to date there is no gold standard. Some of these predictive tools employ imaging; an example is the Mayo Clinic ADPKD calculator, which uses the height-adapted total kidney volume (htTKV) to classify patients into one of five classes (A–E) on the basis of growth rates: less than 1.5%, 1.5–3%, 3–4.5%, 4.5–6%, or more than 6% per year36. Measuring TKV for use as a prognostic biomarker does not require high precision. Measurement by the ellipsoid equation as well as by means of various imaging modalities is possible37. Another imaging approach that can be used to predict PKD progression involves the measurement of renal length. Bhutaniet al. demonstrated that a renal diameter of 16.8 cm is predictive of chronic kidney disease (CKD) stage 3 within 8 years38. An alternative to imaging-based prognostic strategies is the Predicting Renal Outcome in Polycystic Kidney Disease (PROPKD) scoring system, as shown in a large cohort from Brittany, France23. This scoring system uses both clinical and genetic data to stratify risk of disease progression. Patients may reach a score suggestive of rapid progression if they have hypertension or early urinary symptoms before the age of 35 years plus a PT mutation. Male sex is also included in the scoring system. Obvious drawbacks of this predictive tool are the need to perform genetic testing, which may not be available in many centers, and the impossibility of employing it in patients younger than 35 who are asymptomatic. In addition, it is subject to the need to establish a relationship between early symptoms and ADPKD and the limitation that many rapid progressors with PT mutations do not develop early symptoms.The prediction of which patients will have a rapid progression is crucial not only for the decision of whether to use a disease-modifying drug but also for the recruitment of patients to clinical trials39. When this algorithm was tested in a cohort of 305 patients, it was found that 15.7% fulfilled the ERA-EDTA criteria and that the overall proportion of patients with rapid progression rose to 27% upon incorporation of expanded criteria based on data from the REPRISE  trial40 41. This study, together with a recent assessment of an unpublished large series of patients, calls into question the suitability of the algorithm, especially in its first steps when limiting for age/eGFR and using retrospective eGFR. Each country, even region, in Europe has either approved the use of the drug on these terms or proposed some changes. For example, the National Institute for Health and Care Excellence (NICE) decision in the UK was to treat on the basis of these criteria but only patients with CKD stage 2 to 3 without limiting age, whereas in Scotland they approved its use in stage 1. In the US, the preferred approach for definition of a rapid progressor is based on htTKV, age, and eGFR, basically using the Mayo ADPKD calculator42. Risk assessment in ADPKD is an evolving process that will undergo further refinement as new clinical data are obtained and prediction tools are developed. The performance of risk assessment guidelines needs to be evaluated and validated by real-life clinical data, but it is probable that the use of a composite tool including age, renal function, imaging, family history and possibly genetic testing will be able to define rapid progression very accurately.The European Renal Association-European Dialysis and Transplant Association (ERA-EDTA) Working Groups on Inherited Kidney Disorders and European Renal Best Practice (WGIKD/ERBP) developed a hierarchical decision-making algorithm that may offer guidance on the identification and prediction of rapid disease progression in patients with ADPKD and subsequently identify candidates for tolvaptan treatment26. The target should now be 95–110/60–75 mmHg in 15- to 49-year-old patients with an eGFR of more than 60 mL/min/1.73 m2. First-line management of hypertension should include a blocker of the renin–angiotensin–aldosterone system (angiotensin-converting enzyme inhibitors and angiotensin-II receptor blockers)27. Second- and third-line treatments should be diuretics and beta-blockers. Calcium channel blockers (particularly the dihydropyridine class) should be considered if blood pressure is not controlled by the other agents27. The CRISP (Consortium for Radiologic Imaging Studies of Polycystic Kidney Disease) and HALT studies suggest that moderate dietary sodium restriction (2.3–3 g) be recommended to patients with ADPKD22.Based on the HALT-PKD  trial, the recommendations regarding blood pressure control for ADPKD were recently lowered43. Fluoroquinolones remain the first-line antibiotic treatment, but their side effects and the increasing prevalence of fluoroquinolone-resistant Gram-negative bacilli mean that cyst infections frequently represent a very large burden.In patients with cyst infection, a positron emission tomography/computed tomography scan is recommended to enable determination of the location of cysts for the purpose of diagnosis or drainage45, moderate enhancement of hydration, spread out over the course of 24 hours, is recommended with the goal of maintaining an average urine osmolality of not more than 280 mOsm/L46.Until more information becomes available27. Although protein-restricted diets are usually already low in phosphorus, patient education on the need to avoid processed foods that contain readily absorbed inorganic phosphates is appropriate. Sodium bicarbonate supplementation is recommended in order to treat metabolic acidosis, and the aim is to maintain the plasma bicarbonate level at a minimum of 22 mmol/L27.Based on experimental studies and lessons learned from CKD in general, a daily protein intake of 0.8–1.0 g/kg ideal body weight seems appropriate in ADPKD48. This fact, together with evidence that excess food intake has a detrimental effect both in PKD trials and in CKD in general, means that moderation of caloric intake and avoidance of an above-average BMI should be recommended to patients with ADPKD29.Mild to moderate food restriction has been found to markedly reduce cystogenesis in animal models of PKD49, but a post-hoc analysis of the HALT-PKD trials failed to demonstrate a benefit of statin therapy on renal outcomes50. However, evidence in ADPKD31, and in CKD in general, indicates that it is appropriate to keep the low-density lipoprotein (LDL) cholesterol level at not more than 100 mg/dL.A small trial with statins in children has yielded positive results7. Based on these results, the European Medicines Agency in 2015 approved tolvaptan to slow the progression of cyst development and renal insufficiency in ADPKD in adults with CKD stages 1–3 at initiation of treatment and evidence of rapidly progressing disease51. TEMPO 4:4 showed that benefit is sustained over time52.In the TEMPO  3:4 trial, the vasopressin receptor antagonist tolvaptan was shown to slow the growth of cystic kidneys and the deterioration of renal function8.In 2018, tolvaptan was approved by the US Food and Drug Administration for the purpose of slowing decline in kidney function in adults at risk of rapidly progressing ADPKD. This decision was based on additional information provided by the REPRISE trial, which demonstrated slowing in the deterioration of renal function, even in the later stages of renal failure8. In addition, a small proportion of patients develop liver function abnormalities; it appears that whereas some of these abnormalities can be significant, all resolve upon drug discontinuation53. This rare drug-induced liver injury led to the institution of a risk evaluation and mitigation strategy with monthly liver enzyme tests for the first 18 months and then at 3-month intervals.Currently, more than 6,000 patients with ADPKD are being treated with tolvaptan around the world. However, tolvaptan does have side effects. It markedly impairs urinary concentrating ability and therefore patients experience polyuria, nocturia, and polydipsia57. Recommendations for the management of ADPKD are shown inA few other drugs, such as mammalian target of rapamycin (mTOR) inhibitors, somatostatin analogues, and bosutinib, also underwent clinical trials but failed to show any positive effect on disease progressionMany new drugs are being tested, and it is probable that more than one drug will be needed to target the different abnormal pathways found in PKD cells.


Speaking up behavior is a manifestation the culture of safety in an organization; however, withholding voice is commonly observed. Within one academic teaching hospital, it was the aim to assess students’ speaking up behaviors and perceived culture in order to stimulation of the academic development in terms of patient safety.Survey amongst medical students using a validated questionnaire. Data were analysed using descriptive statistics.th- 6th clinical term, 32% were in their 7th-8th term and 31% were in the 9th-12th term. 69% of students had a specific safety concern in the past four weeks, 48% had observed an error and 68% noticed the violation of a patient safety rule. Though students perceived specific patient safety concerns, 56% did not speak up in a critical situation. All predefined barriers seemed to play an important role in inhibiting students’ voicing concerns. The scores on the psychological safety scale were overall moderately favourable. Students felt little encouraged by colleagues and, in particular, by supervisors to speak up.326 individuals completed the questionnaire (response rate 24%). 37% of responders were in their 5Speaking up behaviour of students was assessed for the first time in an Austrian academic teaching hospital. The higher the term the more frequent students reported perceived patient safety concerns or rule violations and withholding voice. These results suggest the need to adapt the curriculum concept of the faculty in order to address patient safety as a relevant topic. Speaking up can be defined as assertive communication of quality and patient safety concerns by a team member through information, questions, or opinions in situations where clinical rules are neglected, forgotten or even unknown by a healthcare professional (HCP) in order to avoid patient harm –9. EffecWithin our academic teaching hospital, more than 7.000 employees care for patients and more than 3.000 employees are responsible for science and teaching. As medical students have to attend several clinical internships in their academic career, they become automatically junior members of care teams and may therefore also play an important role in supporting patient safety . In 2012It is obvious that courage is needed to speak up as it demands the readiness to stand up and to do the right thing at the right time ,16. HoweSo far, in German speaking countries no data are available concerning speaking up behaviors of medical students, in particular, in relation to their study progress and years of training. To assess the speaking up behavior and perceived culture in one academic teaching hospital in Austria a validated questionnaire was used to assess self-reported speaking up related behaviors, climate and perceived barriers. Results may give potential implications on the development of a patient safety curriculum.The Ethics Committee of Medical University of Graz approved the study (vote#: 30–303 ex17/18).A cross-sectional survey study was conducted among medical students doing their clinical terms at one large Austrian academic teaching hospital. The diploma in human medicine prepares students for their future professions as doctors in all specialties. Theoretical and practical skills are taught in an integral, subject-centred and patient-oriented way. The first part of the human medicine study (four terms) provides knowledge and basic understanding of the human organism. Essential components into general medicine, insight into nursing in the context of a ward internship, which ensures early patient contact are part of the first term. In the second part of the human medicine study (six terms) students develop the knowledge about the healthy and sick organism. It is based on topic-centered, patient-centered, interdisciplinary teaching, including clinical presentations and using new forms of teaching such as problem-based learning. Increasing training of medical skills and abilities continuously ensures the practical application of theoretically acquired knowledge. Students have to complete clinical terms for at least of 18 weeks during the second part of the study. Each clinical term lasts for four weeks and they rotate between units. Finally, during the clinical practical year (2 terms) students are actively integrated as learning team members in everyday clinical practice. In total, students must be clinically active for 48 weeks in a teaching hospital. They deepen and acquire the competences listed in the “Austrian competence level catalogue” for medical skills in accordance with European requirements .th to 12th term were included and invited by the Organisational Unit for Studies and Teaching to participate via an online survey. It could have happened that not all students between the 5th and 10th term were recently exposed to a four weeks clinical term. They were instructed that their responses were collected anonymously and used only in aggregate. The survey was open for four weeks. As proposed by Burns et al., weekly reminders were automatically sent by the system to non-responders in order to increase the response rate [In the present study, students in their 5nse rate . There wSpeaking Up about Patient Safety Questionnaire (SUPS-Q)’. Development and psychometric properties of this survey have been reported in detail [We used the ‘n detail . The insn detail , 11. Forn detail . Few minThe SUPS-Q is a brief questionnaire that assesses health care workers’ perceived patient safety concerns, their past speaking up behaviors, perceived barriers for speaking up, their evaluations of the speaking up climate at their workplace and their anticipated speaking up behavior.The frequency of speak up-related behaviours includes three scales addressing 1) the frequency of perceived safety concerns (3 items), 2) the frequency of withholding voice, i.e., not to speak up in specified situations (4 items) and 3) the frequency of speaking up (4 items). Response options for the items in these scales are anchored to “in the last four weeks” and include “never” (0 times in the last 4 weeks), “rarely” (1–2 times), “sometimes” (3–5 times), “often” (6–10 times) and “very often” (more than 10 times during the last 4 weeks). Thus, higher mean scale values indicate higher frequencies of past speaking up and withholding voice behaviours respectively.Barriers for speaking up are assessed with one multiple choice item asking for the relevance of 6 predefined barriers in bringing up patient safety concerns (yes/no).Speak up-related climate is assessed by 11 items organized in 3 sub-scales: 1) the psychological safety for speaking up scale (5 items), 2) the encouraging environment for speaking up scale (3 items) and 3) the resignation scale (3 items). The answers are coded in a 7-point-Likert scale from “strongly disagree with this statement” to “strongly agree with this statement”. Thus, higher mean scale scores indicate higher levels of perceived psychological safety at workplace, higher levels of perceiving the workplace as encouraging speaking up and higher levels of resignation with speaking up respectively. A high level of resignation is indicative of a poorer safety culture. Survey items are presented in the tables in this manuscript.The survey also covers a clinical vignette that describes a hypothetical speaking up situation. The vignette reads “You are on a daily round with several doctors and nurses. During the round, the attending doctor shakes hands with a patient who recently had surgery. He wants to examine the patient’s wound. However, the attending does not apply gloves and/or does not disinfect their hands.” Responders were instructed to consider their anticipated behaviours if they would find themselves in the situation. They were asked to complete four questions addressing the realism of the situation, the potential for patient harm, their discomfort with and likelihood of speaking up. These questions each used a 1–7 response scale with specifically labelled poles. The survey closes with an open-end question inviting responders to provide feedback, personal experiences with speaking up and comments.Descriptive statistics ) are reported for all items. For items organized in scales, mean scale scores were computed as aggregate measures. Cronbach’s alpha was computed as a measure of scale reliability. To determine associations of survey responses with study term  nonparametric test for trend across terms and analysis of variance (ANOVA) were used as appropriate. We used the nonparametric test for trend across ordered groups developed by Cuzick, which is an extension of the Wilcoxon rank-sum test. A correction for ties is incorporated into the test . Regressth- 6th clinical term, 32% were in their 7th-8th term and 31% were in the 9th-12th term. Of participating students, 53% were females. The largest fraction of responders spent their last training in the department of surgery (36%), followed by internal medicine (15%), pediatrics (8%), orthopedics and traumatology (7%), anesthesiology and intensive care (6%), gynecology and obstetrics (6%), and neurology (6%).Of invited students (n = 1.524), 326 individuals completed the questionnaire (response rate 24%). At the time of survey participation, 37% of responders were in their 5th-6th term, 64% responded they had not observed a potentially harmful error during the past four weeks whereas it were 41% of students in their 9th-12th term (test for trend p<0.001). Rule violations were observed at least once during the past four weeks by 57% of students in their 5th-6th term and by 78% of students in their 9th-12th term (test for trend p = 0.002). The majority of students reported at least one episode of withholding voice despite safety concerns during the past four weeks ; mean 7th-8th term: 2.9 (SD 1.7); mean 9th-12th term: 3.7 (SD 1.9); p = 0.0022).When confronted with the missed hand disinfection vignette, most students rated this situation as realistic  and as posing a severe risk to the patient . Students reported a high level of discomfort to speak up in the described situation  and–on average—anticipated their likelihood to speak up as low , despite the perceived potential harm for patients. Anova analysis revealed significant differences in perceived realism and potential for harm relative to study term, but not in level of discomfort and likelihood to speak up .In sum, students gave 90 open-ended responses. Students’ free text feedback was summarized in two main categories: 1) experiences of students and 2) solutions . Within This study is the first to report speaking up behaviours and climate for medical students in one Austrian academic teaching hospital using the validated SUPS-Q in German speaking countries. Overall, medical students reported perceived patient safety concerns, which increased significantly with students’ term. Furthermore, rule violations were also observed more often by students of higher terms. Although students perceived patient safety concerns and rule violations, they remained silent, which was more obvious in students of higher terms. The majority reported that an important role in inhibiting students’ voicing concerns was the reaction of the person causing a concern is not possible to predict. All predefined barriers seemed to also play an important role in withholding voice. Students felt also less encouraged to speak up which was associated with a missing safety culture in the clinical departments during their internships. It was raised that it is difficult to submit patient safety concerns. Students considered the described vignette to be a realistic scenario in a healthcare setting; however, the likelihood to speak up was low and was accompanied by a high level of discomfort to speak up.67% of students had at least once a specific concern about patient safety in the past four weeks, 48% had observed an error and 68% noticed the violation of a patient safety rule. Though students perceived specifics patient safety concerns, 56% did not speak up in a critical situation during the last four weeks. Perceived concerns of students were similar to that of surveyed HCPs the year before using the same survey instrument: 69% of HCPs had at least once a specific concern in the past four weeks, 57% had observed an error, 58% noticed HCPs who ignored a patient safety rule, but 42% remained silent [The vignette was considered as a realistic scenario and was with increasing term even more realistic. However, the higher the term the lower the risk of harm was estimated. Compared to the previous study, the vignette was scored twice as much with regard to the dangerousness of the scenario . HoweverMedical errors are already the third leading cause of death in in the US hospitals, therefore education and training in patient safety needs to be a prerequisite in healthcare systems . Though On the one hand, the curriculum concept has to be adapted with regard to patient safety aspects. The growing importance of patient safety aspect requires medical education programs as already suggested by WHO and others ,25. The On the other hand, it is also necessary to establish a safety culture at the clinic. On the organizational level, academic clinical departments have a rich tradition of improving approaches such as prevention, diagnosis and treatment of disease, however, quality improvement and patient safety expertise has to be incorporated in academic clinical departments . Furtherth and 10th term were recently exposed to a four weeks clinical term and skipped the survey. Moreover, the generalizability is significantly limited as the study was performed in a single institution in Austria. The study only focussed on medical students; however, it should be also of interest how nursing students during their internships perceive speaking up behaviours.This study has several limitations. A major limitation of this survey was that only 24% responded to the survey though students were reminded three times. One reason could have been that the survey was performed in May to June where students have to pass their exams or that not all students between the 5Speaking up behaviours of students was assessed for the first time in a German speaking academic teaching hospital. Higher perceived risk of harm for the patient, past episodes of speaking up and a more favorable climate score were all significantly associated with a higher self-reported likelihood to speak up. All predefined barriers for not speaking up seemed to play an important role and was associated with a missing safety culture in attended clinical units during their internships. These results give hints that there is a need to adapt the curriculum concept with regard to patient safety aspects. Finally, there is also the need to establish a safety culture at the organizational and unit level. Speaking up can only work if HCPs and students perceive an open minded setting in their environment.


Epidemiological studies have shown that diabetes is a well-established independent but modifiable risk factor for stroke. The aim of this post hoc analysis of data from the Steno-2 Study was to examine whether multiple risk factor intervention reduced the risk for stroke in individuals with type 2 diabetes and microalbuminuria.In the Steno-2 Study, 160 individuals with type 2 diabetes and microalbuminuria were randomised to intensified or conventional multiple risk factor intervention, targeting classical cardiovascular disease risk factors for a mean of 7.8 years, and then followed for a total mean of 21.2 years. The primary endpoint in this post hoc analysis was time to first stroke event.p = 0.004). Also, the number of recurrent strokes was significantly reduced with intensive therapy.During follow-up, 30 participants experienced a total of 39 strokes. Individuals randomised to conventional therapy were more likely to experience a stroke than those in the intensive-therapy group, with 29 total strokes occurring in 21 participants (26%) in the conventional-therapy group vs a total of ten strokes in nine participants (11%) in the intensive-therapy group  contains peer-reviewed but unedited supplementary material, which is available to authorised users. A recent publication from the Global Burden of Disease (GBD) Study 2016 estimates the global lifetime risk of stroke to be 24.9% in the total population for both sexes, with an increase in risk of 15.4% for men and 3.2% for women compared with the previous survey, which was conducted in 1990 [In individuals with diabetes in the USA, an increased incidence of stroke from 1990 to 2010 has been demonstrated. Although a 52.7% decrease in the age-standardised rates of stroke was seen, the increase in diabetes prevalence resulted in a 47% increase in the yearly number of strokes [The importance of individualised intensified multifactorial intervention in the treatment of type 2 diabetes has been highlighted in the Steno-2 Study. In this study, an increased median lifespan of 7.9 years with significant risk reductions for major cardiovascular disease (CVD) events, heart failure and microvascular complications was observed with intensified treatment of multiple risk factors vs conventional therapy . Also, iIn the present post hoc analysis, the primary aim was to examine the difference in time to first stroke event between the intensive and conventional treatment groups of the Steno-2 Study.ClinicalTrials.gov registration no. NCT00320008) has been described in detail previously [n = 80 in each group; Fig. The Steno-2 Study  and by the Danish Data Protection Agency (J.Nr. 2015-41-4042). All patients gave their informed consent upon randomisation and confirmed this on follow-up visits.All CVD endpoints in the Steno-2 Study were defined a priory and have been described in detail elsewhere . The defIn the current analysis, we distinguish between stroke and TIA. This was not the case in the original Steno-2 Study where we used a combination of the two conditions in order to increase power due to the shorter duration of follow-up and, thus, a smaller number of expected events.The primary analysis of the present study was time to first stroke event, with the secondary analyses being time to a combined endpoint of stroke and cardiovascular death and time to a combined endpoint of stroke and all-cause mortality. In sensitivity analyses, cases of TIA were added to the endpoints specified above.HRs for the primary and secondary outcomes of this post hoc analysis were calculated using Cox regression. Recurrent strokes were compared using Fisher’s exact test. Statistical analyses were performed using Stata/IC version 15 . For the primary outcome, proportional hazards assumption was checked by the ‘estat phtest’ function of Stata.Baseline values in each of the two original treatment groups are shown in the ESM Table p = 0.004; Fig. The hazard for stroke was reduced by 69% with intensive therapy vs conventional treatment  and for the combined endpoint of stroke and all-cause mortality   out of the 35 participants with a cardiovascular event experienced a stroke as their first manifestation of CVD, whereas this was the case in 14 out of 51 individuals (27%) in the conventional-therapy group.Of the total 39 strokes evaluated in the study, 35 were ischaemic. The remaining four were haemorrhagic strokes, two of which were the first stroke in one patient in each of the two treatment groups, with the last two being a recurrent stroke occurring in two different individuals in the conventionally treated group.p = 0.018).Individuals who were originally randomised to conventional treatment were more likely to experience recurrent strokes than those originally randomised to intensified therapy, with one participant in the intensive-therapy group vs ten participants in the conventional-therapy group experiencing more than one stroke . However, participants in the original intensive-therapy group survived for a significantly shorter period after a stroke than those originally randomised to conventional therapy . This pattern was not observed for cardiovascular events not related to strokes , for which a smaller proportion of individuals randomised to intensive treatment vs conventional treatment died during follow-up after an event (55% vs 82%), with no difference in survival time after the evident event (median 3.4 years vs 3.9 years).In the present post hoc analysis of data from individuals with type 2 diabetes and microalbuminuria from the Steno-2 Study with 21.2 years of follow-up, the hazard for first stroke after randomisation was reduced by 69% and the absolute risk by 15% with intensive therapy vs conventional treatment. Similarly, individuals originally randomised to conventional treatment were more likely to have recurrent strokes than those originally randomised to intensified therapy. Our findings once again emphasise the importance of an intensified multifaceted intervention approach, according to present EASD/ADA guidelines, for reducing CVD in patients with type 2 diabetes.The proportion of individuals with a stroke during the follow-up period was 11% and 26% in the intensive- and conventional-therapy group, respectively. Since all participants in the Steno-2 Study had microalbuminuria  at baseline, the high incidence of stroke emphasises the importance of early screening for vascular risk factors in individuals with type 2 diabetes.A surprising finding in the present post hoc analysis was the significantly shorter median lifespan after the first stroke of 0.3 years in the intensive-therapy group vs 3.1 years in the conventional-therapy group. We have no conclusive explanation for this finding. However, since the treatment algorithm for intensive therapy recommended a daily intake of 150 mg acetylsalicylic acid (ASA), it is reassuring that there was no increase in the number of cerebral haemorrhage events in those originally assigned to the intensive-treatment group.In diabetic nephropathy, the classic Kimmelstiel–Wilson lesions are nodules of hyaline material in the glomerulus that occur due to an increase in mesangial matrix deposition . In the As previously mentioned, it has been reported that the risk for stroke in individuals with type 2 diabetes has declined on a national scale [The major strength in the Steno-2 Study is the completeness of data for individuals enrolled in the trial. Except for one individual who emigrated from Denmark, leading to a lack of data during the observational part of the trial, a complete track of all endpoints and hospital admissions was available from Danish National Registers and applied in all analyses.One major limitation of the study is the small sample size of 160 individuals. Another limitation is that a high-risk group  were selected as the study population. Therefore, the magnitude of risk reduction we demonstrate here might not be attributable to a lower risk population. However, the risk reductions with intensive therapy presented in the current post hoc analysis are in coherence with previous cardiovascular risk reductions reported in the trial, both during its interventional and observational phase. Furthermore, since all participants were offered intensive treatment after the randomised phase, the effects presented may actually be an underestimation of the true effects.Our study demonstrates that stroke is a frequent and fatal complication in individuals with type 2 diabetes and microalbuminuria. An intensified multifactorial intervention significantly reduced the occurrence of this outcome, as well as the number of recurrent cerebrovascular events.ESM(PDF 75 kb)


Introduction: Recently, the branching pattern of the lateral femoral cutaneous nerve (LFCN) named Fan type has been reported that LFCN injury cannot be avoided in surgical dissections that use the direct anterior approach to the hip joint in the cadaveric study. We hypothesized that the Fan type can be identified by ultrasound The aim of this study was to investigate whether LFCN injury occurs in DAA-THA in cases identified as the Fan type based on preoperative ultrasound of the proximal femur.Methods: Ultrasonography of the proximal femur on the surgical side was performed before surgery and the LFCN distribution was judged as the Fan type or Non-Fan type. A self-reported questionnaire was sent to the patients at two months after surgery, and the presence or absence of LFCN injury was prospectively surveyed.Results: After application of exclusion criteria, 45 hips were included. LFCN injury was observed after surgery in 9 of the 10 patients judged as the Fan type based on the ultrasound of the proximal femur , and no LFCN disorder was actually observed in 25 of the 26 patients judged as Non-Fan type (specificity: 96.2%).Conclusions: To prevent injury of the LFCN in patients judged as the Fan type on the ultrasound test before surgery, the risk of direct injury of the LFCN may be reduced through the approach in which an incision is made in the fascia which is opposite to the radial spreading, i.e., between the sartorius and tensor fasciae latae muscles or slightly medial from it. Of approaches for total hip arthroplasty (THA), the direct anterior approach (DAA) is the only soft tissue-preserving approach using the inter-muscular and inter-nervous plane ,2. FunctHowever, DAA-specific complications have also been pointed out –8, and oRegarding the distribution of the LFCN, many studies using ultra sound and cadavers have been reported, in which abundant distribution patterns were observed, showing individual variation –19. In sThus, we hypothesized that the LFCN distribution in the proximal femur can be identified by ultrasound and LFCN injury develops in the Fan type, whereas small femur offset may be involved in LFCN injury in the types other than the Fan type. The aim of this study was to investigate: (1) whether LFCN injury occurs in DAA-THA in cases identified as the Fan type based on preoperative ultrasound of the proximal femur and (2) whether LFCN injury is likely to occur even in cases identified as Non-Fan type with a small femur offset before surgery.DAA-THA was performed in 147 hips between June 2016 and July 2017. Fourteen cases with previous of surgery of the hip joint on the affected side (including 8 cases of revision surgery), 22 cases of femoral neck fracture, 13 cases of Crowe III and IV developmental dysplasia of the hip, 30 cases in which the examiner of ultrasoud was the operator, and 23 cases operated by a hip joint surgeon with experience of 40 or fewer cases of DAA were excluded [Ultrasonography of the proximal femur on the surgical side was performed before surgery and the LFCN distribution was identified as the Fan type or Non-Fan type based on the classification reported by Rudin et al. . The opeAll patients were positioned supine and were scanned on affected side using the LOGIQ e Expert  with a 12 MHz linear array transducer. Ultrasonography was performed following the method reported by Zhu et al. . The ultAs reported previously, a preoperative CT was used in place of the X-ray to obtain more precise offset measurements according to Pasquier et al. . TherefoAll surgeries were performed by 3 hip joint surgeons with experience of 40 or more cases of THA through DAA ,20. DAA p-value < 0.05 were considered statistically significant. All statistical analyses were performed using IBM SPSS Statistics for Mac IBM Corp., Armonk, NY, USA).We used the Mann–Whitney U test to compare differences in each preoperative offset between patients reporting LFCN injury, versus those with no symptoms. Differences with a We examined the proximal femur of the 45 hips by the ultrasound test before surgical treatment with THA through DAA and surveyed the presence or absence of LFCN injury 2 months after surgery.2 (range 18–35 kg/m2). LFCN injury occurred in 19 (42.2%) of the 45 hips (p = 0.03) (p = 0.21) and total offset  between the 2 groups (p = 0.047) (p = 0.54) and total offset  between the 2 groups . Osteoarthritis was diagnosed in 35 hips (%), osteonecrosis in eight hips (%), and Rheumatoid arthritis in two hips (%). There were 18 procedures involving the right hip (40%). The mean body mass index was 24.2 kg/m 45 hips . Ten hip 45 hips . Thirty- 45 hips . The sen = 0.03) . On the 2 groups . In the = 0.047) . no sign2 groups .Since the DAA for THA is a soft tissue-preserving approach using the inter-muscular and inter-nervous plane, functional discovery after surgery is fast compared to those after surgeries through the other approaches, and the dislocation rate is low, being superior in many points –5. HowevWe consider that acquisition of ability to identify the LFCN distribution before surgery is the first step to prevent anterior approach-induced LFCN injury and this is also useful to take preventive measures in the future. Thus, focusing on non-invasiveness of ultrasonography, we tried to identify the LFCN distribution in patients before THA. LFCN injury was observed after surgery in 9 of the 10 patients identified as the Fan type based on the ultrasound of the proximal femur , and no LFCN injury was actually observed in 25 of the 26 patients judged as Non-Fan type (specificity: 96.2%). These findings suggested that when the LFCN distribution on the ultrasound test is identified as the Fan type before surgery, LFCN injury may develop after surgery at a high probability. Rudin et al. reported that there were 3 main patterns of the LFCN distribution in the proximal femur in a cadaveric study . In the Symptoms of LFCN injury were observed in about 29% of the Non-Fan-type cases that should be able to avoid LFCN injury. The femoral offset was significantly smaller in these cases with LFCN injury. In addition, their offset was also significantly smaller than the Fan type cases with LFCN injury.They are indicated that not only the problem with the LFCN distribution, such as the Fan type, but the femoral offset has an influence on the developmental mechanism of LFCN injury, as previously reported . SeveralThere are several limitations of this study. Firstly, the sample size was small. Secondly, only one examiner performed ultrasonography, so that validity, such as the concordance rate among examiners, could not be evaluated. Thirdly, the presence of LFCN injury was identified based on only subjective evaluation in the questionnaire. No objective evaluation or survey of neurologic manifestation derived from lumbar vertebral disease as differential diagnosis was performed. However, no previous study on LFCN injury performed the echo test before surgery or verified the clinical outcomes after surgery. Thus, we believe that the present study is sufficiently valuable as a preliminary study aiming at reduction of these complications.The authors declare that they have no conflict of interest.