The cardiovascular risk equations for diabetes patients from New Zealand and Chinese electronic health records (CREDENCE) study is a unique prospectively designed investigation of cardiovascular risk in two large contemporary cohorts of people with type 2 diabetes from New Zealand (NZ) and China. The study was designed to derive equivalent cardiovascular risk prediction equations in a developed and a developing country, using the same epidemiological and statistical methodology. Two similar cohorts of people with type 2 diabetes were identified from large general population studies in China and New Zealand, which had been generated from longitudinal electronic health record systems. The CREDENCE study aims to determine whether cardiovascular risk prediction equations derived in patients with type 2 diabetes in a developed country are applicable in a developing country, and vice versa, by deriving and validating equivalent diabetes-specific cardiovascular risk prediction models from the two countries. Baseline data in CREDENCE was collected from October 2004 in New Zealand and from January 2010 in China. In the first stage of CREDENCE, a total of 93,207 patients  were followed until December 31st 2018. Median follow-up was 7.0 years  and 5.7 years (China). There were 5926  CVD events in the New Zealand cohort and 3650  in the Chinese cohort. The research results have implications for policy makers, clinicians and the public and will facilitate personalised management of cardiovascular risk in people with type 2 diabetes worldwide.The online version contains supplementary material available at 10.1007/s10654-021-00795-9. Diabetes is a major cause of cardiovascular disease (CVD) and risk prediction equations are increasingly used to identify diabetes patients at particularly high risk of CVD, to inform personalised treatment decisions . A recenCardiovascular Risk Equations for Diabetes patiEnts from New Zealand and Chinese Electronic health records (CREDENCE) study will be the first study to prospectively derive and validate comparable CVD risk prediction equations, for people with type 2 diabetes, simultaneously in a developed and developing country. This study will not only derive contemporary equations that can be applied to people with type 2 diabetes in New Zealand and China, but will also enable a fair comparison of equations derived in very different settings. If the equations perform well in both settings, this will also provide support for the current approach of deriving CVD risk prediction equations largely in developed countries and applying recalibrated versions in developing countries [As far as we are aware, the ountries . The preThe overall study design is shown in Fig. PREDICT is an open cohort, with participants entering following opportunistic risk assessments between 27th October, 2004 and 30th December, 2016. In the PREDICT study, some data items were entered onto the electronic PREDICT template within the EHR by the patient’s general practitioner at the time of the risk assessment, while other items were already in the EHR. The index date used in PREDICT was the date a patient’s CVD risk assessment was completed by their primary care practitioner, when all cardiovascular risk factors required for the risk assessment were available in the EHR. The PREDICT-T2D cohort included: (1) patients whose primary care EHR indicated they had type 2 diabetes at the time of their index CVD risk assessment; (2) patients dispensed oral hypoglycaemic agents or insulin at the time of assessment; or (3) those with diabetes-related ICD codes  in hospital discharge records prior to the index risk assessment. Required variables that were not already available in the EHR (e.g. duration of diabetes) were entered onto the PREDICT electronic template by the patient’s primary care practitioner at the time the risk assessment was completed.In the CHERRY-T2D cohort, we included: (1) patients with type 2 diabetes who had already entered the CHERRY study at an index date set as 1st January 2010; (2) patients with diabetes who entered the CHERRY study after 1st January 2010, and their index date was set as their date of registration for health service; and (3) patients from the original CHERRY study who were newly diagnosed with type 2 diabetes between 1st January 2010 and 31st December 2018, where their index date was the date of diabetes diagnosis  lower than 30 ml/min/1.73 mhave no valid healthcare identifier or had inconsistent or contradictory variables recorded across data sources.In the first stage of the CREDENCE study, we will derive CVD risk prediction equations for patients with diabetes. Patients from the two cohorts will be excluded if they:History of prior CVD or renal dysfunction is defined by corresponding ICD-10 codes . Patients prescribed loop diuretics and digoxin (proxy for heart failure) will be also excluded.We have applied identical ICD codes of relevant disease status used in the New Zealand and Chinese sites, selected after considering the differences in patients’ recruitment methods due to the different health care systems and disease screening programmes. After applying the same inclusion and exclusion criteria to the two studies, both PREDICT and CHERRY T2D cohorts included approximately 46,500 primary care patients with type 2 diabetes aged 30 to 74 years.1c, total and HDL cholesterol, history of atrial fibrillation, blood pressure lowering medication, lipid lowering medication and oral hypoglycaemic medication or insulin , urinarn Tables . The metFor each patient, data was sought on each of the following outcomes and their dates of diagnosis: non-fatal CHD, non-fatal stroke, transient ischaemic attacks (TIA), cause-specific CVD mortality  and other cardiovascular diseases. The primary outcome will be CVD defined according to the ICD-10 codes listed in Table Both PREDICT and CHERRY studies were designed with electronic follow-up procedures. In the PREDICT study, primary care patients are “electronically followed” every 1–2 years through encrypted National Health Index (NHI) number linkage to routine national hospitalisation and mortality databases. In CHERRY, records of CVD, other chronic diseases and deaths are updated annually. In the first stage of CREDENCE, the follow-up time was defined from index assessment  until the first CVD event, death due to other causes, withdrawal from the cohort for various reasons or end of follow-up . We plan to extend the follow-up period in future study stages.We will follow the Transparent Reporting of a multivariable prediction model for Individual Prognosis Or Diagnosis (TRIPOD) guidelines . This fiWe have presented the proportions of missing values for each proposed risk predictors in the two CREDENCE cohorts in Table In PREDICT and CHERRY, all data are linked using unique encrypted identifiers. Sets of logical, technical and administrative controls are implemented. Data access rights are assigned to personnel according to their role in the study. Third-party companies (Enigma Solutions Ltd for PREDICT and Wonders Information for CHERRY) are engaged, where necessary, to handle data extraction, linkage and storage to ensure privacy protection. Currently, data contained in the CREDENCE study are not freely available on a public server. Therefore, scientists who are interested in the CREDENCE study cohorts should contact the study investigators regarding potential collaborations.As shown in Fig. Demographic characteristics of the CREDENCE cohorts, stratified by country and sex, are presented in Table 2 and 24.1 km/m2, respectively). The New Zealand study participants also had, on average, higher levels of HbA1c (62.3 mmol/mol in PREDICT-T2D and 59.8 mmol/mol in CHERRY-T2D), and lower levels of eGFR (89.6 ml/min/1.73m2 in PREDICT-T2D and 95.6 ml/min/1.73m2 in CHERRY-T2D). The prevalence of current smoking was twice as high among Chinese men (36%) versus New Zealand men (17%), whereas fewer than 1% of Chinese women were current smokers, compared with more than 13% of New Zealand women . Proportions of patients on several medications, particularly statins, were also higher in the PREDICT-T2D cohort, and more patients in the New Zealand cohort had atrial fibrillation at index assessment. Other predictors are broadly comparable in the two cohorts.Medical history and baseline measurement of CVD risk factors in the two CREDENCE cohorts are presented in Table Table The CREDENCE study is a unique international comparative study in which patients with type 2 diabetes, meeting specified inclusion and exclusion criteria, have been prospectively identified from EHR-based general population cohort studies in New Zealand and China. In the first stage of the study, we aim to derive and evaluate CVD risk prediction models using the same methodological framework.The prevalence of diabetes is increasing in most countries. CVD risk assessment of patients with diabetes is essential for effective personalised disease management –12. NumeThough models in the first category are simple to implement in clinical practice, many key predictors for patients with diabetes are not included, e.g. diabetes duration, renal function, diabetes pharmacotherapies. A number of attempts have been made to compare the performance of diabetes-specific models versus general population models, with diabetes-specific models generally performing better than general population models . HoweverGiven these issues, it is unknown which CVD risk prediction models will perform best in different populations. Model comparisons and validation studies have also been complicated by between-study variation in inclusion criteria, study timeframes, predictor and outcome definitions, distributions of risk factors, duration of follow-up, as well as differences in background risks of study populations. As a result, few comparative studies can be considered fair comparisons. The CREDENCE study aims to address these issues by adopting a uniform approach to development and validation of risk prediction models, derived in two cohorts from very different settings, that were prospectively defined to ensure they were comparable.The CREDENCE study has a number of strengths. Firstly, the study is a prospectively designed comparative study conducted in New Zealand and Chinese type 2 diabetes patient cohorts, in which equivalent CVD risk prediction equations will be developed. The PREDICT study in New Zealand and the CHERRY study in China, from which the diabetes cohorts were identified, are comparable large-scale population-based cohort studies derived from EHR systems. Although some baseline characteristics of patients with diabetes are different in the two cohorts, that is to be expected and we have applied the same inclusion and exclusion criteria for cohort entry to minimise these differences. Furthermore, we have used comparable definitions of risk predictors and outcomes and we will develop and evaluate CVD risk prediction models using the same methodological framework, including consistent methods of modelling and performance assessment, etc. This also allows the prediction models developed in one cohort to be externally validated in the other, hence providing information on generalisability of models.Secondly, the study can provide contemporary CVD risk prediction equations for use in patients with type 2 diabetes in New Zealand and China, to fill an important research and practice gap. To the best of our knowledge, no diabetes-specific CVD risk prediction model has been developed in mainland Chinese patients with type 2 diabetes in a real-world population. Finally, both cohorts are large, natural population-based cohorts with repeated longitudinal measurements specifically designed to investigate CVD risk management and clinical outcomes. This will facilitate research on how risk factors influence the onset and progression of CVD events in patients with diabetes over the life course.Routinely collected electronic health data has some advantages over data collected in epidemiological research studies in terms of size, generalisability and representativeness, as well as completeness of follow-up when it can be linked to comprehensive outcome databases. According to the TRIPOD guidelines, prediction models using real world data such as data from healthcare records are recommended for both public health and clinical practice. However, this approach generally involves less comprehensive and less precise protocols for data collection compared to traditional research studies.The study also has several other limitations. Firstly, both original cohorts draw information from administrative databases and electronic health records that are designed for healthcare management but not for the epidemiological research. Therefore, there could be possible misclassification regarding the subtypes of diabetes. However, this misclassification is likely to be very small as the incidence of type 2 diabetes is much higher than type 1 diabetes, and the age of diagnosis was over 30 years for most patients (95.7% in PREDICT-T2D and 99.7% in the CHERRY-T2D); most type 1 diabetes is diagnosed at much younger ages [The CREDENCE study is an international collaboration in which equivalent CVD risk prediction equations will be derived from comparable cohorts of patients with type 2 diabetes: one from New Zealand and the other from China. The cross-country nature of the study design, the use of comparable analytical approaches, coverage of the total diabetes populations within defined geographic regions, and the availability of repeated measurements of cardiovascular risk factors and clinical outcomes, make it a unique resource for research on the CVD risk and risk management.Supplementary file 1 (DOCX 42 kb)Below is the link to the electronic supplementary material.


Out of 16 science concepts categorized, the common science concepts causing misconceptions among students were investigated to understand item difficulty patterns using Rasch measurement. The findings of this study evaluated that 32 developed items are valid and reliable whereby the item difficulty estimates ranged from −5.13 logits to 5.06 logits. Chemistry is the scientific discipline with the highest mean logits than other disciplines. There is no significant item difficulty estimate across the science disciplines. We also found DIF issues in one item based on gender and four items based on grade. This study contributes a significant role in mapping and informing item difficulty patterns in science concepts to tackle teachers' problems in assessing and teaching science concepts to improve the students’ science performance. Future studies and limitations are also discussed.Understanding item difficulty in science concepts is essential for teachers in teaching and learning to avoid student misconceptions. This study aims to evaluate the patterns of item difficulty estimates in science concepts exploring student misconceptions across physics, biology, and chemistry and to explore differential item functioning (DIF) items in the developed diagnostic test on the basis of gender and grade. Participants were drawn from 856 students  comprising senior high school students from 11 Item difficulty; Rasch measurement; Misconception; Science; Diagnostic test. The particular science concept provides a practical framework for integrating science disciplines and has a significant impact on the learning process and in the thinking and modeling of natural and technological processes. Several studies reported students' difficulties in learning scientific concepts. Indonesian students' science performances were ranked the lowest in the 2018 PISA report involving 41 countries as target participants . The issThe goal of objective measurement locates at the core of science, and science education research should also attempt to carry the instrument according to objective measurement criteria. Our study evaluates item difficulty estimates using a standardized instrument to assess the distributed science concepts misconceptions to students across the science disciplines using Rasch measurement and explores DIF. Although some research concentrates on students' science conceptions of particular concepts, to what extent students have experienced the ease or difficulty in understanding science concepts has not been fully elucidated using a standardized instrument to measure concepts comprehension across science disciplines. This study will fill the gap in empirical research that provides evidence related to students’ difficulties in understanding science concepts across disciplines, especially science concepts that generate misconceptions in students on the basis of key concepts in the findings of previous research findings by 22.1Misconceptions are defined as misunderstandings and interpretations that are not scientifically accurate, showing inaccurate prior insight and wrong ideas . MisconcNumerous studies have been conducted regarding students; the understanding of science concepts in various disciplines e.g., . StudentAlthough most research is related to student misconceptions in science concepts across disciplines, only a few studies focus on understanding the inherent difficulty level of items in science concepts in various science disciplines e.g., . Recentl2.2Student misconceptions are difficult to identify with traditional methods. Educators have to revise and identify student misconceptions to help students understand new concepts and finally provide opportunities for students to apply these concepts to science problems . To evalResearchers in science majors have used and developed numerous instruments to assess student misconceptions or student conceptual understanding . Two-tie2.3Rasch measurement is a measurement model developed by the Danish mathematician, George Rasch. Rasch measurement is formed on the basis of item–person interactions and probability estimates. Using equations, the interaction between the item and person can be elucidated and described. People who have low ability should not de facto be able to answer items that have a high difficulty level . The proThis study focused on analyzing item difficulty levels for science concepts across disciplines using two-tier multiple-choice diagnostic tests. The Rasch dichotomous model was used to analyze dichotomy data, where 0 was categorized as the misconception and 1 was categorized as the correct answer. The dichotomy data were used to generate the item difficulty level in logits. Rasch measurement was chosen because this method can overcome some limitations in Classical Test Theory (CTT) such as (a) the measurement or data analysis was constructed using interval data not categorical or nominal data; (b) the items' difficulty level and the person's ability are independent; (c) the parameter reliability can measure items and persons and depends on the size of the sample; and (d) the data on the measurement of Rasch explains the response at the individual level, not group-centred statistics .3(1)Are the items on the instrument used valid and reliable?(2)What are the item difficulty patterns measured by diagnostic instruments for assessing student misconceptions on science concepts?(3)To what extent are the item difficulties able to describe the concepts that cause students misconceptions across disciplines and science concepts?(4)Are there any DIF issues based on gender and grade?The study investigates item difficulty patterns, item–person map interaction, and DIF based on gender and grade across science disciplines using the two-tier multiple-choice diagnostic test for assessing student misconceptions. Hence, we set out the following research questions;44.1Participants were drawn from 856 senior high school students and a pre-service science teacher in Pontianak, West Kalimantan province, Indonesia. We selected 11 classes randomly from five different schools in total as representative schools in this area. All participants in this study were students from three different school levels, 10th, 11th, and 12th grades, and pre-service science teachers. The paper-based test was administered at the schools and university. Students and pre-service science teacher spent 120 min completing the test under the supervision of researchers and teachers. 4.24.2.1The background questionnaire was adapted from the Indonesian version of the PISA 2015 SES instrument . The que4.2.2To capture student misconceptions or alternative conceptions, we implemented the developed two-tier multiple-choice diagnostic test. The two-tier test cannot differentiate students who are just guessing answers and related confidence level, and some researchers usually applied CTT analysis and the CRI . OtherwiThe two-tier multiple-choice diagnostic test consists of two-level questions. The first tier question asks about science content, and the second tier question asks about scientific reasoning. Students can choose one choice in the second tier or write down their own reason in the form of an open-ended answer to explain the related science content. 4.3Before applying data collection in schools and universities, researchers asked permission to administer the tests to related institutions and were granted ethical research approval from the university. With the help and supervision of teachers, the paper-based test was implemented in the classroom. For item scoring, the correct answer was scored as 1 point, and an incorrect answer was scored as 0 points for all the items. Students get 1 point if they address the task correctly in the first and second tiers.The Winsteps version 4.8.0 software  and Stat55.1Rasch analysis provided two parameters of reliability; item reliability and person reliability, ranging from 0 to 1. Both the item and person reliability are acceptable in this study at 1.00 and 0.8, respectively , and theValidation criteria based on item fit statistics, standardized mean square residual (ZSTD), and the mean square residual (MNSQ) indicated two items with positive point biserial correlations (PTMA) values: BIO21 (.17) and CHEM23 (.08) do not meet the fit criteria with an outfit MNSQ above 1.6. The ideal outfit and infit MNSQ are 1 based on the Rasch measurement model, but the acceptable values range from 0.5 to 1.5 (approximately 1.6 still acceptable) and infit and outfit ZSTD ranging from −2 to +2 sequentially . If the For the person fit criteria, the mean of outfit and infit MNSQ are 0.95 and 1.01, which is close to the ideal threshold around 1, and the mean of infit and outfit ZSTD are -0.1 and 0.1, which are still acceptable. The result from the person fit criteria confirms that participants in this study are fit based on Rasch measurement.5.2The principal component analysis of Rasch (PCAR) was used to evaluate instrument dimensionality. The two-tier multiple-choice diagnostic test was used to assess student misconception in science, so we assumed that the unidimensionality criteria as a single factor to measure misconception in science as a latent construct. Based on PCAR, a test only measures a dimension if the minimum variance explained by the measure is >30 % . ResultsLocal independence confirms that the performance of one item is independent of the performance of other items, with the raw residual correlation between pairs of the items <0.3 . the ite5.3We calculated the standard deviation (SD) and the mean of average item difficulty measure for each of the three science disciplines, that is, physics, biology, and chemistry, using item difficulty estimates or logits of items . Table 4Additionally, we also calculated the item difficulty estimates (measure) on the basis of the 16 science concepts as shown in A two-way Analysis of Variance (ANOVA) was used to analyze the effect of science concepts and science discipline on item difficulty estimates based on logits. The 2 × 2 ANOVA group in this study achieved the assumption of homogeneity variances based on Levene's test (p > 0.05). To validate the normality data assumption, the Kolmogorov–Smirnov test was run before conducting the two-way ANOVA. The results showed that the item difficulty estimates did not differ significantly from normality (p > 0.05) with kurtosis (2.21) and skewness (−0.14).As presented in 5.4For understanding concepts in science distributing misconception to students, we can inspect the item difficulty estimates results from 5.5DIF analysis was performed to assess differences in item function on the basis of gender and grade on all items in test. DIF analysis investigated item responses on the basis of categorical variables for each item on assessing student misconceptions using a test . Differe6Through the statistical analysis, we have confirmed that all items used in the developed instrument meet the valid and reliable criteria according to the parameters for the Rasch measurement. The 32 developed items have outfit and infit MNSQ ranging from 0.13 to 1.72 see , wherebyThe difficulty item pattern in the 16 science concepts studied had different average item difficulty levels based on three specific disciplines offered in Indonesian schools refer to . The aveThe results of the two-way ANOVA show that there is a significant effect on the difficulty estimates of whole items on each science concept, p < 0.05. There is also a significant interaction between science concepts and disciplines. However, the item difficulty estimates did not differ significantly in the three different science disciplines, p > 0.05. These findings are consistent with previous studies that found the item difficulty estimates in science concepts did not differ by science disciplines . This fiTo investigate the item difficulty estimates for each science concept in the present study, we categorized the average item difficulty estimates for each concept into four categories in DIF confirms that CHEM32 has differences based on gender. In CHEM 32, the item difficulty estimates for females, DIF measure, is 4.69 logits, and for males, the DIF measure is 5.70. These results were in line with previous studies by  that rep7In summary, all items in the developed two-tier multiple choices diagnostic test meet the valid and reliable criteria. Our study confirms that the difficulty level of items on various science concepts is not universally based on science topics, but they are connected or similar across science disciplines, especially in physics, biology, and chemistry. We also found particular items in the science concept may have different difficulty levels based on gender and grade.7.1We accept that there are some limitations in our study such as the items not covering all concepts in science learning, only selecting a few items across disciplines that persistently generate misconceptions, the fact that the dataset used only cross-sectional data, and the lack of racking analysis. Some of the limitations outlined above are the improvements that we must consider in further research. Variations of items studied in the further work should be able to cover all science concepts taught across science disciplines, especially at the senior high school level, so that researchers can map the overall item difficulty level of whole science concepts. Time series data collection or longitudinal research design must be added to explore whether there is a change of item difficulty level with the racking method in the Rasch measurement. Racking analysis allows researchers to evaluate whether there is a change in the difficulty level of the item on the different testing times sequentially . We feelFor a future study, we will explore in more detail person ability estimates to identify students’ misconceptions and investigate whether students are guessing answers or have inconsistent answer patterns. By evaluating student misconceptions in science, teachers can determine the extent to which students experience misconceptions in science learning.Soeharto Soeharto and Benő Csapó: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.10.13039/501100015763University of Szeged Open Access Fund (grant number: 5436).This work was supported by the https://figshare.com/articles/dataset/item_difficulites_pattern_sav/16926115/1.Data associated with this study has been deposited online at The authors declare no conflict of interest.No additional information is available for this paper.


Advancing age causes reduced hippocampal neurogenesis, associated with age-related cognitive decline. The spatial relationship of age-induced alterations in neural stem cells (NSCs) and surrounding cells within the hippocampal niche remains poorly understood due to limitations of antibody-based cellular phenotyping. We established iterative indirect immunofluorescence imaging (4i) in tissue sections, allowing for simultaneous detection of 18 proteins to characterize NSCs and surrounding cells in 2-, 6-, and 12-month-old mice. We show that reorganization of the dentate gyrus (DG) niche already occurs in middle-aged mice, paralleling the decline in neurogenesis. 4i-based tissue analysis of the DG identifies changes in cell-type contributions to the blood-brain barrier and microenvironments surrounding NSCs to play a pivotal role to preserve neurogenic permissiveness. The data provided represent a resource to characterize the principles causing alterations of stem cell-associated plasticity within the aging DG and provide a blueprint to analyze somatic stem cell niches across lifespan in complex tissues. Somatic stem cells divide throughout life to generate progeny that is required for tissue homeostasis and repair . With ad+ cells were poorly correlated between rounds. While staining was impaired for ID4, the quality was improved for MT3, suggesting the elution step may have some efficacy for antigen retrieval.To achieve multiple rounds of 4i on tissue, sections needed to be adequately mounted to minimize loss of samples through detachment without affecting antigenicity. Coating the glass-bottomed well-plates with poly-d-lysine (PDL) was effective to preserve tissue adhesion across multiple cycles of 4i in sections of 3-month-old adult mouse brain , human eAfter establishing a robust protocol for iterative immunostainings in complex tissues, we used 4i to analyze age-associated cellular and molecular alterations in the DG of the hippocampal formation. NSCs generate new neurons throughout life in the DG. However, hippocampal neurogenesis is significantly decreasing during adulthood . To test+ cells decreased with advancing age, KI67+ R cells did not change. Additionally, the density of immature neurons, positive for DCX was strongly reduced at 12 months of age  . As expes of age .In the neurogenic niche, R cells interact with and are regulated by several different cell types that are present within their DG niche . To asseWe next used 4i to analyze the expression levels of selected proteins in the same R cells identified in the quantification of cell density. We focused on HOPX and MT3, which have been identified to be present in R cells and may be related to R cell quiescence . SupportTo further validate the 4i approach on tissue sections, we analyzed age-related expression of LaminB1 (LB1), one of the four intermediate filaments that make up the nuclear lamina, and where reductions with aging and the premature aging phenotype of progeria have been previously described . Confirm+ R cells; however, their contribution to increased GFAP vasculature coverage with advancing age appears unlikely given the substantially reduced coverage of Nestin-expressing cells  of radial processes extending from R cells . While lTo further confirm the findings of the randomized microniche analyses, we took a more targeted approach, measuring the volumetric distribution of niche cells radiating out from Nestin-labeled radial processes and KI67-labeled proliferating cells . ConsistWe here used 4i on tissue sections with the aim to characterize age-related changes in the number and distribution of a variety of cell types within individual tissue sections of the DG. We show that alterations within the neurogenic niche of the DG occur relatively early in life and identify changes in niche cell composition of the BBB and microenvironments surrounding adult NSCs as putative regulators of neurogenic permissiveness throughout life.Reduced levels of neurogenesis in the mammalian DG have been associated with age-related cognitive decline and neurodegenerative diseases, such as Alzheimer’s disease . Thus, pWe here used 18 different antibodies to label identical tissue sections. However, that number may be easily scaled up when required. We show that 4i is applicable to a variety of mouse and human tissues, indicating its potential use in basically all tissues and organs. Thus, 4i will be useful to analyze a plethora of tissues and may turn out to be especially useful when only limited tissues/cells are available. For instance, it is a current fundamental limitation in clonal lineage tracing experiments that daughter cells cannot be definitely phenotyped. Rather, current limitations only allow for exclusion-based phenotyping, at least when starter/mother cells can generate a diverse set of daughter cell fates . Thus, to date, all clonal lineages that generate >2 cell types can only be partially phenotyped and diverse cell types cannot be conclusively classified within the exact same clone . 4i on tWe here demonstrate the applicability of iterative immunostaining in tissue sections using 4i, provide a comprehensive data set of the aging DG with a large set of combined markers, and identify vasculature- and glia-associated changes within the neurogenic niche that may be responsible to mediate the age-related decline of neurogenesis in the mammalian hippocampus.Animal experiments were approved by the Cantonal Commission for Animal Experimentation of the Canton of Zurich, Switzerland in accordance with national and cantonal regulations . In developing the 4i protocol for tissue sections, brain tissue was collected from male and female C57Bl/6 mice ranging between 2 and 4 months of age. To attain embryonic day (E) 14.5 embryonic samples, daily plug checks were used to estimate the date of conception. Embryos were collected 14 days following the observation of a vaginal plug. For the aging study, C57BL/6J males at 2 (n=3), 6 (n=3), and 12 months (n=3) of age were used. All mice were kept in a 12 hr light/dark cycle with food and water provided ad libitum.For adult tissues, animals were transcardially perfused using a peristaltic pump with cold 0.9% saline solution followed by 4% paraformaldehyde (PFA) in phosphate buffer. Brains were collected and post-fixed in 4% PFA at 4°C for 8 hr. The brains were then cryo-protected in 30% sucrose for 2 days prior to sectioning. Adult mouse brain tissue was sectioned into 40 μm coronal sections on a cryotome (Leica SM2010R). For E14.5 samples, embryonic heads from C57Bl/6 mice were washed in phosphate-buffered saline (PBS) and fixed with 4% PFA overnight. Cryoprotection occurred in two steps, first for 2 days in 15% sucrose followed by 2 days in 30% sucrose, before being flash-frozen and embedded in OCT Compound (Tissue-Tek) using liquid nitrogen. Sectioning was done coronally at 40 μm using a cryostat. Sections were collected and washed with PBS to remove the remaining OCT before being transferred to cryoprotectant solution . Forebrain-specific organoids derived from hESCs  were generated as described before , and fixPrior to mounting, glass-bottomed 24-well plates (Cellvis P24-1.5H-N) were coated with 1 mg/ml PDL (Sigma-Aldrich P6407). 75 μl of the PDL solution was transferred into the center of each well and brushed to the edges with a fine paintbrush to ensure total coverage. The plates were rocked on a shaker for 5 min, after which the PDL was collected for reuse. Wells were rinsed three times with deionized water and let dry for at least 2 hr. Tissue sections were washed three times in PBS, loaded into wells containing 500 µl 1× PBS, which was subsequently aspirated allowing the tissue to lie flat and dry for approximately 20 min until there was no visible liquid remaining around the edges of the sections. The tissue was then rinsed for 30 s with 4% PFA and washed in PBS three times for 5 min each. Organoids sections were gently aspirated using a P1000 pipet with a trimmed tip and transferred into a well of a glass bottomed well plate. The sections were allowed to settle onto the glass before carefully removing the excess liquid and allowing to dry. Mounted organoid sections were additionally fixed with 4% PFA for 15 min followed by three washes with PBS. Prior to staining, the organoid sections were washed with the elution buffer three times for 5 min as a form of mild antigen retrieval.Once mounted, sections were blocked for 1 hr at room temperature in a blocking solution containing PBS with 3% donkey serum, 0.5% Triton-X, and 0.025% PFA (PBS++). Sections were incubated in the primary antibodies , dilutedBefore imaging, fresh imaging buffer was prepared by dissolving 0.7 M N-Acetyl-Cysteine in 0.2 M phosphate buffer, using 10 M NaOH to adjust the pH to 7.40. Approximately 500 µl of the imaging buffer was added to each well. All 4i images were taken on a Zeiss LSM800 confocal laser-scanning microscope. Images from tile scans were exported using Carl Zeiss ZEN Blue software and stitched using Fiji . For the2O, then 150 µl of dH2O followed by 150 µl of the elution buffer was added to each well, shaken for 5 min, and repeated for a total of three washes. After elution, the tissue was washed three times for 5 min in PBS, after which the blocking step for the next round of IF could be started.A stock solution containing 0.5 M L-Glycine, 3 M Urea, and 3 M Guanidine hydrochloride was prepared and kept at 4°C. Prior to elution, 0.07 M (0.02 g/ml) TCEP-HCl was added to a volume of the stock solution determined by multiplying the number of wells by the working volume of 150 µl by three washes. The pH of the buffer was then lowered to 2.5 using 5 M HCl to facilitated denaturation of the antibodies. The tissue was first rinsed with dHTo achieve accurate alignment between rounds of staining, images were registered using consistent DAPI intensity patterns. At a high digital zoom, the x and y coordinates, as well as the stack position of pixels in DAPI puncta that were recognizable across cycles and were measured. Images were transformed in the x and y planes to align to the image from the first round, with the canvas size being adjust so no image cropped and data was lost. For alignment in the z-plane, blank frames were added or frames lacking positive fluorescent signal were subtracted from the image stack accordingly using the ‘Add Slice’ and ‘Delete Slice’ functions in ImageJ.Two brain sections from 2-month-old mice were mounted and stained for all 18 antibodies used in the main study. Regions of the DG were imaged with a Zeiss LSM800 confocal laser-scanning microscope at 20× with the same parameters as in the main study. The sections were then subjected to six rounds of elution washes (3×5 min). The brain sections were incubated with the same secondary antibodies used in the first round and re-imaged to confirm successful antibody removal. A subsequent cycle of staining and imaging was performed with the same primary and secondary antibody combinations as in the first round . Images All quantifications were performed on non-projected 3D z-stack images with cells being sampled throughout the entire thickness of the image. All measures of density and intensity were performed on 16-bit images, while volumetric analyses were performed using 8-bit binary representations of the original images.+/SOX2+ soma with a clear vertical process that was also GFAP+ and S100β−. ROIs were created outlining the cell soma using the polygon selection tool in Fiji, which were saved and later used to measure fluorescent intensities of stem cell markers HOPX, and MT3, the intensity of the nuclear envelope protein LaminB1, as well as checking for the presence of the cell cycle marker KI67. NR glia-like cells in cell cycle were identified by being KI67+ and located within the lower granular cell layer or SGZ extending approximately 20 µm beyond the hilar edge of the GCL. Immature neurons were counted using DCX. Positive cells had a clear ring of non-nuclear staining that encircled a significant majority of the cell (approx. 75%). Glial cell types, astrocytes, microglia, and oligodendrocytes, were identified as being S100β+, IBA1+, or NG2+, respectively. All cells were counted throughout the entire images using the Cell Counter plugin in Fiji using localization identifiers for the ML, hilus/CA3, and the supra and infrapyramidal blades of the GCL. The areas of the hippocampal subregions were measured. Cell density was calculated by dividing the number of cells by the regional volume expressed as mm3 (region area [mm2] × tissue thickness [0.04 mm]).Cell counting: R cells were defined by HOPX+ astrocytes were also used to measure the intensities of HOPX and MT3 in astrocytes.Fluorescent intensity was measured in the z-position in which it was brightest for each cell. Measures were normalized to the background intensity, which was averaged from five ROIs per slice containing no positive cells within the GCL. Cells were normalized to the background of the representative slice in which the cells were measured to account for any gradient in fluorescent intensity caused by unequal antibody penetrance. The fluorescent intensity of LaminB1 was measured in R cells, immature neurons, and PV interneurons using the ROIs generated during cell counting. For glial cell types, 100 cells were selected at random throughout the imaged hippocampal structures and somatic ROIs were drawn. To ensure accurate measurement, if necessary, ROIs were adjusted to better fit the LaminB1 outline. Intensity measures were normalized to the background, which was averaged from five ROIs per slice throughout the tissue containing no positive signal. Like with the RGL markers, normalization was slice specific to account for a gradient in intensity across the z-plane. The ROIs made to measure LaminB1 in S100βVasculature dimensions: All measures of vasculature dimensions were conducted using CollagenIV staining within the visible areas of the hippocampus . To calculate vasculature density, volumes were generated based on Cavalieri’s principle, summing the area of blood vessels in each slice of an image stack and dividing by the tissue volume. Vasculature areas were measured by applying a 3D Gaussian blur with pixel radii of 2, 2, and 1.5 in the x, y, and z directions, respectively, thresholding at a fluorescence intensity of 1500, followed by two binary erosions to generate an accurate binary representation of the CollagenIV staining.+ RGL processes into an ROI and clearing the outside. Binary representations were created the same as was done for CollagenIV for CD13, GFAP, and the isolated Nestin processes with thresholding at lower intensity limits of 1800, 5250, and 2000, respectively. For NG2, and IBA1, a median filter with a radius of 2 pixels was applied and thresholds were applied at 5500 and 2000, respectively. ROIs were generated by creating selections around the binary CollagenIV and Nestin, which were then applied to the other stains, measuring the area of positive pixels within the ROIs. The measured areas were normalized to the total ROI areas to get percent of coverage. S100β was excluded from all contact and volumetric analyses as the level of background prevented the creation of accurate binary representations.Cellular interactions: Cellular interactions were estimated by measuring the area of colocalization of cell markers on the vasculature and RGL processes. RGL processes were manually isolated from vasculature in Nestin stains by outlining NestinRandomized niche mapping: Binary representations of DCX, HOPX, SOX2, PV, ARC, and KI67 were generated in the same manner as CollagenIV with thresholds set at 2500, 2200, 1600, 4300, 2500, and 2000, respectively. In generating binaries with intensity thresholding, Arc and Ki67 images had small false positive puncta and required additional processing to remove these background elements. Particle analysis was run to select all for positive cells using a lower area limit of 25 µm for ARC, and 15 µm for Ki67. ROIs were saved and used to draw the cells onto a blank canvas. To generate randomized sample regions, ROIs were created around the GCL using maximum z-projections of DAPI stains, which were then overlaid and filled in white on a blank image. A second equivalent blank image was subdivided by drawing vertical white bars extending the height of the canvas with a width of 1 pixel, spaced 160 pixels apart, equivalent to 50 µm in the microscope image scale. The filled representation of the GCL was multiplied with the image containing the bars resulting in an image with white bars spaced 160 pixels apart, fit to the shape and spanning the width of the blades of the GCL. ROIs were created for each bar individually by particle analysis. On a blank canvas, the bars were filled and an intensity gradient distance map was generated around it. The pixels in the bar had an intensity value of 0, with intensities increasing by one with each pixel radiating away from the origin until a value of 255. The binary representations of ARC, CD13, CollagenIV, DCX, GFAP, HOPX, IBA1, Ki67, Nestin, NG2, PV, SOX2, and S100β, which were divided by 255 so the new intensity value was 1. The binary distance maps for each bar were multiplied against the divided binary images created an image with 8-bit intensity profile representative of the distance from the point of origin. Analyzing the histogram of the resulting images provided the areas and spatial distribution of positive signal for each stain within the distance map. Areas of stains were summed within the distance maps to up to a 160 pixel (50 µm) radius, which provided adequate and contiguous sampling of the GCL, bordering areas of the hilus, and inner ML. The areas were also summed across the z-stack creating volumes. These were normalized by dividing by the total volume sampled by the distance map and reported as a percentage.+ R cell processes, and proliferating cells, and measured against the binary representations of cells the same as was done for the randomized probing. Nestin-labeled R cell processes were measured as a whole per section. Ki67+ cells were isolated using particle analysis on the maximum intensity z-projection. ROIs were created around the cells and individually applied to the 3D binary to clear signal outside of the cell of interest. Stain volumes were binned at 5 µm intervals and divided by the total measured volume and reported as percent coverage.Targeted niche mapping: Distance maps were generated around the Nestin+ astrocytes so they were compared using Welch’s t-tests. One-way ANOVAs were performed to assess differences between ages in vascular densities, blood vessel coverage, randomized microniche volumes, and stem cell contact measures. These analyses were followed by post hoc Tukey’s multiple comparisons tests. When data failed the Brown-Forsythe test for equal variances, Welch’s ANOVA tests were run with Dunnett’s T3 multiple comparisons. For the microniche volumetric measures, Games-Howell’s multiple comparisons were used to account for the large group sizes. In comparing spots identified as young and old, Nestin high and low, and KI67 positive and negative, multiple unpaired t-tests were performed with Holms-Sidak correction for multiple comparisons. Microenvironments surrounding radial processes and proliferating cells were analyzed using mixed-effects model analysis and Tukey’s multiple comparisons tests.For all analyses, an alpha level was set at 0.05. To assess preservation of antigenicity Pearson correlations were performed on the normalized intensity values of isolated cells or ROIs. Unpaired t-tests were used to compare counted cell densities, as well as quiescent cell marker and LaminB1 intensities of DCX-labeled immature neurons and PV interneurons between the 2- and 12-month groups. The standard deviations between age groups were significantly different for the intensities of LaminB1 in R cells, IBA1-labeled microglia, and S100β public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Acceptance summary:This paper describing the development of a new anatomical method in the context of adult hippocampal neurogenesis in the aging brain will have a high impact for the scientific community. It will allow to deepen our understanding of cellular complexity in the normal and disease brain.Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Marianne Bronner as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Tim James Viney (Reviewer #2).Thank you for submitting your article "Characterization of the neurogenic niche in the aging dentate gyrus using iterative immunofluorescence imaging" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:The reviewers are very enthusiastic about your work and the impact of this 4i method for the scientific community. However, more information is needed to improve the manuscript. In particular, we recommend toi) Strengthen the reporting of the methods,ii) Add some control for the consistency in antibody staining , the sensitivity of the elution process, individual section variability etc.iii) To provide high resolution single-channel images.Concerning the main criticism of Reviewer 1, you may as requested add a group of old mice (>18 months) or alternatively remove all references to aging in the title, abstract and discussion.In addition, it would be very beneficial for the community to add in the discussion (or methods/results) comments about the boundaries conditions of the 4i method .Reviewer #1 (Recommendations for the authors):1) This study does not addresses ageing but the transition of the hippocampal niche to an adult or mature stage, the text including the title should be modified to reflect this.2) There is no evidence that staining for the different antibodies used works as well in the 2-month and 12-month-old sections (except for the similar expression levels of LaminB1 in some cell types), expression of a protein that is not expected to change between the two stages should be analysed alongside the proteins analysed, as a control of consistency in antibody staining.3) In contrast with the thorough characterisation of neural stem cells, other cell types in the niche are identified with only one marker. This might reduce the ability of the authors to draw conclusions regarding age-related changes in the niche, particularly for cell types such as astrocytes or microglia that can exist in different subtypes or different activation states. This could be discussed.4) The comparison of the intensity of antibody labelling before and after 6 rounds of elution  is an important validation of the method. However it should be conducted systematically for each antibody used in 4i as different epitopes recognised by different antibodies may differ in their sensitivity to the elution process.5) In the same figure, have the authors verified that not only the secondary antibody but also the primary antibody is eluted in each cycle (by re-incubating the eluted sections with the secondary antibody only)?6) I find it somewhat counterintuitive that microniches from 2-month-old animals segregate into two groups of young-like and old-like spots rather than forming a continuum between these two states . Please discuss the implications.7) Proximity of radial processes with CollagenIV and CD13 diminishes between 2- and 6-months and increases between 6- and 12- months. There is therefore no clear trend in the changes that occur across the lifespan. This cast some doubt on the significance of the analysis and the results obtained. Please comment.8) In Figure 7A-B, the authors conduct an analysis of contact sites of radial stem cells with other cell types in the niche. However the analysis does not seem to have enough spatial resolution to conclude that NSCs contact other niche cells rather than being in proximity but without contact. The conclusion from this analysis should be toned down.Reviewer #2 (Recommendations for the authors):I recommend strengthening the reporting of the methods, including examples of the specific analysis, and providing high resolution single-channel images demonstrating colocalisation and associations. This would significantly improve the ability to interpret the results and assess the conclusions. Essential revisions:The reviewers are very enthusiastic about your work and the impact of this 4i method for the scientific community. However, more information is needed to improve the manuscript. In particular, we recommend toi) Strengthen the reporting of the methods,ii) Add some control for the consistency in antibody staining , the sensitivity of the elution process, individual section variability etc…iii) To provide high resolution single-channel images.Concerning the main criticism of Reviewer 1, you may as requested add a group of old mice (>18 months) or alternatively remove all references to aging in the title, abstract and discussion.In addition, it would be very beneficial for the community to add in the discussion (or methods/results) comments about the boundaries conditions of the 4i method .We thank the reviewing editor and the reviewers for their supportive and constructive comments on our manuscript. We are confident that our new experiments and analyses fully address the concerns previously raised by the reviewers. We feel that newly added data and analyses substantially strengthened our manuscript.Reviewer #1 (Recommendations for the authors):1) This study does not addresses ageing but the transition of the hippocampal niche to an adult or mature stage, the text including the title should be modified to reflect this.We understand the reviewer’s concern and have carefully edited our revised manuscript. We now carefully avoid the term “aged” when describing results for 12-month-old mice. We also more explicitly introduced what we refer to by the term “advancing age”. We had not meant to portray our findings in the context of “aged” mice  but rather in the context of “advancing age”, which we define as functional alteration based on chronological age of an individual/tissue. Maturation until mid-life cannot be ruled out but given that in mice and humans function of cells seems to decline rather early on in life (certainly around the first third of the maximum life span if not much earlier), we do not believe that the changes in neurogenesis  seen in 12-month-old mice purely reflect “maturation” of that brain area. We understand that the use of “aged” is not just semantics. However, we do feel that the use of the term “advanced age” is certainly justified and together with the introductory remarks now included will not mislead the reader.2) There is no evidence that staining for the different antibodies used works as well in the 2-month and 12-month-old sections (except for the similar expression levels of LaminB1 in some cell types), expression of a protein that is not expected to change between the two stages should be analysed alongside the proteins analysed, as a control of consistency in antibody staining.We agree with the reviewer and have now included novel data showing thatHOPX and MT3 – showing altered expression levels in neurogenic progenitors – do not change expression levels in classical astrocytes with age . Furthermore, we do show that changes of LB1 with age are cell type specific .3) In contrast with the thorough characterisation of neural stem cells, other cell types in the niche are identified with only one marker. This might reduce the ability of the authors to draw conclusions regarding age-related changes in the niche, particularly for cell types such as astrocytes or microglia that can exist in different subtypes or different activation states. This could be discussed.We understand the reviewer’s concern and have now extended the discussion accordingly. Please refer to page 15 of the revised manuscript.4) The comparison of the intensity of antibody labelling before and after 6 rounds of elution  is an important validation of the method. However it should be conducted systematically for each antibody used in 4i as different epitopes recognised by different antibodies may differ in their sensitivity to the elution process.This is an excellent suggestion and we have now performed the suggested experiments. The new data are included in the revised extended data Figure 1. Furthermore, we have now added a more detailed description how “new” antibodies should be tested. Please refer to page 5 of the revised manuscript.“To assess potential effects of cyclic staining and repeated elutions on sample antigenicity, adult brain sections were stained with the 18 antibodies used in the present study, imaged, and subsequently subjected to six rounds of elution prior to restaining. Measured fluorescence intensities between the stainings before and after rounds of elution were strongly correlated , indicating that antigenicity is preserved across repeated cycles of iterative immunostainings for most of the antibodies that we used in tissue sections, similar to the high reproducibility that 4i achieves in cultured cells .”5) In the same figure, have the authors verified that not only the secondary antibody but also the primary antibody is eluted in each cycle (by re-incubating the eluted sections with the secondary antibody only)?We have now performed the suggested experiments, again corroborating the results that were obtained using cultured cells . The new data are included in the revised extended data Figure 1.6) I find it somewhat counterintuitive that microniches from 2-month-old animals segregate into two groups of young-like and old-like spots rather than forming a continuum between these two states . Please discuss the implications.Indeed, the microniches in 2-month-old mice do represent a continuum in a PCA (indicated by the stretch on component 1). However, there are already at the 2 months time point microniches that do resemble microniches in the DG of mice with more advanced age. We have discussed this finding now more explicitly. Please refer to page 10 and 11 of the revised manuscript. The split between young and old was manufactured by K-means clustering expecting two groups.7) Proximity of radial processes with CollagenIV and CD13 diminishes between 2- and 6-months and increases between 6- and 12- months. There is therefore no clear trend in the changes that occur across the lifespan. This cast some doubt on the significance of the analysis and the results obtained. Please comment.The reviewer is correct that stem cell contacts with CollagenIV are reduced at 6 months compared to 2-month-old mice and then increased between 6- and 12-month-old mice (CD13 is not different between 2- and 6-month-old mice). These are the data we observed that may indicate that the situation is distinct between 6- and 12-month-old mice. We do not understand how that result may “cast some doubt on the significance of the analysis”: it would be rather unlikely if the changes we observed would all follow a linear trajectory. However, we have extended the presentation of that finding and also followed the reviewer’s advice to tone down conclusions regarding contact sites (please refer to changes on page 11 and 12).“To analyze possible interactions between R cells and other cell types in the niche, the area of cell marker co-localization  was measured. In general, a greater percentage of radial processes in 12-month-old animals were in close proximity with niche cell markers for vasculature, pericytes, and microglia, than in younger mice . R cell processes were preferentially close to the vasculature in areas with low pericyte coverage (negative for CD13) , but co-localization of radial processes with CD13-labeled pericytes was increased at 12 months compared to at 2 months . Further, the number of close proximity potential contact sites with IBA1-labeled microglia increased with age, while proximity between R cells and OPCs remained unchanged .”8) In Figure 7A-B, the authors conduct an analysis of contact sites of radial stem cells with other cell types in the niche. However the analysis does not seem to have enough spatial resolution to conclude that NSCs contact other niche cells rather than being in proximity but without contact. The conclusion from this analysis should be toned down.We agree with the reviewer and have modified the description of our results accordingly. Please refer to page 11 and 12 of the revised manuscript (and to point 7).Reviewer #2 (Recommendations for the authors):I recommend strengthening the reporting of the methods, including examples of the specific analysis, and providing high resolution single-channel images demonstrating colocalisation and associations. This would significantly improve the ability to interpret the results and assess the conclusions.We understand the reviewer’s concerns and have carefully edited our manuscript to provide more methodological details. Further, we have added the single channels for the R Cell quantification .


Although effective, some oral pre-exposure prophylaxis (PrEP) users face barriers to adherence using daily pills, which could be reduced by long-acting formulations. Long-acting cabotegravir (CAB LA) is a potential new injectable formulation for human immunodeficiency virus (HIV) PrEP being tested in phase III trials.We use a mathematical model of the HIV epidemic in South Africa to simulate CAB LA uptake by population groups with different levels of HIV risk. We compare the trajectory of the HIV epidemic until 2050 with and without CAB LA to estimate the impact of the intervention.Delivering CAB LA to 10% of the adult population could avert more than 15% of new infections from 2023 to 2050. The impact would be lower but more efficient if delivered to populations at higher HIV risk: 127 person-years of CAB LA use would be required to avert one HIV infection within 5 years if used by all adults and 47 person-years if used only by the highest risk women.If efficacious, a CAB LA intervention could have a substantial impact on the course of the HIV epidemic in South Africa. Uptake by those at the highest risk of infection, particularly young women, could improve the efficiency of any intervention. Using injectable cabotegravir for HIV prevention could, if efficacious, be a valuable addition to the portfolio of HIV prevention options in South Africa and could have a substantial impact on the progression of the epidemic. There continues to be a substantial number of new human immunodeficiency virus (HIV) infections worldwide, with an estimated 1.7 million people newly infected in 2018 .Several HIV prevention methods are available, including male and female condoms, voluntary medical male circumcision (VMMC), early access to antiretroviral treatment (ART), and tenofovir/emtricitabine (TDF/FTC) daily oral pre-exposure prophylaxis . When these are implemented in combination, declines in HIV incidence can be observed at the population level . HoweverOral PrEP has been a promising addition to the HIV prevention portfolio. When taken as prescribed, it is highly effective at reducing HIV acquisition risk , 7. HoweIn 2016, an intravaginal ring lasting 1 month and containing dapivirine was shown to be moderately effective at reducing HIV incidence, with higher effectiveness among women with evidence of product use, and is now undergoing regulatory review , 12. HowOne promising candidate for an injectable form of PrEP is long-acting cabotegravir (CAB LA). Cabotegravir (formerly GSK1265744) is a potent integrase strand transfer inhibitor with a high barrier to resistance that can be formulated as an oral tablet or a long-acting suspension for injectable intramuscular use . This maTwo phase IIa studies, ÉCLAIR (NCT02076178) and HPTN 077 (NCT02178800), demonstrated that injectable CAB LA for HIV prevention was well tolerated with an acceptable safety profile, despite more frequent injection site reactions among participants in the active arm compared to the placebo arm , 19. A dPhase IIb/III studies HPTN 083 NCT02720094) and HPTN 084 (NCT03164564) are now underway to compare the safety and efficacy of CAB LA with daily oral PrEP among (1) HIV-uninfected cisgender men and transgender women who have sex with men (MSM and TGW) and (2) HIV-uninfected women 094 and H. The HPTLong-acting cabotegravir could, if efficacious, be a valuable addition to the HIV prevention portfolio, particularly in locations and among populations where HIV incidence remains high. South Africa has the highest number of annual new HIV infections of any country worldwide . Annual We calibrated a deterministic compartmental simulation model to represent the ongoing HIV epidemic in South Africa over time using data on demography, age-specific and sex-specific HIV prevalence and incidence, ART and VMMC scale-up, and patterns of male condom use , 24. TheFour existing prevention interventions are simulated in the model. These are as follows: (1) condom use; (2) VMMC; (3) early ART; and (4) oral PrEP.The historical expansion of these interventions is calibrated to available data. From 2019 onwards, we make 2 sets of assumptions about how these interventions continue to be used. This creates 2 counterfactual scenarios to use as baselines against which to introduce CAB LA . In the The model simulates the uptake of CAB LA by HIV-negative adults under 5 scenarios: (1) adults ; (2) men ; (3) women ; (4) high-risk women ; and (5) high-risk young women .We simulate CAB LA uptake by each population group to achieve “effective coverage” levels of 1%, 5%, 10%, and 20%. Effective coverage is defined as the proportion of users who benefit from full adherence to the product. Thus, persons that have received the product but do not use it sufficiently to achieve effectiveness are classed outside this coverage value.Long-acting cabotegravir introduction is simulated in the model from 2023. Scale-up is linear and takes 4 years to achieve the specified effective coverage level. We assume that (1) CAB LA has 90% efficacy at reducing the per-sex-act risk of HIV acquisition for users, (2) a CAB LA injection is given to users every 8 weeks, and (3) the mean duration of continuous CAB LA use is 5 years. We perform a sensitivity analysis reducing CAB LA efficacy to 60% and the mean duration of use to 1 year. When CAB LA is introduced into the Projected Scale-up baseline, we assume that all uptake is among new PrEP users , based on the observation that in the family planning sphere, addition of a new contraceptive method generally expands the overall method usage .We simulate the population of South Africa from 2020 to 2050, and we estimate the number of HIV infections averted by CAB LA, if safe and efficacious, from its introduction in 2023 over 5, 10, and 25 years, compared with both the Constant Coverage and Projected Scale-up baselines. The number of infections averted represents new HIV infections among all 15- to 49-year-old adults and includes indirect infections. We report the person-years (PY) spent on CAB LA and the number of CAB LA injections dispensed in these periods. We calculate the number of PY on CAB LA required to avert 1 HIV infection by dividing the total PY of CAB LA dispensed in a specified time period by the total HIV infections averted in that period (this metric is akin to the “number needed to treat” (NNT) concept of Buchbinder et al  for oralUse of CAB LA by 10% of all women without prioritizing by HIV risk (scenario 3) can reduce HIV incidence by 13% against the Constant Coverage baseline and 15% against the Projected Scale-up baseline, and by 10% and 11%, respectively, when delivered to men (scenario 2). If all adults are eligible to receive CAB LA (scenario 1), HIV incidence could decline by 22% and 24% by 2050 compared with each baseline, when used by 10% of the population. This represents averting 15% of incident infections from 2023 to 2050 for both baselines. The same patterns of results are seen at 1%, 5%, and 20% uptake levels for each population group, with the reductions in incidence scaled accordingly . With a A comparison of Fewer PY of CAB LA use are required to avert 1 infection when calculated over a longer time frame because the initial roll-out period, when CAB LA has not yet reached full coverage, contributes less compared with the total duration. The Constant Coverage baseline shows the same pattern but with fewer PY of CAB LA required to avert 1 infection, because overall incidence is higher .The future cost of CAB LA is unknown, but by assuming different costs we can gain an indication of the potential cost of averting 1 HIV infection based on the PY of CAB LA required. For example, at a total cost (including procurement and delivery) of $300 per year, it would cost $38 000 to avert 1 infection if CAB LA is used by all adults, or $14 000 to avert 1 infection if CAB LA is used only by high-risk young women. If the total cost were $1000 per year, the cost of averting 1 infection would be $127 000 and $47 000, respectively.We have evaluated the potential impact of introducing a long-acting PrEP product, parameterized to represent injectable cabotegravir, to different population groups in South Africa, against a backdrop of either little or extensive scale-up of other prevention technologies. We show that a CAB LA intervention, if the product is efficacious, could have a substantial impact on the course of the HIV epidemic, averting 15% of new infections from 2023 to 2050 when delivered to 10% of the adult population. Use of CAB LA by those at higher risk of infection, particularly young women, could improve the efficiency of any intervention, as measured by the NNT. For example, 127 PY of use are needed to avert 1 infection when used by adults compared to 47 when used by high-risk young women when introduced alongside the scale-up of other prevention technologies. These high-risk groups have a baseline incidence of approximately 3% per PY , which aWe have provided a rough illustration of how the impact and efficiency of a CAB LA intervention will influence its cost-effectiveness. To fully understand the implications requires consideration of the value of an infection averted, which may vary by location and population.Long-acting injectable PrEP for HIV prevention is a promising potential addition to the range of existing prevention options. Although the WHO recommends that oral PrEP should be available to populations at substantial risk of HIV infection, defined as incidence of more than 3%, adherence can be a challenge for some users and current use is limited , 23, 29.Ultimately, combination prevention approaches are likely required to meet the needs of all at-risk individuals, and this may include other novel forms of PrEP such as the dapivirine ring, and eventually antiretroviral implants, as well as CAB LA, oral PrEP, and other existing and future technologies. Some of the challenges of introducing oral PrEP have been to foster demand for the product and then to support continued use with high adherence after initiation, particularly among AGYW . Some leAlthough we find that CAB LA use by those at highest risk of HIV infection is the most efficient way to implement an intervention, our ability to identify those “at risk” in reality may be limited. Women, and particularly AGYW, may not perceive themselves as at risk . HoweverThere are several limitations to this analysis. The major limitation to the modeling approach is the lack of uncertainty in the input epidemiological parameters. Instead, we have parameterized the model with the most recent data available and performed scenario analyses with 2 baselines and varying coverage levels. Uncertainty over costs, delivery channels, user acceptability, and other interventions over a 30-year period are greater than uncertainty in many model parameters, so we have chosen to illustrate scenarios that highlight some key relationships between coverage and impact. Sensitivity analyses show how the quantitative results are moderated by the assumed efficacy and duration of CAB LA use, but the qualitative results hold after minor adjustments to the input parameters, and it is these that are worth emphasising.We have focused on South Africa, but there are many other regions with HIV incidence among particular geographies or key populations for whom CAB LA might also be beneficial. The model does not include MSM or TGW because the burden of the HIV epidemic in South Africa is overwhelmingly among the heterosexual population. Furthermore, there is little data among risk group size and behavior with which to reliably parameterize this model. However, these groups are known to be at very high risk of HIV infection, and their inclusion in any intervention programming would be essential. HIV prevalence of 18.1% has been estimated among MSM in South Africa , and HIVHealth economic modeling is required to elucidate further the contexts in which CAB LA may or may not be cost-effective. Two early studies found long-acting injectable PrEP very cost-effective or cost-saving if prioritized to those at highest risk of infection in South Africa , 36; anoThe introduction of CAB LA could be a valuable addition to the portfolio of HIV prevention options in South Africa and could have a substantial impact on the progression of the epidemic. The efficiency of any intervention, which will determine its cost-effectiveness, will be determined by the ability to identify and support use by those at the highest risk of infection.The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at jiaa296_suppl_Supplementary_MaterialClick here for additional data file.


Participants used a computer-based task to judge the hostility of persons (fictitious characters). Adolescents with ID were found to make more polarizing judgments  and were more likely to be guided by the opinions of a fictitious peer ingroup  compared with adolescents without ID. No such differences were found between adolescents with ID and MA-matched children. The results are discussed in terms of scientific and practical implications.Adolescents with intellectual disability (ID) experience challenges and uncertainty when making judgments about other people’s intentions. In an attempt to achieve certainty, they might exhibit judgment tendencies that differ from those of typically developing adolescents. This study investigated social judgment making in adolescents with ID ( Adolescents with intellectual disability (ID) often experience difficulties adequately judging the intentions of others, especially in ambiguous social situations and concerning hostile intentions . Their lIn order to better understand these processes, this study uses a computer-based task involving fictitious characters to assess the judgment tendencies of adolescents with ID, primary school mental age (MA)-matched children and adolescents without ID. The primary goal of this study is to investigate the extent to which adolescents with ID polarize their social judgments when judging hostile intentions. The results may provide an indication of individual judgment pattern preferences in adolescents with ID in circumstances in which no external references are available. However, social judgments are typically made in a social context in which various opinions conflict with one another. These different opinions are often represented by different social groups: groups who belong  and others who do not . Such intergroup behavior is common in the everyday lives of adolescents  of photographed peers and were more open to peer influence than typically developing adolescents; they performed at levels comparable to children matched for MA  polarizing judgment tendencies, and on (2) susceptibility to ingroup influence (ingroup bias) was developed. The goal of this study is to compare these judgment tendencies of adolescents with ID [experimental group (EG)] with adolescents without ID [comparison group 1 (CG1)]. In addition, this study includes a comparison group of MA-matched children [comparison group 2 (CG2)] to shed more light on the role of MA in these processes cf. .In the present study, participants were asked to complete a computer-based task in one session. They were shown images of unknown people see  and askeHypothesis 1: Adolescents with ID make more polarizing social judgments than adolescents without ID.Hypothesis 2: Adolescents with ID are more open to ingroup influence than adolescents without ID.In accordance with earlier findings , similarExplorative analyzes were used to consider the role of different polarization tendencies as an explanation for the group differences in susceptibility to ingroup influence. In this regard, theoretical approaches point to a preference for a simplifying thinking style  associated with both polarized judgment making and increased ingroup bias .This study was conducted within a larger research project related to peer influence and peer relations in individuals with ID cf. . The insAdolescents with ID were recruited at Swiss special needs schools for students with ID. Participants of the comparison groups attended regular schools. Overall, informed consent was obtained from a total of 324 participants . Due to technical problems and participant absences  data were collected for 300 individuals .General Adaptive Composite (GAC) value was calculated over all items in the social, practical and conceptual domains. The corresponding values can be differentiated into very low (≤70), low (71–79), below average (80–89), average (90–109), above average (110–119), and high (≥ 120). Students were included in the EG if they demonstrated adaptive behavior ranging from very low to below average. Because this study focuses on early- to mid-adolescence, only individuals between 12 and 17 years of age were included in the EG. In order to recruit EG participants, teachers at special needs schools were asked to distribute letters to parents of adolescents who were able to express themselves well verbally, had a good comprehension of language, could write their own name, and had a mild ID (no further specification). Of the 71 participants from special needs schools, 34 satisfied the study’s inclusion criteria for IQ , adaptive behavior , and age . Because the CFT-20R norms provide no differentiation of IQ scores below 54, no specific IQ scores could be specified for the 11 participants with an IQ below 54. It is important to note, however, that these 11 participants had GAC scores  ranging from very low to below average, just like the other participants. Using observation protocols during the experimental task, no apparent motor or sensory deficits were observed and all participants were able to meet the technical requirements of the task.Swiss special needs schools can only be attended by students who have a clinical diagnosis of ID as defined by the ICD-10 code, have been assessed using an IQ test (IQ < 70) and typically have received a clinical rating of adaptive behavior . TherefoM = 100.12, SD = 9.26), age and gender were used as the selection criteria. If no same-gender person with an age difference of up to 0.5 years was available, the next person with the smallest age difference, regardless of gender, was included in the sample. For the selection of the 34 children in CG2, matching was based on the CFT-20R raw score. If several participants had the same CFT-20R raw score, the person with the same gender was selected. Based on the relatively small number of potential children with the same MA for CG2 compared with the adolescents without ID for CG1, the priority in CG2 matching was the CFT-20R raw score but gender was still considered. The CFT-20R does not provide IQ scores for children of this age. Observation protocols indicated that the children in CG2 had no significant difficulties with the tasks or technical facilities.Next, a pairwise matching procedure was conducted to identify participants for CG1 and CG2. To select the 34 participants of CG1 . Consistent with the purpose of matching, the EG and CG1 did not differ significantly in chronological age  and CG1  and CG2  of each virtually simulated person was displayed in each image; the images differed only in gender, hair style and hair color, eye color and clothing . Only after these exercises were completed correctly did the experimental procedure continue.Each of the participants sat at a laptop and was told through headphones that they would rate the perceived hostility of each person depicted in images. Hostility was defined for participants as “lying, teasing, or telling nasty things about others.” The scale for judging hostility took the form of a bar presented below the pictures , participants rated the hostility of the virtually simulated persons on a scale that had the same color as the own group  were dependent on participants’ first ratings (T1). To investigate ingroup orientation, at T2 half of the items (four items) were given an ingroup rating that was placed at a standardized distance  from the participants’ first rating at T1 (see The measured distance between the participant’s rating (T2) and the mean of the ingroup ratings was used to indicate susceptibility to ingroup influence  were included. In these distractors, the judgments of both the ingroup and outgroup at T2 either corresponded to the participants’ judgment at T1 (3x) or varied across the entire scale (3x). Accordingly, participants rated a total of 14 virtually simulated persons at two time points.After conducting the task, a debriefing session with the participants was conducted in which the tasks were explained and the topics of social judgment and external orientation in daily life were discussed.Multilevel analyses were conducted taking into account the nested nature of the data: Participants (level 2) rated multiple experimental stimuli  were calculated using the software SPSS to examine the structure of the data. Next, the data were tested for important statistical assumptions with respect to the primary analyses .polarization of social judgment was measured at T1 for all items  and ingroup orientation (ρIC = 0.504) were calculated using the unconditional model . The results suggest that 20.9% of the variance in the polarization of social judgment was due to differences between individuals; for ingroup orientation 50.4% of the variance is due to differences between individuals.Intraclass correlations cf.  were calp < 0.001) and ingroup orientation (p < 0.001). Furthermore, the residuals were tested for normality using a Kolmogorov–Smirnov test. For both polarization of social judgment and ingroup orientation, the residuals of the three groups were not normally distributed (p < 0.001). To identify outliers within the three groups regarding polarization of social judgment and ingroup orientation, boxplots were analyzed: mild outliers were characterized as being more than 1.5 times but less than three times the interquartile range, and extreme outliers were characterized as being more than three times the interquartile range. No outliers were found in polarization of social judgment in any of the three groups. Additionally, there were no outliers in the data collected from adolescents with ID and MA-matched children. In contrast, for ingroup orientation four adolescents without ID exhibited mild outliers on some single ratings, and two adolescents without ID exhibited extreme outliers on two ratings. No participant exhibited consistent outlier behavior across all items. Although these analyses reveal that some assumptions of the implemented models were violated, such deviations in samples of this size should not be a concern  were not equal with respect to polarization of social judgment ( concern . In addi concern . The MLR concern .median = 38.5). In addition to this tendency toward polarizing social judgment, a smaller clustering of ratings toward the middle of the scale is also evident. MA-matched children exhibit a similar distribution pattern (median = 21.5). Among adolescents without ID, about 50% of the ratings were located in the middle third of the scale (median = 16.0).Hypothesis 1 tested whether adolescents with ID make significantly more polarizing social judgments than typically developing adolescents. The descriptive results are listed in polarization of social judgment (see 2(0) = 0.001, p < 0.001; RMSEA = 0.000; CFI = 1.000; TLI = 1.000). Consistent with Hypothesis 1, significantly more polarizing social judgments were found for the EG compared with the CG1  indicated a moderate effect size . The size of this effect was small using the standardized regression coefficient .The two-level means-as-outcomes model (Model 1) included ment see , T1 as aect size . No signmedian = 9) and MA-matched children (median = 10) exhibited clustering of their judgments close to the ingroup. At the same time, a smaller clustering of ratings near the original judgment at T1 (T1 = 40) is visible for these two groups. Adolescents without ID exhibited 50% of judgments at 39 (median), which reveals that many judgments were made close to the original judgment at T1.Hypothesis 2 tested whether adolescents with ID exhibit a significantly stronger susceptibility to ingroup influence compared with typically developing adolescents. distance to the ingroup vs. distance to the outgroup) was zero (p < 0.001). In order to test Hypothesis 2, a means-as-outcomes model (Model 2) was conducted in accordance with the procedure of distance to the ingroup (see 2(0) = 0.000, p < 0.001; RMSEA = 0.000; CFI = 1.000; TLI = 1.000). As expected, participants in the EG exhibited a significantly stronger susceptibility to ingroup influence compared with participants in CG1 . The size of this effect was high . When the individual groups  were analysed separately, the Spearman correlation between polarization of social judgment and ingroup orientation was also not significant . These results suggest that these two constructs are not related.Since adolescents with and without ID differ in their polarization of social judgment and susceptibility to ingroup influence, it is of interest to analyze whether polarization of social judgment is correlated with ingroup orientation. No significant Spearman correlation was found between polarization and ingroup orientation in social judgment within the entire sample  was predicted by group membership  and polarization of social judgment on level 2 (model fit: (χ2(1) = 0.836, p = 0.361; RMSEA = 0.000; CFI = 1.000; TLI = 1.025). The EG exhibited a significantly stronger susceptibility to ingroup influence compared with participants in CG1  may be due to a common underlying cognitive thinking style that consists of a preference for simplicity and a need to avoid ambiguity in information processing and judgment . AccordiTo the best of the author’s knowledge, this study is the first to investigate the minimal group paradigm in adolescents with ID and matched comparison groups. Using a new experimental task, this research extends prior findings regarding the role of individual and contextual factors in social judgment making of adolescents with ID. However, it also has limitations.In the computer-based task, the participants rated the hostility of virtually simulated people based on minimal information  provided. In this judgment situation, no explicit indications about each person’s actual hostility  were available and the task was therefore ambiguous for all participants. One might suggest that no accurate answer was possible. However, the focus of this study was on polarizing social judgment style in social judgment situations with minimal information , as is common in everyday first impressions cf. , rather In general, adolescents with ID and MA-matched children may have had more difficulties solving the task because it was ambiguous and no obviously correct answer was possible. This situation could have led to more polarizing judgments in adolescents with ID and MA-matched children. In addition, it is possible that adolescents with ID and MA-matched children had more difficulties using the rating scale for social judgment making compared with adolescents without ID. Although participants received training about using the scale, an additional alternative form of judgment making would be useful in future studies to determine the influence of the use of the scale on the results. The present study aimed to investigate the influence of an ingroup among adolescents with ID within an intergroup context. For this purpose, the opinions of the in- and outgroup  were presented simultaneously. Additional follow-up research could add to the present findings by investigating outgroup influence and the interplay between in- and outgroup influence in intergroup contexts. Furthermore, it would be interesting to better understand group processes in adolescents with ID in different social contexts .Despite certain limitations, these study results have several implications. It was found that adolescents with ID tend to polarize judgments in situations where behavioral traits  are derived only from external characteristics of the target persons. Judgment of personality traits based on external characteristics  plays a significant role when forming first impressions . In suchbefore they have to make an important decision. Doing so may involve using visualized symbols such as a visual decision-making aid  and no additional knowledge about other characteristics was introduced, it is remarkable that adolescents with ID strongly oriented toward this group. This increased susceptibility to social influence may contribute to heightened social risks in adolescents with ID: an unknown group may promise group membership if an adolescent with ID acts in a specific way, for example. However, an orientation toward an ingroup (in contrast with an outgroup) can also be considered a social learning opportunity for adolescents with ID. Accordingly, in certain contexts such as schools teachers may create affiliations by referring to similarities between an individual and positive social groups in order to promote a positive ingroup influence cf. .In summary, this study contributes to the still-scarce knowledge base of how adolescents with ID make social judgments. The insights gained point to the social vulnerabilities associated with ID and can contribute to providing perspectives for better support of adolescents with ID.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the institutional research committee of the Department of Special Needs Education at the University of Fribourg. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.SE developed the experimental task in collaboration with Philipp Nicolay and Christian Huber from the University of Wuppertal (Germany) and Christoph Michael Müller , organized and took part in the recruitment of participants, data collection, conducted all data analyses, and wrote the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.