GeneticsMakie.jl: a versatile and scalable toolkit for visualizing locus-level genetic and genomic data.

SUMMARY: With the continued deluge of results from genome-wide association and functional genomic studies, it has become increasingly imperative to quickly combine and visualize different layers of genetic and genomic data within a given locus to facilitate exploratory and integrative data analyses. While several tools have been developed to visualize locus-level genetic results, the limited speed, scalability and flexibility of current approaches remain a significant bottleneck. Here, we present a Julia package for high-performance genetics and genomics-related data visualization that enables fast, simultaneous plotting of hundreds of association results along with multiple relevant genomic annotations. Leveraging the powerful plotting and layout utilities from Makie.jl facilitates the customization and extensibility of every component of a plot, enabling generation of publication-ready figures. AVAILABILITY AND IMPLEMENTATION: The GeneticsMakie.jl package is open source and distributed under the MIT license via GitHub (https://github.com/mmkim1210/GeneticsMakie.jl). The GitHub repository contains installation instructions as well as examples and documentation for built-in functions. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Integrase Strand Transfer Inhibitors Are Associated With Incident Diabetes Mellitus in People With Human Immunodeficiency Virus.

BACKGROUND: Integrase strand transfer inhibitors (INSTIs) are associated with weight gain in people with HIV (PWH). Less is known about the risk of other metabolic outcomes such as diabetes mellitus and hyperglycemia. METHODS: IBM® MarketScan® databases for commercially and Medicaid-insured adults were used to identify PWH newly initiating antiretroviral therapy (ART). The primary outcome was a composite of new-onset diabetes mellitus/hyperglycemia in the 6 months following ART initiation and was identified using International Classification of Disease, Ninth revision, Clinical Modification (ICD-9-CM) and ICD-10-CM diagnosis and procedure codes and Current Procedural Terminology, 4th Edition (CPT-4) codes. To examine the relationship between INSTI use and the composite outcome, we estimated the risk using Cox proportional hazards models with calendar time-specific standardized mortality ratio weights. RESULTS: Of 42 382 PWH who initiated ART between 1 July 2007 and 30 June 2018, 22 762 (54%) were treated with INSTI-based regimens. Mean age was 38 years, 74% were male, and 19% were Medicaid insured. PWH on INSTIs were 31% more likely to develop new-onset diabetes mellitus/hyperglycemia (hazard ratio [HR], 1.31; 95% confidence interval [CI], 1.15-1.48]) compared with those who initiated non-INSTI-based regimens. When examined individually, the highest risk was associated with elvitegravir (HR, 1.54; 95% CI, 1.32-1.97; P < .001) and the lowest risk with raltegravir (HR, 1.19; 95% CI, 1.03-1.37; P = .02). CONCLUSIONS: INSTI use was associated with increased risk of new-onset diabetes mellitus/hyperglycemia in the 6 months following ART initiation.

Sensitivity of a bedside reagent strip for the detection of spontaneous bacterial peritonitis in ED patients with ascites.

STUDY OBJECTIVE: To determine the sensitivity of a highly sensitive bedside leukocyte esterase reagent strip (RS) for detection of spontaneous bacterial peritonitis (SBP) in emergency department (ED) ascites patients undergoing paracentesis. METHODS: We conducted a prospective, observational cohort study of ED ascites patients undergoing paracentesis at two academic facilities. Two practitioners, blinded to each other's results, did a bedside RS analysis of the peritoneal fluid in each patient and documented the RS reading at 3-min according to manufacturer-specified colorimetric strip reading as either "negative", "trace", "small", or "large". The primary outcome measure was sensitivity of the RS strip for SBP (absolute neutrophil count ≥ 250 cells/mm3) at the "trace" threshold (positive equals trace or greater). RESULTS: There were 330 cases enrolled, with 635 fluid analyses performed. Of these, 40 fluid samples had SBP (6%). Bedside RS had a sensitivity, specificity, positive predictive value, and negative predictive value of 95% (95% CI 82%-99%), 48% (95% CI 44%-52%), 11% (95% CI 10%-11%), and 99% (95% CI 97%-99%) respectively at the "trace" threshold for the detection of SBP. CONCLUSION: Bedside use of the RS in ED ascites patients demonstrated high sensitivity for SBP. Given the wide confidence intervals, we cannot currently recommend it as a stand-alone test. We recommend further study with a larger number of SBP patients, potentially combining a negative RS result with low clinical suspicion to effectively rule out SBP without formal laboratory analysis.

Incidence and predictors of 30-day postoperative readmission in children.

BACKGROUND: Hospital readmissions are being used as a quality metric for hospital reimbursement without a clear understanding of the factors that contribute to readmission. OBJECTIVE: The objective of this study was to report the incidence of 30-day postsurgical readmission in children, identify the predictors for readmission, and create an algorithm to identify high-risk children. METHODS: Data from the 2012-2014 Pediatric database of the American College of Surgeons National Surgical Quality Improvement Program were analyzed using univariable and multivariable logistical regression analysis. RESULTS: Among 182 589 children included in the 2012-2014 American College of Surgeons National Surgical Quality Improvement Program Pediatric database, 4.8% (8815/182 589) experienced a readmission within 30 days. Four significant predictors were retained in the multivariable logistic regression model: American Society of Anesthesiologists physical status ≥ 3 (OR: 1.9, 95% CI: 1.8-2.0), presence of congenital heart disease (OR: 1.66, 95% CI: 1.31-2.11), inpatient status at time of surgery (OR: 3.5, 95% CI: 3.3-3.7), and at least 1 postoperative complication (neurologic, renal, wound, cardiac, bleeding, or pulmonary) (OR: 3.14, 95% CI: 2.92-3.34). The multivariable logistic regression model showed reasonably good discrimination in predicting 30-day readmissions with receiver operating characteristic area under the curve of 0.747 (95% CI: 0.73-0.75) and good calibration (Brier score: 0.044). We created a predictive algorithm of 30-day readmission based on the 4 significant predictors. CONCLUSION: Children with congenital heart disease, high American Society of Anesthesiologist physical class, inpatient status, and at least 1 postoperative complication of any kind are at high risk for postsurgical readmissions. We provide an algorithm for quantifying this risk with the goal of reducing the number of readmissions, improving the care of patients with complex chronic illnesses, and reducing hospital costs.

Development and Validation of a Risk Stratification Score for Children With Congenital Heart Disease Undergoing Noncardiac Surgery.

BACKGROUND: Children with major and severe congenital heart disease (CHD) undergoing noncardiac surgery are at increased risk of mortality. The objective of this study was to identify the predictors for in-hospital mortality, and to develop a risk stratification score that could be used to help decision making and the development of perioperative management guidelines. METHODS: We included all children with major (eg, tetralogy of Fallot with wide open pulmonary insufficiency, hypoplastic left heart syndrome including stage 1 repair) or severe CHD (eg, children with uncorrected CHD, children with documented pulmonary hypertension, children with ventricular dysfunction requiring medications, or children listed for heart transplant) recorded in the 2012 and 2013 American College of Surgeons National Surgical Quality Improvement Program Pediatric databases in a derivation cohort, and those recorded in the 2014 database in a validation cohort. The primary outcome variable for our analysis was the incidence of in-hospital mortality. We used univariable and multivariable logistic regression to determine the preoperative predictors for in-hospital mortality and designed the risk stratification score. RESULTS: Among the 183,423 children included in the 2012, 2013, and 2014 American College of Surgeons National Surgical Quality Improvement Program database, we included 4375 children with major or severe CHD in the derivation cohort (mortality: 4.7% [204/4375]) and 2869 in the validation cohort (morality: 4.0% [115/2869]). Eight preoperative predictors were retained in the final multivariable logistic regression model: emergency procedure (odds ratio [OR]: 1.66, 95% confidence interval [CI]: 1.19-2.31, P = .003), severe CHD (OR: 1.65, 95% CI: 1.15-2.39, P = .007), single-ventricle physiology (OR: 1.83, 95% CI: 1.10-3.06, P = .020), previous surgery within 30 days (OR: 2.01, 95% CI: 1.40-2.89, P < .001), inotropic support (OR: 2.05, 95% CI: 1.40-3.01, P < .001), preoperative cardiopulmonary resuscitation (OR: 2.46, 95% CI: 1.32-4.57, P < .004), acute or chronic kidney injury (OR: 4.42, 95% CI: 2.00-9.75, P < .001), and mechanical ventilation (OR: 7.80, 95% CI: 5.42-11.21, P < .001). We created a risk stratification score ranging from 0 to 10 that showed very good calibration and discrimination in the validation cohort (area under the curve: 0.831 [95% CI: 0.787-0.875]), corresponding to an optimism-corrected area of 0.826. Scores ≤ 3 are associated with low risk of mortality (OR: 1.54, 95% CI: 0.78-3.04), scores ranging from 4 to 6 associated with medium risk (OR: 4.19, 95% CI: 2.56-6.87), and scores ≥ 7 associated with high risk (OR: 22.15, 95% CI: 15.06-32.59). CONCLUSIONS: Our study demonstrates that, in addition to preoperative markers of critical illness (eg, inotropic support, mechanical ventilation, preoperative cardiopulmonary resuscitation, and acute or chronic kidney injury), the type of lesion (eg, single-ventricle physiology) and the functional severity of the heart disease (eg, severe CHD) are strong predictors of in-hospital mortality in children undergoing noncardiac surgery.

Developmental isoform diversity in the human neocortex informs neuropsychiatric risk mechanisms.

RNA splicing is highly prevalent in the brain and has strong links to neuropsychiatric disorders; yet, the role of cell type-specific splicing and transcript-isoform diversity during human brain development has not been systematically investigated. In this work, we leveraged single-molecule long-read sequencing to deeply profile the full-length transcriptome of the germinal zone and cortical plate regions of the developing human neocortex at tissue and single-cell resolution. We identified 214,516 distinct isoforms, of which 72.6% were novel (not previously annotated in Gencode version 33), and uncovered a substantial contribution of transcript-isoform diversity-regulated by RNA binding proteins-in defining cellular identity in the developing neocortex. We leveraged this comprehensive isoform-centric gene annotation to reprioritize thousands of rare de novo risk variants and elucidate genetic risk mechanisms for neuropsychiatric disorders.

Brain cell-type shifts in Alzheimer's disease, autism, and schizophrenia interrogated using methylomics and genetics.

Few neuropsychiatric disorders have replicable biomarkers, prompting high-resolution and large-scale molecular studies. However, we still lack consensus on a more foundational question: whether quantitative shifts in cell types-the functional unit of life-contribute to neuropsychiatric disorders. Leveraging advances in human brain single-cell methylomics, we deconvolve seven major cell types using bulk DNA methylation profiling across 1270 postmortem brains, including from individuals diagnosed with Alzheimer's disease, schizophrenia, and autism. We observe and replicate cell-type compositional shifts for Alzheimer's disease (endothelial cell loss), autism (increased microglia), and schizophrenia (decreased oligodendrocytes), and find age- and sex-related changes. Multiple layers of evidence indicate that endothelial cell loss contributes to Alzheimer's disease, with comparable effect size to APOE genotype among older people. Genome-wide association identified five genetic loci related to cell-type composition, involving plausible genes for the neurovascular unit (P2RX5 and TRPV3) and excitatory neurons (DPY30 and MEMO1). These results implicate specific cell-type shifts in the pathophysiology of neuropsychiatric disorders.

Cross-ancestry atlas of gene, isoform, and splicing regulation in the developing human brain.

Neuropsychiatric genome-wide association studies (GWASs), including those for autism spectrum disorder and schizophrenia, show strong enrichment for regulatory elements in the developing brain. However, prioritizing risk genes and mechanisms is challenging without a unified regulatory atlas. Across 672 diverse developing human brains, we identified 15,752 genes harboring gene, isoform, and/or splicing quantitative trait loci, mapping 3739 to cellular contexts. Gene expression heritability drops during development, likely reflecting both increasing cellular heterogeneity and the intrinsic properties of neuronal maturation. Isoform-level regulation, particularly in the second trimester, mediated the largest proportion of GWAS heritability. Through colocalization, we prioritized mechanisms for about 60% of GWAS loci across five disorders, exceeding adult brain findings. Finally, we contextualized results within gene and isoform coexpression networks, revealing the comprehensive landscape of transcriptome regulation in development and disease.

Leveraging CD4 Cell Count at Entry Into Care to Monitor Success of Human Immunodeficiency Virus Prevention, Treatment, and Public Health Programming in the Greater St Louis Area Between 2017 and 2020.

CD4 cell count at entry into human immunodeficiency virus (HIV) care is a useful indicator of success of multiple steps in HIV public health programming. We demonstrate that CD4 cell count at care initiation was stable in St Louis between 2017 and 2019 but declined in 2020. Missouri efforts in the Ending the HIV Epidemic plan should focus on rapidly identifying individuals with undiagnosed HIV infection.

Isoform-level transcriptome-wide association uncovers genetic risk mechanisms for neuropsychiatric disorders in the human brain.

Methods integrating genetics with transcriptomic reference panels prioritize risk genes and mechanisms at only a fraction of trait-associated genetic loci, due in part to an overreliance on total gene expression as a molecular outcome measure. This challenge is particularly relevant for the brain, in which extensive splicing generates multiple distinct transcript-isoforms per gene. Due to complex correlation structures, isoform-level modeling from cis-window variants requires methodological innovation. Here we introduce isoTWAS, a multivariate, stepwise framework integrating genetics, isoform-level expression and phenotypic associations. Compared to gene-level methods, isoTWAS improves both isoform and gene expression prediction, yielding more testable genes, and increased power for discovery of trait associations within genome-wide association study loci across 15 neuropsychiatric traits. We illustrate multiple isoTWAS associations undetectable at the gene-level, prioritizing isoforms of AKT3, CUL3 and HSPD1 in schizophrenia and PCLO with multiple disorders. Results highlight the importance of incorporating isoform-level resolution within integrative approaches to increase discovery of trait associations, especially for brain-relevant traits.

Developmental isoform diversity in the human neocortex informs neuropsychiatric risk mechanisms.

RNA splicing is highly prevalent in the brain and has strong links to neuropsychiatric disorders, yet the role of cell-type-specific splicing or transcript-isoform diversity during human brain development has not been systematically investigated. Here, we leveraged single-molecule long-read sequencing to deeply profile the full-length transcriptome of the germinal zone (GZ) and cortical plate (CP) regions of the developing human neocortex at tissue and single-cell resolution. We identified 214,516 unique isoforms, of which 72.6% are novel (unannotated in Gencode-v33), and uncovered a substantial contribution of transcript-isoform diversity, regulated by RNA binding proteins, in defining cellular identity in the developing neocortex. We leveraged this comprehensive isoform-centric gene annotation to re-prioritize thousands of rare de novo risk variants and elucidate genetic risk mechanisms for neuropsychiatric disorders. One-Sentence Summary: A cell-specific atlas of gene isoform expression helps shape our understanding of brain development and disease. Structured Abstract: INTRODUCTION: The development of the human brain is regulated by precise molecular and genetic mechanisms driving spatio-temporal and cell-type-specific transcript expression programs. Alternative splicing, a major mechanism increasing transcript diversity, is highly prevalent in the human brain, influences many aspects of brain development, and has strong links to neuropsychiatric disorders. Despite this, the cell-type-specific transcript-isoform diversity of the developing human brain has not been systematically investigated.RATIONALE: Understanding splicing patterns and isoform diversity across the developing neocortex has translational relevance and can elucidate genetic risk mechanisms in neurodevelopmental disorders. However, short-read sequencing, the prevalent technology for transcriptome profiling, is not well suited to capturing alternative splicing and isoform diversity. To address this, we employed third-generation long-read sequencing, which enables capture and sequencing of complete individual RNA molecules, to deeply profile the full-length transcriptome of the germinal zone (GZ) and cortical plate (CP) regions of the developing human neocortex at tissue and single-cell resolution.RESULTS: We profiled microdissected GZ and CP regions of post-conception week (PCW) 15-17 human neocortex in bulk and at single-cell resolution across six subjects using high-fidelity long-read sequencing (PacBio IsoSeq). We identified 214,516 unique isoforms, of which 72.6% were novel (unannotated in Gencode), and >7,000 novel exons, expanding the proteome by 92,422 putative proteoforms. We uncovered thousands of isoform switches during cortical neurogenesis predicted to impact RNA regulatory domains or protein structure and implicating previously uncharacterized RNA-binding proteins in cellular identity and neuropsychiatric disease. At the single-cell level, early-stage excitatory neurons exhibited the greatest isoform diversity, and isoform-centric single-cell clustering led to the identification of previously uncharacterized cell states. We systematically assessed the contribution of transcriptomic features, and localized cell and spatio-temporal transcript expression signatures across neuropsychiatric disorders, revealing predominant enrichments in dynamic isoform expression and utilization patterns and that the number and complexity of isoforms per gene is strongly predictive of disease. Leveraging this resource, we re-prioritized thousands of rare de novo risk variants associated with autism spectrum disorders (ASD), intellectual disability (ID), and neurodevelopmental disorders (NDDs), more broadly, to potentially more severe consequences and revealed a larger proportion of cryptic splice variants with the expanded transcriptome annotation provided in this study.CONCLUSION: Our study offers a comprehensive landscape of isoform diversity in the human neocortex during development. This extensive cataloging of novel isoforms and splicing events sheds light on the underlying mechanisms of neurodevelopmental disorders and presents an opportunity to explore rare genetic variants linked to these conditions. The implications of our findings extend beyond fundamental neuroscience, as they provide crucial insights into the molecular basis of developmental brain disorders and pave the way for targeted therapeutic interventions. To facilitate exploration of this dataset we developed an online portal ( https://sciso.gandallab.org/ ).

Cross-ancestry, cell-type-informed atlas of gene, isoform, and splicing regulation in the developing human brain.

Genomic regulatory elements active in the developing human brain are notably enriched in genetic risk for neuropsychiatric disorders, including autism spectrum disorder (ASD), schizophrenia, and bipolar disorder. However, prioritizing the specific risk genes and candidate molecular mechanisms underlying these genetic enrichments has been hindered by the lack of a single unified large-scale gene regulatory atlas of human brain development. Here, we uniformly process and systematically characterize gene, isoform, and splicing quantitative trait loci (xQTLs) in 672 fetal brain samples from unique subjects across multiple ancestral populations. We identify 15,752 genes harboring a significant xQTL and map 3,739 eQTLs to a specific cellular context. We observe a striking drop in gene expression and splicing heritability as the human brain develops. Isoform-level regulation, particularly in the second trimester, mediates the greatest proportion of heritability across multiple psychiatric GWAS, compared with eQTLs. Via colocalization and TWAS, we prioritize biological mechanisms for ~60% of GWAS loci across five neuropsychiatric disorders, nearly two-fold that observed in the adult brain. Finally, we build a comprehensive set of developmentally regulated gene and isoform co-expression networks capturing unique genetic enrichments across disorders. Together, this work provides a comprehensive view of genetic regulation across human brain development as well as the stage-and cell type-informed mechanistic underpinnings of neuropsychiatric disorders.

Infectious Diseases Consultation Is Associated With Decreased Mortality in Enterococcal Bloodstream Infections.

BACKGROUND: Enterococcus species frequently cause health care-associated bacteremia, with high attributable mortality. The benefit of consultation with infectious disease (ID) specialists has been previously illustrated with Staphylococcus aureus bacteremia. Whether ID consultation (IDC) improves mortality in enterococcal bacteremia is unknown. METHODS: This is a retrospective cohort single-center study from January 1, 2015, to June 30, 2016, that included all patients >18 years of age admitted with a first episode of Enterococcus bacteremia. Patients were excluded if death or transfer to palliative care occurred within 2 days of positive blood culture. RESULTS: Two hundred five patients were included in the study, of whom 64% received IDC. Participants who received IDC were more likely to undergo repeat cultures to ensure clearance (99% vs 74%; P < .001), echocardiography (79% vs 45%; P < .001), surgical intervention (20% vs 7%; P = 0.01), and have appropriate antibiotic duration (90% vs 46%; P < .001). Thirty-day mortality was significantly higher in the no-IDC group (27 % vs 12 %; P < .007). In multivariate analysis, 30-day in-hospital mortality was associated with both E. faecium bacteremia (adjusted odds ratio [aOR], 2.39; 95% confidence interval [CI], 1.09-5.23) and IDC (aOR, 0.35; 95% CI, 0.16-0.76). CONCLUSIONS: Patients who received IDC for Enterococcus bacteremia had significantly lower 30-day mortality. Further prospective studies are necessary to determine if these outcomes can be validated in other institutions for patients who receive IDC with Enterococcus bacteremia.

Noma surgery.

OBJECTIVE: Noma is a devastating and destructive disease of the face for which there is a dearth of information regarding surgical options. Herein, we describe the facial deformities and patient characteristics in a patient population affected by noma and the surgical approaches used in treatment. METHODS: Retrospective case series of a Doctors Without Borders (Médecins Sans Frontières) intervention at Sokoto Children's Noma Hospital in northern Nigeria, the highest-volume noma hospital in the world. RESULTS: Twenty-two procedures were performed on 18 patients with noma, 44% of whom were children. The majority of patients (n = 10, 55.5%) were made aware of surgical care through a Doctors Without Borders outreach program. Patients' reasons for seeking care included functional (61.1%, n = 11), appearance (61.1%, n = 11), and social stigma (66.7%, n = 12). The majority (83.3%, n = 15) had lip involvement. Four patients (22.2%) underwent staged flap procedures including prelamination, flap delay, or pedicle division. The mean duration of surgical procedure was 87 minutes (range 5-306 minutes). The minor complication rate was 16%. There were no major complications or deaths. CONCLUSION: Noma is a mutilating disease of the face that occurs in settings of extreme malnutrition. A total of 55.5% of noma patients were made aware of surgical care through outreach programs. The majority of noma patients seek care to improve function (61.1%) and appearance (61.1%), and to address social stigma (66.7%). A total of 83.3% of noma patients had lip involvement. Facial reconstructive surgeons must rely on principles of congenital, traumatic, and oncologic deformity repair while focusing on safe, reliable procedures for low-resource settings. LEVEL OF EVIDENCE: 4 Laryngoscope, 129:96-99, 2019.

Anesthesia for patients with subglottic cysts.

Acquired subglottic cysts can cause rapid development of respiratory distress. Subglottic cysts are a disease of premature infants and other pathologies of prematurity should be anticipated. Perioperative success is dependent on communication between surgeon and anesthesiologist. Contingency plans for an emergency surgical airway should be in place in the event of total airway obstruction.

Post-Operative Outcomes in Children With and Without Congenital Heart Disease Undergoing Noncardiac Surgery.

BACKGROUND: Significant advances have been made in the diagnosis and treatment of children with congenital heart disease (CHD), allowing for longer life expectancies and an increasing number who will require noncardiac surgery. OBJECTIVES: This study sought to compare the incidence of mortality and major adverse post-operative outcomes following noncardiac surgery in children with and without CHD. METHODS: Data from the 2012 pediatric database of the American College of Surgeons National Surgical Quality Improvement Program were analyzed. After propensity score matching, and stratification by severity of CHD, mortality and adverse post-operative outcomes were compared between controls and children with CHD. RESULTS: Among the 51,008 children included in the database, 4,520 children with CHD underwent noncardiac surgery. After propensity score matching, we included 2,805 children with minor CHD, 1,272 with major CHD, and 417 with severe CHD. Children in each subgroup were matched and compared with controls without CHD who underwent noncardiac surgery of comparable complexity. The incidence of overall mortality was significantly higher in children with moderate (3.9%) and severe (8.2%) CHD compared with their controls (respectively, 1.7% [p < 0.001] and 1.2% [p = 0.001]). Both 30-day and overall mortality were significantly increased in children with severe CHD (odds ratio [OR]: 8.43, 95% confidence interval [CI]: 2.52 to 28.21; p < 0.001; OR: 7.32, 95% CI: 2.83 to 18.90; p < 0.001) compared with their matched controls. Overall mortality was also significantly increased in children with major CHD compared with their controls (OR: 2.28; 95% CI: 1.37 to 3.79; p = 0.002), whereas no difference was observed between children with minor CHD and their matched controls. CONCLUSIONS: Children with major and severe CHD, undergoing noncardiac surgery, have an increased risk of mortality compared with children without CHD. Further studies need to identify the optimal environment for surgical procedures, develop trained multidisciplinary teams to care for children with CHD, and define management strategies for improving outcomes in this high-risk population.

Daily online cone beam computed tomography to assess interfractional motion in patients with intact cervical cancer.

PURPOSE: To quantify interfraction motion in patients with intact cervical cancer and assess implications for clinical target volume (CTV) coverage and required planning margins. METHODS AND MATERIALS: We analyzed 10 patients undergoing external beam radiotherapy using online cone beam computed tomography (CBCT) before each fraction. CTVs were contoured on the planning CT and on each CBCT. Each CBCT was rigidly registered to the planning CT with respect to bony anatomy. The CTV from each CBCT was projected onto the planning CT and compared to the CTV from the planning CT. Uniform three-dimensional expansions were applied to the planning CTV to assess required planning margins. For each fraction, the minimum margin required to encompass the CTV was calculated, and the volume of CTV (on the CBCT) encompassed by the PTV was determined as a function of margin size. RESULTS: A uniform CTV planning treatment volume margin of 15 mm would have failed to encompass the CTV in 32% of fractions. The mean volume of CTV missed, however, was small (4 cc). The mean planning margin (across patients and fractions) required to encompass the CTV was 15 mm. Variation in margin estimates was high, with interpatient variation being the predominant component. Increased rectal volume was associated with posterior (p < 0.0001) and superior (p = 0.0004) shifts in the CTV, whereas increased bladder volume was associated with superior shifts (p < 0.0001). CONCLUSIONS: Interfraction motion results in a high probability of missing the CTV using conventional planning margins, but the volume of CTV missed is small. Adaptive radiotherapy approaches are needed to improve treatment accuracy.