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Glioblastoma (GBM) presents a significant public health challenge as the deadliest and most common malignant brain tumor in adults. Despite standard-of-care treatment, which includes surgery, radiation, and chemotherapy, mortality rates are high, underscoring the critical need for advancing GBM therapy. Over the past two decades, numerous clinical trials have been performed, yet only a small fraction demonstrated a benefit, raising concerns about the predictability of current preclinical models. Traditionally, preclinical studies utilize treatment-naïve tumors, failing to model the clinical scenario where patients undergo standard-of-care treatment prior to recurrence. Recurrent GBM generally exhibits distinct molecular alterations influenced by treatment selection pressures. In this review, we discuss the impact of treatment-surgery, radiation, and chemotherapy-on GBM. We also provide a summary of treatments used in preclinical models, advocating for their integration to enhance the translation of novel strategies to improve therapeutic outcomes in GBM.

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TLRs initiate innate immune signaling pathways via Toll/IL-1R (TIR) domains on their cytoplasmic tails. Various bacterial species also express TIR domain-containing proteins that contribute to bacterial evasion of the innate immune system. Bacterial TIR domains, along with the mammalian sterile α and TIR motif-containing protein 1 and TIRs from plants, also have been found to exhibit NADase activity. Initial X-ray crystallographic studies of the bacterial TIR from Acinetobacter baumannii provided insight into bacterial TIR structure but were unsuccessful in cocrystallization with the NAD+ ligand, leading to further questions about the TIR NAD binding site. In this study, we designed a Course-Based Undergraduate Research Experience (CURE) involving 16-20 students per year to identify amino acids crucial for NADase activity of A. baumannii TIR domain protein and the TIR from Escherichia coli (TIR domain-containing protein C). Students used structural data to identify amino acids that they hypothesized would play a role in TIR NADase activity, and created plasmids to express mutated TIRs through site-directed mutagenesis. Mutant TIRs were expressed, purified, and tested for NADase activity. The results from these studies provide evidence for a conformational change upon NAD binding, as was predicted by recent cryogenic electron microscopy and hydrogen-deuterium exchange mass spectrometry studies. Along with corroborating recent characterization of TIR NADases that could contribute to drug development for diseases associated with dysregulated TIR activity, this work also highlights the value of CURE-based projects for inclusion of a diverse group of students in authentic research experiences.

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Fibrotic interstitial lung diseases (fILDs) have poor survival rates and lack effective therapies. Despite evidence for immune mechanisms in lung fibrosis, immunotherapies have been unsuccessful for major types of fILD. Here, we review immunological mechanisms in lung fibrosis that have the potential to impact clinical practice. We first examine innate immunity, which is broadly involved across fILD subtypes. We illustrate how innate immunity in fILD involves a complex interplay of multiple cell subpopulations and molecular pathways. We then review the growing evidence for adaptive immunity in lung fibrosis to provoke a re-examination of its role in clinical fILD. We close with future directions to address key knowledge gaps in fILD pathobiology: (1) longitudinal studies emphasizing early-stage clinical disease, (2) immune mechanisms of acute exacerbations, and (3) next-generation immunophenotyping integrating spatial, genetic, and single-cell approaches. Advances in these areas are essential for the future of precision medicine and immunotherapy in fILD.

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There remains a large need for a greater understanding of the metastatic process within the prostate cancer field. Our research aims to understand the adaptive - ergo potentially metastatic - responses of cancer to changing microenvironments. Emerging evidence has implicated a role of the Polyaneuploid Cancer Cell (PACC) state in metastasis, positing the PACC state as capable of conferring metastatic competency. Mounting in vitro evidence supports increased metastatic potential of cells in the PACC state. Additionally, our recent retrospective study of prostate cancer patients revealed that PACC presence in the prostate at the time of radical prostatectomy was predictive of future metastatic progression. To test for a causative relationship between PACC state biology and metastasis, we leveraged a novel method designed for flow-cytometric detection of circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) in subcutaneous, caudal artery, and intracardiac mouse models of metastasis. This approach provides both quantitative and qualitative information about the number and PACC-status of recovered CTCs and DTCs. Collating data from all models, we found that 74% of recovered CTCs and DTCs were in the PACC state. In vivo colonization assays proved PACC populations can regain proliferative capacity at metastatic sites following dormancy. Additional direct and indirect mechanistic in vitro analyses revealed a PACC-specific partial Epithelial-to-Mesenchymal-Transition phenotype and a pro-metastatic secretory profile, together providing preliminary evidence that PACCs are mechanistically linked to metastasis.

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Glioblastoma (GBM) is the most aggressive brain cancer. To model GBM in research, orthotopic brain tumor models, including syngeneic models like GL261 and genetically engineered mouse models like TRP, are used. In longitudinal studies, tumor growth and the treatment response are typically tracked with in vivo imaging, including bioluminescence imaging (BLI), which is quick, cost-effective, and easily quantifiable. However, BLI requires luciferase-tagged cells, and recent studies indicate that the luciferase gene can elicit an immune response, leading to tumor rejection and experimental variation. We sought to optimize the engraftment of two luciferase-expressing GBM models, GL261 Red-FLuc and TRP-mCherry-FLuc, showing differences in tumor take, with GL261 Red-FLuc cells requiring immunocompromised mice for 100% engraftment. Immunohistochemistry and MRI revealed distinct tumor characteristics: GL261 Red-FLuc tumors were well-demarcated with densely packed cells, high mitotic activity, and vascularization. In contrast, TRP-mCherry-FLuc tumors were large, invasive, and necrotic, with perivascular invasion. Quantifying the tumor volume using the HALO® AI analysis platform yielded results comparable to manual measurements, providing a standardized and efficient approach for the reliable, high-throughput analysis of luciferase-expressing tumors. Our study highlights the importance of considering tumor engraftment when using luciferase-expressing GBM models, providing insights for preclinical research design.

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Fibrosis drives end-organ damage in many diseases. However, clinical trials targeting individual upstream activators of fibroblasts, such as TGFß, have largely failed. Here, we target the leukemia inhibitory factor receptor (LIFR) as a "master amplifier" of multiple upstream activators of lung fibroblasts. In idiopathic pulmonary fibrosis (IPF), the most common fibrotic lung disease, we found that lung myofibroblasts had high LIF expression. Further, TGFß1, one of the key drivers of fibrosis, upregulated LIF expression in IPF fibroblasts. In vitro anti-LIFR antibody blocking on human IPF lung fibroblasts reduced induction of profibrotic genes downstream of TGFß1, IL-4 and IL-13. Further, siRNA silencing of LIFR in IPF precision cut lung slices reduced expression of fibrotic proteins. Together, we find that LIFR drives an autocrine positive feedback loop that amplifies and sustains pathogenic activation of IPF fibroblasts downstream of multiple external stimuli, implicating LIFR as a therapeutic target in fibrosis. Significance Statement: Fibroblasts have a central role in the pathogenesis of fibrotic diseases. However, due to in part to multiple profibrotic stimuli, targeting a single activator of fibroblasts, like TGFß, has not yielded successful clinical treatments. We hypothesized that a more effective therapeutic strategy is identifying a downstream "master amplifier" of a range of upstream profibrotic stimuli. This study identifies the leukemia inhibitory factor receptor (LIFR) on fibrotic lung fibroblasts amplifies multiple profibrotic stimuli, such as IL-13 and TGFß. Blocking LIFR reduced fibrosis in ex vivo lung tissue from patients with idiopathic pulmonary fibrosis (IPF). LIFR, acting as a master amplifier downstream of fibroblast activation, offers an alternative therapeutic strategy for fibrotic diseases.

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BACKGROUND: Misuse of prescription opioids is a well-established contributor to the US opioid epidemic. The primary objective of this study was to identify which level of care delivery (ie patient, prescriber, or hospital) produced the most unwarranted variation in opioid prescribing after common surgical procedures. STUDY DESIGN: Electronic health record data from a large multihospital healthcare system were used in conjunction with random-effect models to examine variation in opioid prescribing practices after similar inpatient and outpatient surgical procedures between October 2019 and September 2021. Unwarranted variation was conceptualized as variation resulting from prescriber behavior unsupported by evidence. Covariates identified as drivers of warranted variation included characteristics known to influence pain levels or patient safety. All other model variables, including prescriber specialty and patient race, ethnicity, and insurance status were characterized as potential drivers of unwarranted variation. RESULTS: Among 25,188 procedures with an opioid prescription at hospital discharge, 53.5% exceeded guideline recommendations, corresponding to 13,228 patients receiving the equivalent of >140,000 excess 5 mg oxycodone tablets after surgical procedures. Prescribing variation was primarily driven by prescriber-level factors, with approximately half of the total variation in morphine milligram equivalents prescribed observed at the prescriber level and not explained by any measured variables. Unwarranted covariates associated with higher prescribed opioid quantity included non-Hispanic Black race, Medicare insurance, smoking history, later hospital discharge times, and prescription by a surgeon rather than a hospitalist or primary care provider. CONCLUSIONS: Given the large proportion of unexplained variation observed at the provider level, targeting prescribers through education and training may be an effective strategy for reducing postoperative opioid prescribing.

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BACKGROUND: Studies have indicated that patients infected with the SARS-CoV-2 virus fare worse clinically after a traumatic injury, especially those who are older and have other comorbidities. OBJECTIVE: This study aims to understand the effects of Corona Virus Disease 19 (COVID-19) diagnosis on patients undergoing surgery for hip fractures. METHODS: This is a retrospective review of the 2021 American College of Surgeons (ACS) National Surgical Quality Improvement Program (NSQIP) Targeted Hip Fracture database for patients who underwent surgery. Two cohorts were formed based on patients' preoperative COVID-19 status, as tested within 14 days prior to the operation. Several clinical factors were compared. RESULTS: The COVID-positive cohort consisted of 184 patients, all of whom had a laboratory-confirmed or clinically suspected SARS-CoV-2 infection, while the COVID-negative cohort consisted of 12,211 patients with no infection. A lower proportion of COVID-positive patients had an emergent operation compared to the COVID-negative cohort (58.70% vs. 73.09%, p < .001). Preoperatively, the COVID-positive cohort showed higher rates of coagulopathy/bleeding disorders (22.83% vs. 14.12%), congestive heart failure (16.30% vs. 9.84%), diabetes mellitus (28.26% vs. 19.24%), and dementia (42.39% vs. 28.07%), with p ≤ .005 for all. Postoperatively, a higher proportion of COVID-positive patients died (9.78% vs. 5.40%) or had pneumonia (8.70% vs. 3.65%), hospital readmission within 30 days (10.87% vs. 6.76%), and pressure sores (8.15% vs. 4.55%), with p ≤ .033 for all. CONCLUSION: The diagnosis of COVID-19 in hip fracture patients was associated with higher rates of postoperative complications, including mortality, when compared to COVID-negative patients, indicating the severity of the viral infection.

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BACKGROUND: There is no consensus on whether laparoscopic experience should be a prerequisite for robotic training. Further, there is limited information on skill transference between laparoscopic and robotic techniques. This study focused on the general surgery residents' learning curve and skill transference within the two minimally invasive platforms. METHODS: General surgery residents were observed during the performance of laparoscopic and robotic inguinal hernia repairs. The recorded data included objective measures (operative time, resident participation indicated by percent active time on console or laparoscopy relative to total case time, number of handoffs between the resident and attending), and subjective evaluations (preceptor and trainee assessments of operative performance) while controlling for case complexity, patient comorbidities, and residents' prior operative experience. Wilcoxon two-sample tests and Pearson Correlation coefficients were used for analysis. RESULTS: Twenty laparoscopic and forty-four robotic cases were observed. Mean operative times were 90 min for robotic and 95 min for laparoscopic cases (P = 0.4590). Residents' active participation time was 66% on the robotic platform and 37% for laparoscopic (P = < 0.0001). On average, hand-offs occurred 9.7 times during robotic cases and 6.3 times during laparoscopic cases (P = 0.0131). The mean number of cases per resident was 5.86 robotic and 1.67 laparoscopic (P = 0.0312). For robotic cases, there was a strong correlation between percent active resident participation and their prior robotic experience (r = 0.78) while there was a weaker correlation with prior laparoscopic experience (r = 0.47). On the other hand, prior robotic experience had minimal correlation with the percent active resident participation in laparoscopic cases (r = 0.12) and a weak correlation with prior laparoscopic experience (r = 0.37). CONCLUSION: The robotic platform may be a more effective teaching tool with a higher degree of entrustability indicated by the higher mean resident participation. We observed a greater degree of skill transference from laparoscopy to the robot, indicated by a higher degree of correlation between the resident's prior laparoscopic experience and the percent console time in robotic cases. There was minimal correlation between residents' prior robotic experience and their participation in laparoscopic cases. Our findings suggest that the learning curve for the robot may be shorter as prior robotic experience had a much stronger association with future robotic performance compared to the association observed in laparoscopy.

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Rapid and reliable circulating tumor cell (CTC) and disseminated tumor cell (DTC) detection are critical for rigorous evaluation of in vivo metastasis models. Clinical data show that each step of the metastatic cascade presents increasing barriers to success, limiting the number of successful metastatic cells to fewer than 1 in 1,500,000,000. As such, it is critical for scientists to employ approaches that allow for the evaluation of metastatic competency at each step of the cascade. Here, we present a flow cytometry-based method that enables swift and simultaneous comparison of both CTCs and DTCs from single animals, enabling evaluation of multiple metastatic steps within a single model system. We present the necessary gating strategy and optimized sample preparation conditions necessary to capture CTCs and DTCs using this approach. We also provide proof-of-concept experiments emphasizing the appropriate limits of detection of these conditions. Most importantly, we successfully recover CTCs and DTCs from murine blood and bone marrow. In Supplemental materials, we expand the applicability of our method to lung tissue and exemplify a potential multi-plexing strategy to further characterize recovered CTCs and DTCs. This approach to multiparameter flow cytometric detection and analysis of rare cells in in vivo models of metastasis is reproducible, high throughput, broadly applicable, and highly adaptable to a wide range of scientific inquiries. Most notably, it simplifies the recovery and analysis of CTCs and DTCs from the same animal, allowing for a rapid first look at the comparative metastatic competency of various model systems throughout multiple steps of the metastatic cascade.

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Importance: Excess opioid prescribing after surgery can result in prolonged use and diversion. Email feedback based on social norms may reduce the number of pills prescribed. Objective: To assess the effectiveness of 2 social norm-based interventions on reducing guideline-discordant opioid prescribing after surgery. Design, Setting, and Participants: This cluster randomized clinical trial conducted at a large health care delivery system in northern California between October 2021 and October 2022 included general, obstetric/gynecologic, and orthopedic surgeons with patients aged 18 years or older discharged to home with an oral opioid prescription. Interventions: In 19 hospitals, 3 surgical specialties (general, orthopedic, and obstetric/gynecologic) were randomly assigned to a control group or 1 of 2 interventions. The guidelines intervention provided email feedback to surgeons on opioid prescribing relative to institutionally endorsed guidelines; the peer comparison intervention provided email feedback on opioid prescribing relative to that of peer surgeons. Emails were sent to surgeons with at least 2 guideline-discordant prescriptions in the previous month. The control group had no intervention. Main Outcome and Measures: The probability that a discharged patient was prescribed a quantity of opioids above the guideline for the respective procedure during the 12 intervention months. Results: There were 38 235 patients discharged from 640 surgeons during the 12-month intervention period. Control-group surgeons prescribed above guidelines 36.8% of the time during the intervention period compared with 27.5% and 25.4% among surgeons in the peer comparison and guidelines arms, respectively. In adjusted models, the peer comparison intervention reduced guideline-discordant prescribing by 5.8 percentage points (95% CI, -10.5 to -1.1; P = .03) and the guidelines intervention reduced it by 4.7 percentage points (95% CI, -9.4 to -0.1; P = .05). Effects were driven by surgeons who performed more surgeries and had more guideline-discordant prescribing at baseline. There was no significant difference between interventions. Conclusions and Relevance: In this cluster randomized clinical trial, email feedback based on either guidelines or peer comparison reduced opioid prescribing after surgery. Guideline-based feedback was as effective as peer comparison-based feedback. These interventions are simple, low-cost, and scalable, and may reduce downstream opioid misuse. Trial Registration: ClinicalTrials.gov NCT05070338.

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Immunophenotyping of out-of-hospital cardiac arrest (OHCA) patients is of increasing interest but has challenges. Here, we describe steps for the design of the clinical cohort, planning patient enrollment and sample collection, and ethical review of the study protocol. We detail procedures for blood sample collection and cryopreservation of peripheral blood mononuclear cells (PBMCs). We detail steps to modulate immune checkpoints in OHCA PBMC ex vivo. This protocol also has relevance for immunophenotyping other types of critical illness. For complete details on the use and execution of this protocol, please refer to Tamura et al. (2023).1.

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Introduction: Fatigue, brain fog, and sleep disturbance are among the most common symptoms of postacute sequelae of SARS-CoV-2 infection (PASC). We sought to determine the impact of sleep disruption on cognition and quality of life in patients with neurologic manifestations of PASC (Neuro-PASC). Methods: Thirty-nine patients were recruited from Neuro-COVID-19 clinic. Mean age was 48.1 years, 71.8% were female, and 82% were never hospitalized for COVID-19. Patients were evaluated via clinical assessment, quality-of-life measures in domains of cognitive function, fatigue, sleep disturbance, anxiety, and depression, NIH Toolbox cognitive tests, and 7 days of wrist actigraphy. Results: The median number of neurologic symptoms attributed to PASC was 6, with brain fog being the most common in 89.7%. Regarding non-neurologic symptoms, 94.9% complained of fatigue and 74.4% of insomnia. Patients reported significant impairment in all quality-of-life domains and performed worse in a task of attention compared to a normative US population. Actigraphy showed Neuro-PASC patients had lower sleep efficiency, longer sleep latency (both p < 0.001), and later sleep midpoint (p = 0.039) compared to 71 age-matched healthy controls with no PASC history. Self-reported cognitive symptoms correlated with the severity of fatigue (p < 0.001), anxiety (p = 0.05), and depression (p < 0.01). Objective evidence of sleep disruption measured by wakefulness after sleep onset, sleep efficiency, and latency were associated with decreased performance in attention and processing speed. Conclusion: Prospective studies including larger populations of patients are needed to fully determine the interplay of sleep disruption on the cognitive function and quality of life of patients with PASC.

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Septal Myectomy (SM) and Alcohol Septal Ablation (ASA) improve symptoms in patients with Hypertrophic Cardiomyopathy with outflow tract obstruction (oHCM). However, outcomes data in this population is predominantly from specialized centers. The National Inpatient Database was queried from 2011 to 2019 for relevant international classification of diseases (ICD)-9 and -10 diagnostic and procedural codes. We compared baseline characteristics and in-hospital outcomes of patients with oHCM who underwent SM vs ASA. A p-value < 0.001 was considered statistically significant. We identified 15,119 patients with oHCM who underwent septal reduction therapies, of whom 57.4% underwent SM, and 42.6% underwent ASA. Patients who underwent SM had higher all-cause mortality (OR: 1.8 (1.3-2.5)), post-procedure ischemic stroke (OR: 2.3 (1.7-3.2)), acute kidney injury (OR: 1.4 (1.2-1.7)), vascular complications (OR: 3.6 (2.3-5.3)), ventricular septal defect (OR: 4.4 (3.2-6.1)), cardiogenic shock (OR: 1.7 (1.3-2.3)), sepsis (OR: 3.2 (1.9-5.4)), and left bundle branch block (OR: 3.5 (3-4)), compared to ASA. Patients who underwent ASA had higher post-procedure complete heart block (OR: 1.3 (1.1-1.4)), right bundle branch block (OR: 6.3 (5-7.7)), ventricular tachycardia (OR: 2.2 (1.9-2.6)), supraventricular tachycardia (OR: 1.6 (1.4-2)), and more commonly required pacemaker insertion (OR: 1.4 (1.3-1.7)) (p < 0.001 for all) compared to SM. This nationwide analysis evidenced that patients undergoing SM had higher in-hospital mortality and periprocedural complications than ASA; however, those undergoing ASA had more post-procedure conduction abnormalities and pacemaker implantation. The implications of these findings warrant further investigation regarding patient selection strategies for these therapies.

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This study investigated the impact of initial culture media pH on the antibacterial properties and metabolic profile of cell-free supernatants (CFSs) from Lactobacillus acidophilus BIOTECH 1900 (LAB1900). The CFSs harvested from LAB1900 grown in de Man, Rogosa, Sharpe broth with initial pH of 5.5 (CFS5.5) and 6.6 (CFS6.6) were tested. The two CFSs elicited varying degrees of activity against three gram-negative bacteria. In the agar-well diffusion against Pseudomonas aeruginosa, CFS5.5 and CFS6.6 recorded 14.36 ± 1.34 and 13.06 ± 1.29 mm inhibition, respectively. Interestingly, against Klebsiella pneumoniae, CFS5.5 showed 14.36 ± 1.56 mm inhibition which was significantly higher than the 12.22 ± 1.31 mm inhibition of CFS6.6 (p = 0.0464). While against Acinetobacter baumannii, significantly higher inhibition of 10.66 ± 0.51 mm was observed in CFS6.6 compared to the 7.58 ± 1.93 mm inhibition of CFS5.5 (p = 0.0087). Nonetheless, both CFSs were bactericidal, with a minimum inhibitory and bactericidal concentration range of 3.90625-7.8125 mg/mL. The varied antibacterial activities may be attributed to the metabolite compositions of CFSs. A total of 152 metabolites driving the separation between CFSs were noted, with the majority upregulated in CFS5.5. Furthermore, 15 were putatively identified belonging to acylcarnities, vitamins, gibberellins, glycerophospholipids, and peptides. In summary, initial culture media pH affects the production of microbial metabolites with antibacterial properties.

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BACKGROUND: We sought to evaluate the unique benefits and challenges the virtual recruitment and interviewing platform had on general surgery residency applicants. METHODS: Applicants who interviewed for a categorical position at our institution during the 2021 and 2022 Match season were contacted to participate in the anonymous online survey focused on applicant behavior related to the virtual interview format. Data were analyzed using chi-square and paired t-tests. RESULTS: A response rate of 56.7 â€‹% (n â€‹= â€‹135) was achieved. Applicants accepted a median of 17 (IQR 13-20) interviews in 2021 and 15 (IQR 11-19) interviews in 2022. More than half (54 â€‹%) of applicants indicated they applied to more programs, and 53 â€‹% accepted more interviews, because of the virtual format. The greatest advantages of the virtual interviews as cited by applicants were saving money (96.3 â€‹%), saving time (49.6 â€‹%), and avoiding travel risks (43.7 â€‹%). The top limitations of virtual interviews were less exposure to current residents and faculty (61.5 â€‹%), to the city or location of the program (58.5 â€‹%), and difficultly comparing programs (57.8 â€‹%). The 2022 Match cycle included use of the supplemental application; however, 85 â€‹% of applicants did not feel that the supplemental improved their overall application. Some applicants (20 â€‹%) who "signaled" programs did not receive an interview offer from any of the programs they signaled. CONCLUSION: The transition to virtual interviews saved applicants time and money but limited their exposure. Future efforts to maintain virtual interviews will need to be balanced against the intangible benefit of human interaction and observing a program's culture.

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BACKGROUND: Little is known about the distribution and outcomes of hip fractures in pediatric patients during the COVID-19 pandemic. OBJECTIVE: To study the clinical outcomes of both pediatric and adult patients who underwent hip fracture surgeries and determine the effects of changes surrounding the COVID-19 pandemic. METHODS: Both pediatric and adult surgical hip fracture cases were analyzed from the pandemic year (2020) and the control year (2019) using the American College of Surgeons National Surgical Quality Improvement Program database. RESULTS: Between the prepandemic (control) and pandemic years, a total of 2,438 pediatric and 28,180 adult cases were compared. Pediatric patients had similar perioperative characteristics and outcomes between the two years. Significantly fewer hip fractures were reported among adults during the pandemic (p < .001). Preoperatively, more adult patients had ventilator dependence (p = .020), transfusions (p = .029), and systemic inflammatory response syndrome (p < .001) in 2020. Adult operations were more likely to be emergent in 2020 (p < .001) and adults had more severe disease states. Length of stay (p < .001) and the time from operation to discharge (p < .001) were significantly longer for the adult cohort in 2020. Mortality was also higher for adults during the first year of the pandemic (p = .003), and superficial surgical site infections became more common (p = .036). CONCLUSION: Pediatric hip fracture patients had similar clinical outcomes between 2019 and 2020. Adults with hip fractures presented in more serious clinical conditions, which resulted in higher mortality in 2020. Further studies could better clarify the reasons as to why adult hip fracture patients had markedly worse clinical course during the COVID year than pediatric patients.

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