Journal of Leukocyte Biology

Objective: To characterize genome-wide DNA methylation patterns associated with sepsis using the Infinium Methylation EPIC v2.0 platform and to evaluate the feasibility of pooled methylation profiling in a pilot critical care cohort. Design: Single-center pilot epigenome-wide association study using pooled whole-blood genomic DNA and pool-level bioinformatic analysis. Setting: Academic medical center. Patients: Fifty critically ill adults enrolled within 48 hours of illness onset and 20 healthy controls. Interventions: None. Measurements and Main Results: Critically ill patients required mechanical ventilation and/or vasopressor support. Sepsis was defined according to Sepsis-3 criteria. Seventy individual samples were organized into 14 intended pools of 5 individuals each: 7 sepsis pools, 3 critically ill non-septic pools, and 4 healthy-control pools. One critically ill non-septic pool was excluded because of poor DNA quality, yielding 13 analyzable pools. For the primary pooled comparison, 7 sepsis pools were compared with 6 non-sepsis comparator pools comprising 2 critically ill non-septic and 4 healthy-control pools. After quality control and preprocessing with SeSAMe, 876,094 CpG sites were retained. The initial pool-level screen identified 170,897 candidate differentially methylated regions. Application of stringent secondary filters (false discovery rate <= 1%, absolute delta-beta >= 7.5%, and >= 5 CpGs per region) yielded a high-confidence subset with marked directional skewing, including 155 hypomethylated and 32 hypermethylated regions in sepsis. Differentially methylated region-associated genes were enriched in myeloid leukocyte activation, myeloid leukocyte-mediated immunity, defense response to bacterium, neutrophil granule biology, and hematopoietic cell lineage pathways. Additional signals involved microRNA-associated targets, ribosome biogenesis, RNA processing, long noncoding RNAs, and previously uncharacterized loci. Conclusions: In this pilot pooled EPIC v2.0 study, sepsis was associated with a biologically coherent, predominantly hypomethylated methylation signature enriched in myeloid and host-defense pathways. These findings support the feasibility of pooled methylation profiling for discovery-oriented sepsis biobank studies but should be interpreted as hypothesis-generating given the pool-level design, limited effective sample size, heterogeneous comparator group, and lack of direct validation against individual-level methylation profiles.

Abstract Background: Mean platelet volume (MPV) is a simple, low-cost biomarker that reflects platelet activation. Its prognostic value in septic shock remains controversial. We aimed to determine whether MPV at intensive care unit (ICU) admission is associated with hospital mortality in patients with septic shock. Methods: Retrospective cohort study of consecutive adults with septic shock (Sepsis-3 criteria) admitted to a single ICU. MPV, severity scores (SOFA, APACHE II, SAPS II), procalcitonin, and clinical data were collected. The primary outcome was in-hospital mortality. Spearman correlation, univariate and multivariate logistic regression (with Firth's correction), ROC curves, and subgroup analyses were performed. Results: Fifty-eight patients were included; mortality was 58.6%. MPV did not differ between non-survivors and survivors (13.09 {+/-} 1.37 vs. 12.66 {+/-} 1.45 fL, p = 0.259). MPV showed a weak correlation with procalcitonin ({rho} = 0.394, p = 0.002) but not with severity scores. In multivariate analysis adjusting for age, sex, SOFA and comorbidity count, MPV was not an independent predictor of mortality (OR 1.075, 95% CI 0.682-1.755, p = 0.749). The area under the ROC curve for MPV was 0.598 (95% CI 0.444-0.752), significantly lower than that of SOFA (0.837) and procalcitonin (0.836). Subgroup analyses showed no significant association between MPV and mortality in any stratum. Conclusions: In this cohort of septic shock patients, MPV at ICU admission was not associated with hospital mortality and had poor discriminative ability. Widely used severity scores and procalcitonin remain superior prognostic markers. MPV should not be used as a prognostic tool in septic shock. Keywords: Septic shock, Mean platelet volume, Mortality, SOFA, Procalcitonin, Biomarker

Background: Accurate early identification of sepsis remains a major clinical challenge due to its heterogeneous presentation and overlap of clinical signs with the non-infectious systemic inflammatory response syndrome (SIRS). Timely differentiation is crucial for improving patient outcomes, meeting sepsis bundle requirements and reducing inappropriate antimicrobial use. We hypothesized that clinical and laboratory data available within the first 3 hours of patient presentation could be used to identify patients with sepsis to an actionable level of accuracy, in lieu of traditional microbiology results which would not become available until at least 12-24 hours. Data from two independent studies were used to quantify the diagnostic value of demographic, vital, clinical-laboratory, and microbiological data available at three time points for distinguishing retrospectively diagnosed critically ill patients with either sepsis or non-infectious SIRS. A particular focus of this work was an assessment of the utility of SeptiCyte RAPID (Immunexpress Inc., Seattle, Washington, USA) as an aid to sepsis diagnosis, producing actionable data within 1 hour. Methods: Data from two independent study cohorts were analysed. The 510k cohort consisted of 419 adult patients in intensive care (ICU) (MARS, VENUS, and NEPTUNE trials). The Andalusian cohort consisted of 353 ICU patients from the PANGEA study. Logistic regression models, selected by a greedy search algorithm and validated by repeated cross-validation, were used to determine the contributions of different variables to diagnostic accuracy. Diagnostic performance was quantified by area under the receiver operating characteristic curve (AUC). Results: For the 510k cohort, a baseline AUC of 0.69-0.73 was observed using 5-7 vital and demographic variables assessed immediately upon presentation (time T1). The addition of clinical-laboratory variables, in particular SeptiCyte RAPID, within 1-3 hours post-presentation (time T2) increased the AUC to 0.83-0.85). Finally, the addition of microbiological data 12-24 hours post-presentation (time T3) further improved the AUC to 0.90-0.91. Similar results were obtained for the Andalusian cohort. AUC values at the three time points were as follows: At time T1, AUC = 0.67 based solely on vital signs and demographics; at time T2, AUC = 0.87 based on vitals + demographics + SeptiCyte RAPID or other clinical laboratory data; at time T3, AUC = 0.93 based on vitals + demographics + SeptiCyte RAPID or other clinical laboratory data + microbiology results). For both cohorts, the most significant variables included temperature, mean arterial pressure, respiratory rate, suspected infection site; SeptiCyte RAPID, procalcitonin, confirmed bacterial infection and positive blood culture confirmation. Conclusions: Accuracy of identification of sepsis increases markedly as demographics and vital signs are supplemented with clinical-laboratory information, and ultimately with microbiological culture results. The fastest improvement occurs within the first 3 hours when laboratory data, and in particular SeptiCyte RAPID results, become available. Integrating rapid host-response testing with SeptiCyte RAPID into time-based diagnostic frameworks may enhance early sepsis recognition, improve antimicrobial stewardship, and support guideline-driven clinical decisions.

Background: Chronic rhinosinusitis (CRS) is a heterogeneous inflammatory airway disease associated with impaired mucociliary clearance and persistent inflammation. While prior work has focused on inflammatory and molecular pathways, the physicochemical properties of mucus itself remain poorly characterized. This study aimed to define compositional and biophysical features of CRS mucus that may contribute to dysfunction. Methods: A prospective cross-sectional study was conducted in 15 adults undergoing endoscopic sinus surgery (11 CRS, 4 controls). Mucus was collected from the middle meatus. Hydration was measured by lyophilization. Ionic composition was quantified using mass spectrometry. Viscoelasticity was assessed via oscillatory shear rheology. Total protein, total carbohydrate, sialic acid (Sia) and fucose (Fuc) content were quantified using enzymatic and chemical assays. Statistical comparisons were performed using nonparametric tests. Results: CRS mucus exhibited significantly higher Ca2+; and Mg2+; concentrations (approximately two-fold; p<0.05) and increased variability in hydration and ion content compared to controls. Rheology showed greater heterogeneity and a non-significant trend toward increased viscoelasticity in CRS. Total protein and carbohydrate content were not significantly different; however, the carbohydrate-to-protein ratio was significantly reduced in CRS (p=0.04). Sia content and Sia-to-carbohydrate ratio were significantly elevated in CRS (p=0.04 and p=0.002), particularly in CRS with nasal polyps. Fuc content did not differ between groups. Conclusions: CRS mucus demonstrates coordinated alterations in ionic composition and glycosylation, characterized by increased cation content, hypersialylation, and reduced carbohydrate-to-protein ratios. These changes may contribute to altered mucus properties and impaired mucociliary clearance, highlighting mucus composition as a potential therapeutic target in CRS.

Overview: SeptiCyte RAPID is an FDA-cleared gene expression test that quantifies host immune response to aid in the diagnosis of sepsis. The test yields a score (the SeptiScore) ranging from 0-15, distributed across four bands (1-4) based on increased likelihood of sepsis. Each band can be characterized by average positive and negative likelihood ratios (LR+, LR- respectively) for the discrimination of sepsis versus the non-infectious systemic inflammatory response syndrome (SIRS). Methods: A retrospective analysis of prospectively collected data from a combined cohort of critically ill patients suspected of sepsis (N=889), recruited across 19 hospitals in the USA and Europe. The analysis quantified the LR+ and LR- parameters as a function of SeptiScore, for discrimination of sepsis vs. SIRS in patients admitted to ICU. Hypotheses: (1) The likelihood ratio (LR) framework provides a clinically useful interpretive approach that complements the previously used SeptiScore banding scheme; (2) Low Band 1 SeptiScores are associated with sufficiently small LR- to support the use of SeptiCyte RAPID as a rule-out test for sepsis; (3) High Band 4 SeptiScores are associated with sufficiently large LR+ to support the use of SeptiCyte RAPID as a rule-in test for sepsis; and (4) SeptiScore-derived LR+ and LR- values can be combined with estimates of pre-test probability (derived from patient characteristics and/or other diagnostic tests) to generate individualized, patient-specific post-test probabilities of sepsis. Results: The SeptiCyte RAPID test demonstrates strong diagnostic performance in distinguishing sepsis from SIRS. The likelihood ratios across different score bands provide clear clinical utility: the median LR+ was 3.26 (range 2.57-4.24) for Band 3, and 6.97 (range 4.35-15.57) for Band 4 providing evidence toward ruling in sepsis at high SeptiScores. Conversely, the median LR- was 0.16 (range 0.14-0.20) for Band 2 and 0.085 (range 0.014-0.16) for Band 1, providing evidence toward ruling out sepsis at low SeptiScores. A higher-resolution analysis of SeptiCyte RAPID performance confirmed these trends by evaluating LR+ and LR- at specific values within each band. The sepsis group was further stratified according to whether patients were classified as blood-culture positive (BC+) or blood culture negative (BC-), and the detailed LR+ and LR- analyses were repeated. A monotonic increase in likelihood ratio with increasing SeptiScore was consistently observed, independent of whether sepsis patients were culture-positive, culture-negative, or unstratified with respect to blood culture status. Conclusion: High SeptiScores have correspondingly high LR+ values, and low SeptiScores have correspondingly low LR- values, both of which may have clinical utility. High likelihood ratios for band 4 SeptiScores, which precede traditional microbiology results, may provide clinicians with early confidence of a sepsis diagnosis and microbiology diagnostic stewardship. Low likelihood ratios for band 1 SeptiScores may prompt clinicians to consider an alternate diagnosis to sepsis. Such results, obtained early in the diagnostic workup process, may lead to fewer missed diagnoses and more efficient use of hospital resources.

Clonal hematopoiesis of indeterminate potential (CHIP) represents the age-related expansion of hematopoietic stem cells with preleukemic mutations. However, its association with all-cause and cause-specific mortality has not been well characterized in older adults. We aimed to evaluate whether CHIP is associated with all-cause and cause-specific mortality in a population of older women in the United States. Our study included 6,704 participants in the Women?s Health Initiative Long Life Study (WHI-LLS) without hematologic malignancy. The co-primary exposures were any CHIP (variant allele frequency [VAF] [&ge;] 2%) and large CHIP (VAF [&ge;] 10%), and the primary outcome was all-cause mortality. Multivariable-adjusted Cox proportional hazards models tested the associations of CHIP and CHIP subtypes with all-cause and cause-specific mortality. Any CHIP and large CHIP were independently associated with all-cause mortality, with multivariable-adjusted hazard ratios (aHRs) of 1.12 (95% confidence interval [CI] 1.04-1.21; P = 0.003) and 1.28 (95% CI 1.15-1.43; P < 0.001), respectively. In gene-specific analyses, non-DNMT3A CHIP was associated with all-cause mortality (aHR: 1.22 [95% CI: 1.12-1.34], P < 0.001), while DNMT3A CHIP was not (aHR: 1.07 [95% CI: 0.98-1.18], P = 0.13). Furthermore, large CHIP was associated with cardiovascular (aHR: 1.29 [95% CI: 1.08-1.55], P = 0.006), cancer (aHR: 1.49 [95% CI: 1.11-2.02], P = 0.009), and neurologic (aHR: 1.40 [95% CI: 1.07-1.84], P = 0.02) death. In this cohort of older women, CHIP, particularly large clones and non-DNMT3A CHIP, was associated with all-cause and cause-specific mortality. These findings suggest that clonal size and subtype may differentially influence mortality risk.

Background: Community-acquired pneumonia (CAP) remains a major global health burden disproportionately affecting older adults and people with comorbidities, with Streptococcus pneumoniae as one of the leading bacterial causes in Europe. The Hungarian Occurrence and Burden of PnEumonia (Hungarian-HOPE) study examined the incidence, hospitalization rates, and mortality of CAP between 2016 and 2020 in Hungary. Methods: The National Health Insurance Fund database was used to identify adult CAP patients (all-cause) based on ICD-10 codes J10-18. Outcomes included CAP incidence, 0-15-day hospitalization, and 0-30-day mortality after hospitalization, stratified by age, sex, and comorbidities (chronic obstructive pulmonary disease [COPD], asthma, cardiovascular disease [CVD], and type 1 and 2 diabetes [T1DM, T2DM]). Risk maps visualized relative risk gradients across population strata. Results: During the pre-pandemic period (2016-2019), over 100,000 CAP cases and more than 50,000 hospitalizations were recorded annually. In 2020, recorded cases fell to approximately 98,000, while hospitalizations increased to 66,200. Hospitalization rates increased from 25.1% in 2016 to 29.1% in 2019, then increased to 43.1% in 2020. The 30-day mortality among hospitalized patients rose from 22.7% in 2016 to 23.6% in 2019. Incidence, hospitalization, and mortality all increased with age. Relative to healthy males aged 30-39 years, CAP risk escalated steeply in the [&ge;]80 years cohort (incidence 5-15-fold; hospitalization >3-fold; mortality 11-24-fold) and was further amplified by COPD, CVD, or T2DM, with a lesser effect for T1DM. Conclusions: The results highlight the substantial age- and comorbidity-driven CAP burden in Hungary and support prioritization of preventive strategies including pneumococcal vaccination for older adults and high-risk groups.

Objectives To examine the acute effects of a single bout of high-intensity interval training (HIIT) on fetal blood flow distribution during the third trimester of pregnancy. Methods Thirty-four healthy pregnant participants (mean age 31.6 years, standard deviation (SD) 4.1; gestational week 33.8 (SD 0.4) completed eight 30-second high-intensity cycling work-bouts interspersed with 2-minute rest periods. Fetal heart rate (FHR), maternal blood pressure, and Doppler-derived blood flow indices in the middle cerebral artery, umbilical artery and vein, and ductus venosus were assessed before and after exercise. We estimated fetal liver blood flow and the ratio of umbilical vein flow to ductus venosus. Maternal heart rate (HR) and FHR were recorded throughout exercise. Paired t-tests compared pre- and post-exercise values. Results No significant changes were observed in fetal blood flow indices or distribution following exercise. Average maternal HR and FHR during the work-bouts were 158 bpm (SD 16) and 152 bpm (SD 12), respectively. Following HIIT, maternal systolic blood pressure increased by 5 mmHg (95% CI 1 to 8, p=.014), maternal HR by 22 bpm (95% CI 15 to 28, p<.001), and FHR by 13 bpm (95% CI 10 to 17, p<.001). We recorded 16 instances of FHR above normal range during HIIT. Conclusion A single HIIT session in late pregnancy increased maternal blood pressure and HR and transiently elevated FHR but did not affect fetal blood flow indices or distribution. Brief episodes of fetal tachycardia were observed but appeared to be clinically insignificant. Future research should investigate the effects of repeated HIIT exposure during pregnancy.

INTRODUCTION: Cognitive trajectories may clarify how type 2 diabetes (T2D) and impaired fasting glucose (IFG) relate to dementia risk, but longitudinal associations remain unclear, particularly in the context of stroke. METHODS: Data from 5,631 dementia- and stroke-free older adults (mean age 75 years) from 7 international population-based cohorts were analyzed. Linear mixed-effects models estimated cognitive trajectories during stroke-free and post-stroke follow-up. Glucose status was defined by fasting glucose and prior T2D diagnosis. RESULTS: Over 6.6 years of follow-up (4.5% with incident stroke), T2D was associated with lower baseline cognitive performance compared with normal fasting glucose (-0.14 SD, 95% CI -0.21 to -0.07), but not with faster cognitive decline during stroke-free or post-stroke follow-up. IFG was not associated with lower cognitive performance or faster decline. DISCUSSION: In older adults, T2D was associated with persistently lower cognitive performance but not faster decline, suggesting adverse cognitive effects may be established before late life.

We present a compact pump-and-probe mid-infrared Optothermal Spectrometer (OTHES) equipped with Spatial Probing and Autocorrection (SPAC) optimized for robust intravital application in humans. SPAC-OTHES facilitates alignment stability and spectral comparability across different measurement sessions involving different skin types. Contrary to state-of-the-art, SPAC-OTHES uses camera-based beam detection and an auto-calibration mechanism that enables ca. 73% better spectral reproducibility in intravital measurements in human volunteers than non-calibrated readouts. Moreover, SPAC-OTHES has the potential to lower the glucose quantification error, as demonstrated here in artificial skin phantoms, where an improvement of 52% compared to conventional diode-based detection was observed. The compactness of OTHES, combined with reliable SPAC-readout, has the potential to accelerate commercialization and broad application of biosensors based on mid-infrared spectroscopy.

Background: Antenatal care (ANC) utilization is critical for improving maternal and neonatal health outcomes. Despite the World Health Organization recommendation of at least eight ANC contacts during pregnancy and the implementation of free maternal healthcare policies in Ghana, significant geographic and socioeconomic disparities in ANC utilization persist. This study therefore assessed the spatial distribution and geographically varying determinants of ANC utilization among women in Ghana. Methods: A cross sectional analytical study was conducted using women data from the 2022 Ghana Demographic and Health Survey. The analysis included women aged 15 to 49 years with an index child younger than five years preceding the survey. Descriptive statistics were computed using Stata version 18, while spatial analyses were conducted in QGIS version 3.44. Global Morans I was used to assess spatial autocorrelation, whereas Local Morans I and Getis Ord Gi analyses identified spatial clusters, hotspots, and coldspots of ANC utilization. Ordinary Least Squares (OLS) regression and Geographically Weighted Regression (GWR) models were fitted to assess global and local determinants of ANC utilization. Results: Overall, only 26.0% of women achieved adequate ANC utilization, while 74.0% reported inadequate ANC attendance. Adequate ANC utilization was higher among women with higher education (42.0%) and those from the richest households (41.3%) compared with women without formal education (19.1%) and those from the poorest households (17.6%). Regional disparities were observed, with Western (48.8%), Eastern (48.0%), and Greater Accra (47.3%) regions recording the highest ANC utilization, whereas Savannah (24.7%), Northern (25.8%), and North East (26.8%) regions recorded the lowest utilization levels. Global Morans I demonstrated significant positive spatial autocorrelation (Morans I = 0.457, p = 0.044), indicating geographic clustering of ANC utilization across Ghana. Getis Ord Gi analysis identified significant coldspots within Northern, Savannah, and North East regions, while Central Region demonstrated significant hotspot clustering. OLS regression showed that maternal education (B = 0.284, p = 0.003) and household wealth (B = 0.191, p = 0.011) positively influenced ANC utilization, whereas distance to health facility negatively influenced utilization (B = -0.156, p = 0.019). The GWR model demonstrated improved explanatory performance (Adjusted R-squared = 0.71), confirming substantial spatial heterogeneity in ANC determinants across Ghana. Conclusion: Adequate ANC utilization in Ghana remains low and geographically unequal. Maternal education, household wealth, and geographic accessibility significantly influence ANC utilization, with pronounced disparities concentrated within Northern Ghana. Spatially targeted maternal health interventions aimed at improving education, reducing socioeconomic inequalities, and enhancing healthcare accessibility are required to improve equitable ANC utilization across Ghana.

Wearable digital health technologies may complement traditional gait assessments in amyotrophic lateral sclerosis (ALS) by sensitively capturing real-world mobility changes. In this study, we validated six digital gait metrics derived from ankle-worn sensors in a natural history cohort of 182 individuals with ALS. Investigated metrics correspond to various aspects of gait, including volume, speed, intensity, similarity, variability, and fragmentation. Longitudinal analyses showed significant declines in step count, peak cadence, stride intensity, and stride similarity, with increasing stride duration variability and walking fragmentation over 52 weeks. Many participants exhibited greater relative change in the gait metrics than the self-reported ALS Functional Rating Scale-Revised (ALSFRS-RSE). Stratified analyses revealed that digital metrics captured significant functional decline even in participants with stable walking scores on the ALSFRS-RSE. These findings support the potential utility of these metrics for disease monitoring in ALS clinical care and trials.

Background: The rising prices of cancer medicines have intensified concerns about treatment access and health system sustainability particularly in low- and middle-income settings. Systematic facility level evidence on what medicines is actually available, at what prices, and at what cost to patients remains scarce, constraining evidence-based policy reform. Methods: Using adapted WHO/Health action international methodology, we conducted a cross-sectional survey of 52 cancer medicines across five therapeutic classes at five health facilities in Kisumu County, Kenya. Availability was measured as the proportion of facilities stocking each medicine. Affordability was assessed using days' wages required for the lowest-paid government worker to purchase standard treatment regimens, calculated per one chemotherapy cycle and maximum possible cycles. Results: Overall medicine availability was 48.1%, with marked inter-facility variation. Affordability analysis revealed severe financial barriers. The breast cancer AC regimen required 19.6-47.4 days' wages per full course; cervical cancer cisplatin, 19.8-49.2 days' wages; colorectal FOLFOX, 80.0-303.6 days' wages; and prostate docetaxel reached 437 days' wages at the highest-cost facility. The Social Health Authority's (SHA) KES 550,000 annual ceiling adequately covered cytotoxic regimens for common cancers at competitive prices but was exceeded by 24-116% for HER2-positive breast cancer requiring trastuzumab, with further strain for recurrent cervical and metastatic prostate cancers. Conclusions: Cancer medicines in Kisumu County are inconsistently available and highly variable in price resulting in inequitable access. We call for urgent retail price markup regulation, expanded pooled procurement through KEMSA, inclusion of priority targeted therapies on the Kenya Essential Medicines List, and SHA benefit packages redesigned around full-course regimen costs.

Background: RNA sequencing (RNA-seq) is increasingly recognized as a complementary tool to DNA-based sequencing for improving the diagnostic yield in Mendelian disorders. However, how the diagnostic performance of RNA-seq varies across molecularly and phenotypically distinct patient subgroups remains poorly defined. This study aimed to evaluate and compare the diagnostic utility of RNA-seq across three stratified groups of patients with non-diagnostic exome sequencing. Methods: We performed RNA-seq on whole blood samples from 90 patients with suspected Mendelian disease in whom clinical exome or whole-exome sequencing had failed to establish a molecular diagnosis. Patients were prospectively stratified into three groups of 30: (i) patients with a candidate variant of uncertain significance (VUS) with predicted splicing impact (Group 1), (ii) patients with a specific clinical pre-diagnosis but no identified pathogenic variant (Group 2), and (iii) patients without a specific pre-diagnosis or candidate variant (Group 3). Aberrant splicing, gene expression outliers, and allele-specific expression were analyzed using multiple bioinformatic tools and compared against a GTEx-derived control cohort. Results: RNA-seq contributed to a molecular diagnosis in 29 of 88 evaluable patients (32.9%). Diagnostic yield differed substantially across groups: 82.8% (24/29) in Group 1, 6.9% (2/29) in Group 2, and 10% (3/30) in Group 3. In Group 1, RNA-seq enabled reclassification of candidate VUS through direct demonstration of aberrant splicing events. In Group 2, RNA-seq identified a somatic mosaic ACTB variant missed by exome sequencing and reclassified a previously deprioritized APPL1 VUS. In Group 3, a deep intronic pseudoexon-activating variant in IGBP1 was identified in two siblings with severe microcephaly, providing evidence for a candidate X-linked microcephaly gene, and a pathogenic RNU4-2 variant was detected in a patient with ReNU syndrome, a non-protein-coding gene not captured by standard exome sequencing. Conclusions: RNA-seq has the highest diagnostic utility when applied to evaluate candidate splice variants identified by prior DNA testing but also provides independent diagnostic value in patients without candidate variants. The systematic comparison across stratified patient groups supports the integration of RNA-seq into clinical genomic workflows and highlights the need for standardized analytic frameworks.

Background and Objective: Access to real-world electronic health records (EHRs) remains limited by privacy, governance and annotation constraints, hindering the development of clinical natural language processing models. Realistic synthetic progress notes may provide EHR-like corpora that preserve clinically rigorous information on diagnoses, treatments, symptoms, imaging, laboratory findings and therapeutic trajectories without relying directly on sensitive patient records. This study evaluates whether large language models (LLMs) can generate realistic Spanish prostate cancer progress notes from published case reports, preserving clinical content, temporality and hospital-style conventions.

People living with HIV (PLWH) are known to maintain a degree of immune deficiency despite efficient antiretroviral therapy and may exhibit diminished responses to vaccines. In this study, we assessed the immune response to SARS-CoV-2 infection and vaccines in two geographically distinct PLWH populations. PLWH and HIV-negative (HIV-) participants were recruited from Qu&bec City (QC), Canada, and Bobo-Dioulasso (BD), Burkina Faso, for two visits at 24-week intervals during the predominance of the Omicron variant, from May 2022 to September 2023. Blood samples were collected at each visit for the detection of antibodies against spike (anti-S) and nucleocapsid (anti-N) proteins of SARS-CoV-2 in platelet-free plasma. A total of 360 participants were enrolled. We detected anti-S antibodies in 99% of participants, indicating that nearly all had prior exposure to the SARS-CoV-2 spike antigen, either through vaccination or prior infection. Anti-S titers showed no difference between PLWH and HIV& participants in each location, while significantly higher titers were observed in participants from QC compared to BD. In contrast, anti-N antibodies, indicative of prior infection, were detected in 39% and 86% of the participants in QC and BD, respectively, suggesting that the virus circulated largely in the latter population. No difference in anti-N levels was observed between PLWH and HIV& participants in BD. However, participants in QC had significantly lower titers compared to HIV participants. Overall, this study shows that PLWH develop robust antibody responses to SARS-CoV-2 vaccination, comparable to those observed in HIV& participants. Significant geographic differences were observed in anti-S titers, irrespective of HIV status, with participants from QC displaying higher titers. In contrast, participants from BD had higher anti-N antibody prevalence and titers, reflecting more SARS-CoV-2 infections in BD than in QC. Finally, analysis of anti-S antibody titers against several circulating variants revealed significantly lower levels in unvaccinated participants and in those vaccinated with monovalent vaccines in BD. No significant difference was observed between monovalent and bivalent vaccines administered in QC. All authors have seen and approved the manuscript.

Background Oropouche virus (OROV) is an emerging vector-borne virus with rapidly expanding geographic range, increasing case counts, and growing evidence of severe outcomes including neuroinvasive disease and vertical transmission. Because OROV infection presents with nonspecific febrile illness that overlaps clinically with other viruses including dengue, zika, and chikungunya, accurate molecular diagnostics are essential for patient care and surveillance. Yet existing assays rely on single genomic targets and are vulnerable to detection failure as the virus evolves and reassorts. Methodology/Principal Findings To support diagnostic capacity, we developed and clinically validated a multiplexed qPCR assay targeting three regions of the OROV S segment, incorporating redundancy to preserve sensitivity across viral diversity while enabling robust clinical interpretation. The multiplex also includes an assay targeting RNaseP as an internal sample control to ensure adequate sample processing. We evaluated assay performance using both historical and contemporary OROV strains and validated the assay on contrived serum, plasma, and cerebrospinal fluid samples, assessing linearity, limit of detection (LOD), accuracy, specificity, precision, and sample stability. The assay met or exceeded all predefined acceptance criteria for clinical testing and achieved an LOD as low as 6 copies per reaction for contemporary outbreak strains. We further implemented a logic-based interpretation matrix that reduced false-positive risk while maintaining sensitivity near the analytical LOD. Conclusions/Significance Our assay sensitively and specifically detects OROV RNA in serum, plasma, and cerebrospinal fluid while incorporating safeguards against viral evolution and reassortment. The assay has been approved for use by CLIA at Nexus Medical Labs in 49 U.S. states, expanding access to timely OROV diagnostics in the United States and providing a durable framework for molecular detection of reassorting, rapidly evolving viruses as OROV continues to spread into new regions.

Rationale: Efficacious dose selection for anti-tuberculosis drugs has traditionally relied on achieving plasma exposures above the minimum inhibitory concentration, but this approach has not consistently aligned with clinical outcomes. Objectives: We sought to identify early pharmacokinetic-pharmacodynamic targets most predictive of clinical efficacious dose. Methods: We conducted a back-translational, pharmacokinetic-pharmacodynamic simulation-based analysis of 15 anti-tuberculosis drugs. Using pharmacokinetic data from multiple biological matrices and a range of pharmacodynamic metrics, we established candidate exposure-response targets for attainment. We systematically evaluated the predictive accuracy of each target pair against established clinical doses to formulate a decision-making framework linking key drug properties to the most predictive targets. Measurements and Main Results: Depending on the target used, projected clinical doses varied widely - both within and across compounds - highlighting the importance of target selection for dose projection and go/no-go decisions. In general, targeting cellular lesion-level drug exposures relative to in vivo preclinical potency provided an effective approach for early dose selection. However, for highly penetrating drugs, targeting site-of-action therapeutic exposures in the caseum was more predictive of clinical dose. Based on these findings, we developed a preliminary dose prediction tool that enables drug developers to estimate clinically relevant dose ranges of compounds using in vitro and early in vivo data. Conclusions: This work establishes and validates a simple, evidence-based framework to standardize early translational decision-making on dose selection of anti-tuberculosis candidates in development.

Background: Regular exercise is a highly effective yet underutilized strategy to reduce cardiometabolic disease burden. Whether brief structured exercise programs confer lasting cardiometabolic benefits remains unclear. The STRRIDE-Prediabetes Reunion study examined legacy effects of exercise training on cardiorespiratory fitness, body composition, and cardiometabolic health. Methods: Seventy-three participants (71.3 {+/-} 7.2 years; 64% women; 77% White) completed Reunion assessments ~11 years after completing one of four 6-month interventions differing in exercise amount, intensity, and inclusion of diet-induced weight loss. Linear mixed effects models evaluated longitudinal trajectories; secondary analyses examined baseline-adjusted associations among short-term intervention response and Reunion outcomes. Results: Abdominal adiposity improved across all groups from baseline to Reunion, with waist circumference decreasing ~3 cm over the follow-up period. In contrast, cardiorespiratory fitness and fat-free mass declined significantly. A significant group by time interaction was observed for total fat mass (p=0.01), with continued fat mass reductions observed in women randomized to high amount exercise. After baseline adjustment, greater short-term intervention response was associated with more favorable Reunion outcomes across fitness, body composition, and cardiometabolic domains; fat-free mass showed the strongest association ({beta}=0.84, p<0.0001). Conclusions: In older adults with prediabetes, the STRRIDE-Prediabetes interventions produced several legacy health effects persisting more than a decade later. Legacy effects differed by sex and exercise dose, and short-term intervention response relative to baseline was associated with long-term outcomes, supporting targeted exercise strategies to preserve cardiometabolic health and functional independence with aging.

Abstract Aims To examine which demographic groups nicotine pouch advertisers chose to target on social media, and which groups Meta's algorithms actually delivered the adverts to. Design Cross-sectional analysis of advert-level data from the Meta Ad Library. Setting Meta social media platforms (including Facebook and Instagram) in the UK. Cases A random sample of 741 nicotine pouch adverts shown in the 12 months up to December 2025, and a comparison sample of 1,125 general adverts. Analyses of reach were restricted to adverts eligible for all genders and adult ages (444 pouch adverts; 674 general). Measurements Outcomes were advertiser-set gender and age-group targeting criteria (i.e., groups eligible to be shown each advert) and estimated advert reach to each group (i.e., number of people who saw each advert). Male-to-female reach ratios within age groups, and reach ratios comparing age groups, were calculated per advert and summarised using geometric means. To assess whether patterns were pouch-specific, comparisons with general adverts were made using ratios of reach ratios (RRR). Findings Advertisers of nicotine pouches targeted a broad sample; most adverts (79.1%; 586/741) were eligible to be shown to all genders, the remainder were restricted to men only. All were restricted to adults (minimum age 18 years) and most (95.6%; 708/741) had no upper age limit. Despite this, of pouch adverts eligible to be shown to all adults, adverts were more likely to reach men, particularly among younger men. Among 18-24-year-olds, pouch adverts reached around ten times as many men as women (RR 10.0, 95% CI 8.7-11.5), compared with a slight skew towards women for general adverts (RR 0.81, 95% CI 0.71-0.94), corresponding to an RRR of 12.3 (95% CI 10.0-15.1). Pouch adverts also showed a skew in reach towards younger age groups. Relative to those aged 35-44 years, reach was higher among 18-24-year-olds for nicotine pouch adverts (RR 1.33, 95% CI 1.17-1.51) but much lower for general adverts (RR 0.19, 95% CI 0.17-0.21), corresponding to an RRR of 7.0 (95% CI 6.0-8.2). Conclusions Nicotine pouch adverts on social media are often eligible to be shown broadly to all demographic groups but are disproportionately delivered to young men.