Clinical Chemistry and Laboratory Medicine (CCLM)

Introduction: Blood neurofilament light chain (NfL) is an accessible biomarker of neuroaxonal injury across a broad range of neurological disorders, but its clinical implementation requires robust cross-platform analytical and clinical comparability. The objective of this study was to evaluate the analytical and clinical comparability of plasma NfL measurements using Simoa and Lumipulse across different neurological conditions, by assessing cross-platform agreement and the ability of both assays to distinguish neurological diseases from healthy controls. Paired CSF analyses were performed in a subset of participants to biologically anchor plasma findings to the central compartment. Methods: 383 individuals were included, comprising healthy controls and patients with neurodegenerative conditions, multiple sclerosis and stroke. Plasma NfL was measured in all participants using both Simoa and Lumipulse, with paired CSF analyses in a subset of 92 individuals The Lumipulse testing intermediate precision and between-day repeatability was assessed as by the CLSI EP15. Cross-platform agreement for plasma NfL was evaluated using correlation analyses, Passing-Bablok regression and Bland-Altman analysis. Associations between plasma/CSF NfL concentrations were assessed using Spearman's rank correlation analysis for each platform, separately. Age-adjusted cross-diagnostic differences were evaluated using permutation ANCOVA and multiple linear regression models for each platform, separately. Results: Plasma NfL measured by Simoa and Lumipulse showed strong cross-platform concordance in the whole cohort ({rho}=0.90), with similarly strong concordance observed for CSF NfL in the subset with paired samples ({rho}=0.90). Method-comparison analyses in plasma demonstrated consistent agreement between platforms, with identifiable constant and proportional bias, alongside systematically higher absolute plasma NfL values measured by Lumipulse. Within-platform analyses showed significant correlations between plasma and CSF NfL concentrations ({rho}=0.72 for Simoa; {rho}=0.78 for Lumipulse). Noteworthy, Lumipulse NfL CSF and Blood kits exhibited high precision and analytical accuracy. Across both assays, plasma NfL increased with age and was significantly elevated in patients with neurological disorders compared with healthy controls. Discussion: Simoa and Lumipulse capture a consistent biological signal in plasma across patients with neurological disorders, although their absolute NfL values differ, supporting the use of platform-specific reference ranges in clinical practice.

Brain-derived tau (BD-tau) is an emerging blood-based biomarker for neurodegeneration, yet there are currently limited well validated BD-tau assays available for research and clinical use. To enhance access to this vital biomarker for neurological disorders including traumatic brain injury (TBI), we developed a novel blood-based immunoassay for BD-tau on the ultra-sensitive Quanterix HD-X platform using Single Molecule Array technology. Analytical validation assessed dilution linearity, specificity, precision, detection limits, and spike recovery, each recording robust metrics in agreement with international expert recommendations. The assay demonstrated robust validation metrics, achieving between-run stability of 95% when analyzing aliquots from six independent plasma and serum samples across five analytical runs. It also showed strong dilution linearity when diluted four-fold and achieved over 90% recovery when spiked with cerebrospinal fluid. Next, we evaluated the clinical utility of the assay in cohorts of individuals with traumatic brain injury (TBI), where strong performances were recorded whether using the 2-step or 3-step assay formats ({rho}= 0.94; p < 0.0001). Furthermore, plasma BD-tau distinguished samples from TBI patients based on time from injury and severity (AUC=0.93). Plasma BD-tau differentiated between favorable and unfavorable functional outcomes in the acute-severe group. Our findings underscore the significant potential of the BD-tau assay as a biomarker for TBI in the severe phase.

Background: Multiple myeloma (MM) remains incurable despite therapeutic advances, reflecting limited understanding of the molecular mechanisms underlying disease initiation and progression. MM develops through asymptomatic precursor stages, monoclonal gammopathy of undetermined significance (MGUS) and smouldering multiple myeloma (SMM). This study aimed to investigate protein changes associated with disease progression and, through a further integrative approach, to highlight molecular changes of potential predictive and/or therapeutic value. Methods: We performed a comparative proteomic analysis of 94 bone marrow-derived CD138+-selected plasma cell samples (29 MGUS, 20 SMM, and 45 MM) using LC-MS/MS. Differential protein abundance was assessed using pairwise Mann-Whitney U tests between groups, with Benjamini-Hochberg correction. Pathway enrichment, protein-protein interaction, and co-expression network analyses were also conducted. Selected proteins were further evaluated using public transcriptomic datasets and experimentally validated in independent samples by flow cytometry and enzyme-linked immunosorbent assay (ELISA). Results: Following data processing, proteomic analysis identified 6,203 proteins. Pairwise comparisons revealed significant proteomic differences across disease stages, with 370 differentially abundant proteins exhibiting monotonic changes during disease progression. Pathway analysis showed that monotonically upregulated proteins were mainly associated with gene expression and cell proliferation, whereas downregulated proteins were linked to immune-related processes. Further co-expression network analysis, combined with criteria including detection frequency, biological relevance, and translational potential, highlighted a group of prioritised proteins. Representative examples include nucleolin (NCL) and U3 small nucleolar ribonucleoprotein IMP3 (IMP3), involved in nucleolar organisation, ribosome biogenesis and rRNA processing, as well as the immune-associated lactotransferrin (LTF) and serine protease cathepsin G (CTSG). Transcriptomic support and independent experimental validation by flow cytometry and ELISA confirmed the relevance of selected candidates. Conclusions: Taken together, our findings highlight coordinated changes in immune regulation, RNA processing and ribosome biogenesis during MM progression and identify candidate proteins and their networks, including the emerging pharmacologically tractable target NCL and the underexplored IMP3 of potential therapeutic relevance, opening new avenues for further investigation.

Blood-based biomarkers are increasingly used to investigate brain health, but collecting venous blood is difficult in remote and field settings. Capillary microsampling offers a practical alternative, although the ability to delay processing and its agreement with gold-standard venous blood require validation. We evaluated Tasso+, a minimally invasive upper-arm capillary blood collection system, for measuring neurological and host-response biomarkers in plasma and serum during an exercise-based protocol. Sampling occurred before, immediately after, and approximately 24-to-36 hours after exercise; Tasso+ samples were processed with or without a 72-hour room-temperature delay. Tasso+ samples were compared with matched venous blood, and Capitainer SEP10 dried plasma spots were also evaluated, using Quanterix Simoa and Alamar Biosciences NULISAseq CNS panel. Tasso+ enabled reliable measurement of several key biomarkers, including GFAP and NfL, even after delayed processing. These findings support capillary microsampling for neurological biomarker studies where venepuncture is challenging, including field-based research and participant-led remote sampling.

Background: Growing evidence suggests that urinary {beta}-amyloid precursor protein (A{beta}PP) fragments can serve as an early screening biomarker for mild cognitive impairment and dementia. However, in reality, older adults, regardless of the presence of cognitive decline, often suffer from multiple age-related conditions and are on multiple medications. How these comorbidities and treatments affect the performance of early diagnostic biomarkers remains unclear. Methods : This study further validated the sensitivity, specificity, and clinical value of the Qankorey (R) urinary {beta}-amyloid protein detection kit in early dementia screening through a randomized community screening (n=51187) conducted in Changsha, and a multicenter case-control study conducted at Yuquan Hospital (Tsinghua University), Tiantan Hospital (Capital Medical University), Beijing Friendship Hospital, Zibo 148 Hospital (Shandong), and the Third People's Hospital of Yunnan Province. The multicenter case-control study included 898 participants, comprising 266 healthy, age-matched controls without any comorbidities, 167 patients with mild cognitive impairment/Alzheimer's disease (MCI/AD), and 465 non-AD patients with various comorbidities and age-related diseases. Results: The kit showed a significant age-dependent positive rate in both men and women in Changsha, increasing from 6.29% to 15.40%. The number of weakly positive/positive/negative individuals in the healthy group, non-AD group, and MCI/AD group were 8/12/246 (positive rate 7.52%), 41/16/409 (12.23%), and 77/44/46 (72.46%), respectively, with a Kappa value of 0.669, indicating that the method performed well in the clinical diagnosis of MCI/AD, consistent with previously published results. Among the 8 weakly positive healthy subjects, 6 were found to have brain abnormalities by MRI/CT examination. Comorbidity analysis showed that memory decline was the most significant risk factor (P=9.6 x 10^-23, Fisher's exact test), followed by dizziness (P=1.3 x 10^-14;) , hyperlipidemia (P=3.2 x 10^-12) , history of stroke (P=0.0011), and hypertension (P=0.0058). Treatment analysis showed that cardiovascular drugs and antithrombotic drugs significantly reduced the risk of dementia (P values were 0.0061 and 0.0081, respectively), followed by hypoglycemic drugs (P=0.0358). For AD patients, those receiving only memantine showed a slightly lower positive test rate (P=0.0532). Conclusion: Our findings confirm the diagnostic value of urinary {beta}-amyloid protein detection in MCI and AD-related dementia. Furthermore, this kit can be used in practical clinical applications to assess the risk of cognitive decline and treatment efficacy across various diseases.

Background Thalassemia is one of the most common monogenic disorders worldwide, current screening strategies combining hematological testing with molecular assays still carry a risk of missed diagnoses and undesirable efficiency, particularly for complex structural variants and rare mutations. Methods In this prospective double-blind, multicenter cohort study of 3,842 participants (3,362 pregnant women and 480 male partners), we conducted a head-to-head comparison to systematically evaluate the incremental clinical value and detection performance of single-molecule nanopore sequencing in thalassemia (SMITH) against conventional hematological testing and next-generation sequencing (NGS). Findings The overall concordance rate between NGS and SMITH was 98.6% (3789/3842). The discrepant cases (n=53) were directly attributed to the superior detection capabilities of SMITH, which successfully identified complex structural rearrangements-including 45 -globin gene triplications and four HK alleles-that were missed by NGS. Furthermore, SMITH accurately detected four rare variants (c.134_135insT/, c.-22(C>T)/, {beta}N/{beta}c.316-290delinsAGGGCAATAATTT and {beta}3.5 kb deletion/{beta}N ) and resolved ten trans and three cis configurations within the globin gene allele. Clinically, these technical advantages translated to a 9.3% (5/54) increase in the detection rate of high-risk prenatal couples, effectively preventing one birth affected by moderate-to-severe thalassemia. Additionally, SMITH corrected a diagnostic discrepancy in one case (HK vs. -3.7), sparing the couple from an unnecessary invasive procedure. Interpretation Our findings demonstrate that SMITH provides a powerful platform for resolving globin gene rearrangements, detecting rare variants, and enabling direct haplotype phasing. By effectively eliminating diagnostic blind spots, SMITH is expected to become an optimal method for thalassemia prevention programs. Funding This study was supported by Chinese National Natural Science Foundation Projects 81760037 and 82271894.

Purpose: To characterize peripheral immune alterations in treated birdshot uveitis (BU) patients using high-dimensional mass cytometry and multiplex serology. Design: Cohort study. Subjects: 36 BU patients on immunomodulatory treatment (IMT) and 31 healthy controls (HCs). Methods: Detailed ophthalmologic examinations were performed, and peripheral blood and serum samples were collected for immune profiling using mass cytometry and multiplex cytokine analysis. Main Outcome Measures: Imaging-based indicators of ocular inflammation; peripheral immune cell frequencies; serum cytokine levels. Results: Compared to HCs, BU patients showed increased frequencies of Th17, CD146+ T cells, intermediate effector/central memory T cells co-expressing CXCR3 and CCR4, CD56dim NK cells and elevated IL-18 levels. Patients were clinically stratified by an expert ophthalmologist into three disease activity groups: Inactive, Active (comprising combinations of surface retina, deep retina and choroid activity) and Burned-out. Inactive patients harbored more quiescent effector T cells, e.g. Tim-3+ Tc17-Tc22 intermediates and more CD8+ TSCM, potentially representing a resting pool of autoimmune T cells. Active patients exhibited increased in vivo activation of relevant T cells, with stronger HLA-DR, CD38 or PD-1 expression, and highest levels of CD56dim NK cells. Immune profiles were also linked to treatment subgroups: csDMARDs (conventional synthetic disease-modifying antirheumatic drugs) were associated with higher CD56bright NK frequencies, and absence of therapy showed elevated PD-1/SLAMF7 Tc17+1 and PD-1CD57 CD8 TEMRA cells. IL-6R blockade (tocilizumab) resulted in loss of IL-6R T-cells accompanied by increased SLAMF7 T cells, due to epitope masking. Conclusions: Peripheral CyTOF profiling anchored to thorough clinical stratification revealed disease activity-associated immune signatures and therapy-associated imprints in BU.

Background: Modern therapy for childhood and adolescent leukemia requires accurate risk classification of genomic subtype. Although short-read next-generation sequencing (NGS)- based approaches provide comprehensive clinical diagnostics in limited, highly resourced settings, they remain expensive, slow, and inaccessible to most children worldwide. Transformative approaches are needed to improve diagnostic classification for leukemia globally. Methods: We simultaneously continued to develop an analytical pipeline NASVar (Nanopore variant calling for adaptive sampling), and conducted a multicenter, type-two hybrid clinical validation study of an Oxford Nanopore Technologies (ONT) adaptive-sampling whole-genome sequencing (asWGS) assay across hospitals with varying diagnostic resources. In preparation for implementation, a global panel developed a leukemia-based standardized gene set and consensus laboratory-developed test (LDT) validation guidelines. Measures of assay effectiveness compared to both conventional and orthogonal NGS methods, where available, were simultaneously collected with data to measure the implementation outcomes of feasibility, fidelity, appropriateness, and cost. Results: All four centers successfully completed the LDT validation, with minimal adaptations required for regulatory compliance. A total of 457 specimens were sequenced (331 B-ALL, 83 AML, 43 T-ALL). For the 210 B-ALL cases with locally resolved genomic subtypes defined by DNA alterations, asWGS was 100% concordant (210/210). Cases locally defined as B-other were resolved via asWGS with disease-defining DNA alterations in 47% (49/105) of cases. An additional 41% (43/105) of locally defined B-other cases were classified by incorporation of DNA methylation, and all 16 B-ALL patient-derived xenograft controls were correct, for a total of 96% (318/331) of all B-ALL cases in the cohort resolved with single assay asWGS. For AML, 97% (56/58) of cases with locally resolved genomic subtypes were identified by automated asWGS analysis, while an additional two cases were identified after targeted manual review. At Indus Hospital in Pakistan, the B-ALL and AML diagnostic genomic subtype yield increased from 28% with local standard of care diagnostic testing, to 84% with asWGS. The cost of reagents and consumables in the United States, assuming pooled three-plexing, was $343/sample. Based on the combined hybrid validation results, all centers are independently preparing for clinical return of results. Conclusions: ONT asWGS was successfully validated as a clinical assay in four diverse hospital settings. As a single, multi-omic platform that delivers value across the continuum of high-resource to resource-limited contexts, the approach offers a disruptive solution to address the global equity gap in cancer diagnostics.

Background: Neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP), established biomarkers of neuroaxonal injury and astroglial pathology, are frequently only assessed in blood, which limits conclusions regarding their origin. Bi-compartmental analyses of CSF and serum may help differentiate central or peripheral origin of biomarker elevation. Moreover, studies on NfL and GFAP in distinct neuroinfectious disease (NID) phenotypes are limited. Methods: This retrospective monocentric study analyzed CSF and serum from patients with (meningo-)encephalitis/myelitis (TI+; n=48), meningitis (TI-; n=80), (cranial) nerve palsies/polyradiculitis (PND; n=61), and 113 non-neuroinflammatory/non-neurodegenerative controls. A bi-compartmental model using scatter plots and simple linear regression was applied to assess the origin of blood biomarker levels and discriminate between central and peripheral pathology. Results: CSF and serum NfL and GFAP z-scores were significantly higher in TI+ compared with TI- (CSF-GFAP p<0.001/sGFAP p=0.0083; CSF-NfL p=0.003/sNfL p=0.0004). TI+ and PND differed only in GFAP levels, which were higher in TI+ (CSF-GFAP p=0.0049/sGFAP p=0.003). Bi-compartmental analysis revealed simultaneous elevation of CSF and serum NfL in TI+, indicating predominantly central origin, whereas PND demonstrated a shift toward higher sNfL levels suggesting peripheral origin. Higher clinical severity (modified Rankin Scale 3-5) was associated with elevated serum and CSF GFAP and NfL (sGFAP p=0.012/sNfL p=0.002; CSF-GFAP p<0.0001/CSF-NfL p=0.0001), which also predicted unfavorable outcome at discharge (sGFAP p=0.006/sNfL p=0.004; CSF-GFAP p=0.003/CSF-NfL p=0.012). Conclusions: NfL and GFAP were associated with brain/myelon involvement in NID, predominantly reflecting central pathology. Despite strong CSF-serum correlations, bi-compartmental approaches provide additional insight into biomarker origin and disease compartment.

Transfusion-ready red blood cells can be cultured ex vivo from hematopoietic progenitors. Despite its promising outlook, a cultured transfusion unit cannot be produced at competitive costs. Large media volumes are required to maintain a maximum erythroblast cell density of 1-2.106 cells/mL during the erythroblast proliferation stage. To identify the origin of the cell density limitation, we investigated the cellular support and metabolomic phenotype using different media formulations and feeding regimens. Media that were exposed to an increasing density of erythroblasts (termed spent media) displayed a proportional decrease in erythroblast proliferation support. A 1:1 combination of spent media with fresh media (not previously exposed to the cells) restored growth for all tested conditions. Filtering both fresh and spent media with a 3 kDa cut-off filter, and subsequent recombination of the two fractions, indicated that exhaustion of the small molecular weight fraction (<3 kDa) was primarily responsible for growth limitation. We performed targeted and untargeted metabolomics analysis, for both the intra- and extracellular compartments, following seeding in fresh medium (12, 24, 36 h). We observed degradation of nucleosides, depletion of amino acids, and a decrease in intermediates of the glutathione-ascorbate, {gamma}-glutamyl and cysteine-methionine cycles. The latter compounds suggested an increase in oxidative stress in high density erythroblast cultures. Elimination of nucleosides from the medium led to a lower accumulation of purine salvage intermediates, and a 30% increase in cell productivity. In conclusion, we demonstrate that high-density erythroid cultures are subject to metabolic stress, defining critical constraints for scalable culture expansion.

In view of the outstanding progress of machine learning (ML) and growing cost of health systems, it is a current challenge to incorporate artificial intelligence tools into actual medical practice. Here we explored the feasibility and reliability of using machine learning to perform an important immunological investigation that currently requires experienced biologists : Anti-nuclear cytoplasmic antibodies (ANCAs) are important markers for vasculitis and they may be evidenced by microscopic examination of cells labeled with patients' sera. The use of a reliable ML classifier to discriminate between positive and negative samples would increase the rapidity and decrease the cost of immunofluorescence-based ANCA detection. Here, we tested seven well-documented ML algorithms, ranging from simple models such as k nearest neighbors to more complex convolutional neural networks involving millions of adjustable parameter. We studied the feasibility and reliability of classifying 1114 serum samples that had been collected for about 3 years and assayed with conventional procedure. We compared four strategies consisting of assaying either whole microscope fields or individual cell images, and natural images or histograms. The following conclusions were obtained : (i) Several different strategies allowed us to build models stable enough to discriminate between positive and negative samples collected during about 27 months, with a comparison to human classification yielding a kappa index of about 0.7, that may be considered as fairly good and intermediate between the performance of junior and senior biologists. (ii) Simpler ML models combined with theoretical thinking might provide the most rapid and efficient way of developing a reliable test within the framework of a single institution. (iii) In addition, the interpretability of the simplest model provided some theoretical insight into important classification parameters. (iv) An important point and caveat is that the multiplicity and versatility of currently available tools make it an essential requirement to test repeatedly a given model, that must be chosen as simple as possible, to achieve a reliability compatible with medical use. It is concluded that our study provides a strong incentive to incorporate ML tools in well defined medical tests, which might reduce the risk of human errors and pave the way to fully automatic procedures.

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

Introduction: Synthetic oligopeptides provide a rapid and cost-efficient approach to developing antibodies and diagnostics for emerging viral variants. Methods: This study computationally and experimentally characterized a synthetic peptide analog of the SARS-CoV-2 spike subdomain 2 major disulfide loop (SD2MDL), designated S621 (CPVAIHADQLTPTWRVYSTC). Binding affinity was computationally estimated using the Heuristic Affinity Prediction Tool for Immune Complexes (HAPTIC), while experimental validation was performed using enzyme-linked immunosorbent assay (ELISA) with rabbit-derived antipeptide antibodies. Clinical diagnostic accuracy testing was done using plasma samples from RT-PCR-confirmed COVID-19 patients and pre-COVID-19 controls. Results: S621 demonstrated nanomolar binding affinity (Kdapp = 1.14 nM) and high avidity (3.67 nM), closely matching HAPTIC predictions (3.54 nM). Diagnostic evaluation yielded a sensitivity of 89.92% and specificity of 27.79%, corresponding to an overall accuracy of 71.79%. Discussion: These findings demonstrate that a single synthetic peptide derived from a conserved spike subdomain can function as a high-affinity surrogate for full-length antigens, supporting its potential application in rapid peptide-based immunodiagnostics.

Tacrolimus is an immunosuppressant drug commonly used in transplantation. Although multiple studies have demonstrated that polymorphisms in the CYP3A5 gene impact the metabolism of tacrolimus, routine pre-transplant testing for these markers is still not broadly implemented. TacroType - a new laboratory developed test implemented by One Lambda Laboratories - utilizes a qPCR-based six-plex assay for CYP3A5 genotyping and detects the three most common genetic variants (*3, *6 and *7) associated with loss of CYP3A5 protein function and reduced tacrolimus metabolism. TacroType was optimized to address known sources of protocol, technical or sample variability to achieve accurate and reproduceable genotyping results. An analytical performance study was completed following CLSI guidelines. Accuracy was confirmed for each possible CYP3A5 genotype involving 6 target alleles using 32 well-characterized reference samples. TacroType exhibited accurate performance within a broad range of DNA concentrations and quality. Precision studies indicated consistent genotyping results across 4 operators, 2 instrument types and 5 lots of reagents. Accurate and reproducible assay performance was demonstrated using whole blood from 100 and buccal swabs from 70 donors. The analytical performance of TacroType was evaluated in 4014 total qPCR reactions, with a report rate of 99.8% and genotyping accuracy of 100% (95% confidence interval of 99.9%).

Successive dengue virus (DENV) outbreaks can progressively reshape population immunity influencing disease expression and diagnostic performance. Objectives The aim was to evaluate the impact of secondary infections across sequential outbreaks on clinical severity, serotype dynamics and diagnostic concordance. Methods This retrospective study analyzed 976 febrile-stage samples from three sequential outbreaks in Misiones, Argentina. For serotyping and clinical analyses, 869 viremic samples confirmed by at least one direct method were included (2016: n=512; 2019: n=148; 2024: n=209). Additionally, 318 samples, including 107 non-viremic cases, were used to compare NS1 rapid diagnostic tests (NS1 Ag) and RT-PCR. Viral serotyping and clinical and laboratory markers of disease severity were evaluated. Results Secondary infections increased from 31.05% (2016) to 43.24% (2019) and 53.87% (2024) (p<0.0010). Serotype distribution shifted from DENV-1 predominance in 2016 (95.12%), DENV-1/DENV-4 co-circulation in 2019 (60.71%/39.29%), and DENV-2 predominance in 2024 (97.60%). Secondary infections were associated with more severe disease manifestations, particularly in 2024, with higher hematocrit (p=0.0120) and hemoglobin (p=0.0080), lower white blood cells (p=0.020) and platelet counts (p=0.0030), and elevated AST (p=0.0007) and ALT (p=0.0130). Concordance between NS1 Ag and RT-PCR was lower in secondary infections (k=0.457 vs k=0.759, p=0.0013). Conclusions The rising frequency of secondary infections may affect both clinical severity and diagnostic performance during outbreaks. The clinical impact was more evident in 2024, likely associated with the introduction of a new serotype. These findings highlight the need for optimized surveillance and diagnostic strategies to improve case detection and patient management during epidemics.

Abstract Purpose:Comparison of ocular parameters (ACD, AL, LT, VL, CCT, ASD, LC, LT/ACD) in preterm infants with retinopathy after treatment, those with spontaneous regression, and those without retinopathy, at postmenstrual (ages of 0 (40 weeks), 3 , and 6 months. Methods: Cross-sectional study. This research involved 297 premature infants assigned to three groups based on fundus results and intravitreal injection therapy: an ROP post-injection group, an ROP spontaneous regression group, and a non-ROP group. Axial length (AL), anterior chamber depth (ACD), l e n s t h i c kn e s s (LT), and vitreous length (VL) were assessed in all three groups using a corneal thickness meter at po st menstrual age s (PMA) of 0, 3, and 6 months. Derived parameters--ASD ((ACD + LT), LC ((ACD + LT/2), and LT/ACD--were subsequently calculated. A one-way ANOVA analysis revealed statistically significant differences in these ocular parameters among the groups (P < 0.05). Results: Significant differences e m e r g e d in anterior chamber depth (ACD) and l e n st h i c k n e s s ( LT) between the ROP post-injection group, ROP spontaneous regression group, and non-ROP group at 0, 3, and 6 (months postmenstrual age (PMA). At 0 months PMA: ACD(F=4.33, P=0.014), LT (F=5.45, P=0.005). At 3 months PMA: ACD (F=17.20, P<0.01), LT(F=15.23, P<0.01). At 6 months PMA: ACD (F=17.89, P<0.01), LT (F=17.21, P<0.01). Central corneal thickness (CCT) also differed significantly among the three groups at 0 months PMA(P <0 .0 1 ). All ocular parameters correlated significantly with Postmenstrual Age, with CCT and LT showing a negative correlation. Before 6 months PMA, axial length (AL) and vitreous length (VL) increased significantly, and ACD deepened significantly across all groups (P <0 .05 ). However , LT exhibited no significant change within the ROP group (post-injection group P=0.4; spontaneous regression group P=0 .33). No significant differences existed in any ocular parameters between the ROP post-injection group and the ROP spontaneous regression group (P>0.05). Conclusions: Before 6 months of postmenstrual age (PMA), axial length (AL), vitreous length (VL), and anterior chamber depth (ACD) were increased between the ROP group and non-ROP group; lens thickness (LT) remained unchanged in the ROP group but increased in the non-ROP group. The injection group and the spontaneous regression group showed no significant differences. The primary factors influencing anterior segment development were birth weight (BW), gestational age (GA), and postmenstrual age (PMA).

Background Dry eye disease (DED) is a multifactorial condition marked by tear film instability and ocular inflammation, causing symptoms like grittiness and blurred vision. The global prevalence of Dry eye disease among pregnant women ranges from 27.4% to 89.3% and in Africa it ranges between 20% and 50%. Hormonal changes, advanced maternal age, late pregnancy and prolonged screen time play an important part in its development. Methodology A hospital-based cross-sectional study among 380 pregnant women. Systematic sampling technique was used for recruitment at the antenatal clinic in Mnazi Mmoja Hospital in Dar es Salaam. Clinical dry eye tests were performed along with the administration of a symptom questionnaire that included demographic characteristics and the OSDI tool where OSDI >13 is the threshold for diagnosis of DED. Data were analyzed using Stata version 17.0 and Modified Poisson analyses identified factors associated with dry eye disease, with statistical significance set at p-value<0.05. Results A total of 380 pregnant women were recruited and analyzed with the mean age 31.7{+/-}6.7, 196 (51.6%) were aged 31-46 years. Most were married 273 (71.8%) and 211 (55.5%) had completed secondary education. Dry eye disease (DED) prevalence was 53.2% (48.8%-58.2%). Among those with DED (n=202), 112 (55%) had mild symptoms, 26 (13%) moderate, and 64 (32%) severe. The most common DED subtype was unclassified 72 (35.6%), followed by mixed (67, 33.2%), evaporative 50 (24.8%), and aqueous deficient 13 (6.4%). Significant associations with DED were: advanced gestation age (aPR=2.18 (1.550-3.072), p<0.001), being a housewife (aPR=1.48(1.179-1.857), p=0.001), use of visual display terminals (aPR=1.36(1.219-1.845), p=0.048), working in low humidity (aPR=2.62(1.698-4.045), p=0.001), and working in air-conditioned rooms (aPR=2.40(1.685-3.415), p=0.001). Secondary education was protective against DED (aPR = 0.668 (0.466-0.958), p = 0.028). Conclusion Approximately half of pregnant women have DED, with unclassified DED being the predominant subtype. Late gestation age, occupation, extended screen time, and working environment are significantly associated factors. It is essential to integrate DED screening into antenatal care and establish standardized protocols on DED management. Also, it is essential to promote lifestyle modifications such as reduction of screen time and avoiding dry environments.

Introduction: To investigate the epidemiological characteristics of chronic kidney diseases (CKD) in China in 2021 and its trends between 1990 and 2021, in the context of significant population growth and lifestyle changes over the past 30 years that have likely influenced the CKD spectrum. Methods: Data on CKD prevalence, mortality, disability-adjusted life-years (DALY), and risk factors were obtained from the Global Burden of Disease Study 2021. The estimated decadal percentage changes were calculated to evaluate changes in trends in prevalence, mortality and disease burden. Results: In 2021, an estimated 118.4 (95% UI 109.4 to 127.5) million people in China were affected by CKD, contributing to 204 230 (95% UI 164 736 to 246 372) deaths and 6.13 (95% UI 5.18 to 7.21) million DALY. Although CKD due to diabetes mellitus and hypertension accounted for less than a quarter of all cases, they were responsible for over 90% of CKD-related deaths. Over the past three decades, CKD mortality and DALY rates have steadily increased, although the prevalence has stabilized in the last decade. Diabetes mellitus type 2 and hypertension have emerged as key drivers of CKD burden in China. Conclusions: The CKD burden in China shows a dual pattern of rising incidence and high mortality from diabetes and hypertension-related chronic kidney disease, alongside persistently high years lived with disability from glomerulonephritis and other causes.

Manufacturer-defined signal-strength indices are frequently employed as quality benchmarks for automated optical coherence tomography analysis, yet their empirical relationship with deep learning segmentation accuracy remains unclear. Because these metrics were originally developed for conventional image-processing pipelines, their ability to predict modern model-based segmentation accuracy has not been empirically validated. To address this gap, we evaluated the Heidelberg Spectralis Q-score against U-Net segmentation performance across 5,047 B-scans from 103 eyes for three anatomical boundaries of the posterior segment of the eye: the Ellipsoid Zone (EZ), Bruch's Membrane (BM), and Choroid Outer Boundary (COB). Alongside standard boundary agreement metrics (MAE, MSE, Dice Similarity Coefficient), we adapted the Earth Mover's Distance (EMD) from optimal transport theory as a boundary evaluation metric. Unlike column-wise averages, EMD quantifies boundary agreement as a 2-D geometric displacement, directly measuring residual spatial displacement between the model segmented boundary and the ground-truth boundary. Our results demonstrate that the Q-score - originally designed to gate image-processing-based automated analysis - is a poor predictor of deep learning boundary segmentation accuracy, with explained variance (R2) failing to exceed 1.4% across all three boundaries. We further observed a monotonically increasing error hierarchy with anatomical depth (EZ < BM < COB), consistent across metrics, which is unexplained by the signal strength. At the COB, correlations were paradoxically positive, explained by a B-scan-level mediation chain: higher Q-scores correspond to greater choroidal thickness (r=0.113, {rho}=0.158), which in turn predicts higher COB segmentation error (r=0.165, {rho}=0.191) - a localization difficulty that global signal strength cannot capture. Collectively, these findings challenge the implicit assumption that signal-strength-based quality thresholds are a reliable proxy for deep learning model performance, and motivate a shift toward task-specific acquisition quality criteria calibrated to model performance rather than signal interpretability.

Background: Inherited Retinal Diseases (IRDs) are a group of genetically heterogeneous blinding conditions. Major global genomic reference databases are disproportionately enriched for individuals of European ancestry. This underrepresentation creates a significant bias that impedes the accuracy of genetic diagnosis in the Chinese population. This study aims to address this limitation by constructing a comprehensive genetic landscape of IRDs using large-scale deep-sequencing data from a large Chinese cohort. Methods: The study leveraged variant data primarily from 10,588 individuals in the China Metabolic Analytics Project (ChinaMAP) and cross-referenced findings against multiple national and international databases. We systematically curated variants within a targeted panel of 291 IRD-associated genes. Variant pathogenicity was assessed using a comprehensive pipeline integrating InterVar-automated classification based on 2015 American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines, ClinVar evidence (review status [&ge;] 1 star), and manual literature curation. We delineated the mutational spectrum, identified population-enriched pathogenic/likely pathogenic (P/LP) variants, and analyzed the distribution characteristics of IRD-associated highly-mutated genes. Furthermore, we calculated the carrier frequencies (CF) and genetic prevalence (GP) of autosomal recessive(AR)-IRD genes in the Chinese population. Results: The study revealed a highly concentrated genetic landscape for AR-IRDs in the Chinese population, with ABCA4 and USH2A emerging as the primary drivers of the genetic burden. This finding aligns with previous Chinese cohorts but contrasts with global databases, where genes such as the X-linked RPGR are more prevalent. In contrast, autosomal dominant (AD)-IRDs exhibited high locus heterogeneity, with pathogenic variants dispersed across numerous genes (e.g., COL2A1 and MFN2). We identified a series of P/LP variants that were either high-frequency or significantly enriched in the Chinese population, such as CNGB1 (p.P530R) and specific recurrent alleles in ABCA4 and CYP4V2. The estimated cumulative CF for AR-IRDs was 1 in 5.60, and the theoretical total GP was 1 in 2,624.67, based on the ChinaMAP data. Conclusion: By integrating the ChinaMAP dataset with diverse genomic resources, this study provides a genetic landscape of IRDs in the Chinese population. Our analysis shows a concentrated mutational spectrum in AR-IRDs, contrasting with the pronounced heterogeneity in AD-IRDs. These findings, including population-specific pathogenic variants and refined prevalence estimates, provide a resource for precision diagnostics, genetic counseling, expanded carrier screening (ECS), and public health policy development in China.