Endoplasmic Reticulum Stress Delays Choroid Development in the HCAR1 Knockout Mouse.

The subretina, composed of the choroid and the retinal pigment epithelium (RPE), bears a critical role in proper vision. In addition to phagocytosis of photoreceptor debris, the RPE shuttles oxygen and nutrients to the neuroretina. For their own energy production, RPE cells mainly rely on lactate, a major by-product of glycolysis. Lactate, in turn, is believed to convey most of its biological effects via the hydroxycarboxylic acid receptor 1 (HCAR1). Here, the lactate-specific receptor, HCAR1, is found to be exclusively expressed in the RPE cells within the subretina, and Hcar1-/- mice exhibit a substantially thinner choroidal vasculature during development. Notably, the angiogenic properties of lactate on the choroid are impacted by the absence of Hcar1. HCAR1-deficient mice exhibit elevated endoplasmic reticulum stress along with eukaryotic initiation factor 2α phosphorylation, a significant decrease in the global protein translation rate, and a lower proliferation rate of choroidal vasculature. Strikingly, inhibition of the integrated stress response using an inhibitor that reverses the effect of eukaryotic initiation factor 2α phosphorylation restores protein translation and rescues choroidal thinning. These results provide evidence that lactate signalling via HCAR1 is important for choroidal development/angiogenesis and highlight the importance of this receptor in establishing mature vision.

Novel variants in GALE cause syndromic macrothrombocytopenia by disrupting glycosylation and thrombopoiesis.

Glycosylation is recognized as a key process for proper megakaryopoiesis and platelet formation. The enzyme uridine diphosphate (UDP)-galactose-4-epimerase, encoded by GALE, is involved in galactose metabolism and protein glycosylation. Here, we studied 3 patients from 2 unrelated families who showed lifelong severe thrombocytopenia, bleeding diathesis, mental retardation, mitral valve prolapse, and jaundice. Whole-exome sequencing revealed 4 variants that affect GALE, 3 of those previously unreported (Pedigree A, p.Lys78ValfsX32 and p.Thr150Met; Pedigree B, p.Val128Met; and p.Leu223Pro). Platelet phenotype analysis showed giant and/or grey platelets, impaired platelet aggregation, and severely reduced alpha and dense granule secretion. Enzymatic activity of the UDP-galactose-4-epimerase enzyme was severely decreased in all patients. Immunoblotting of platelet lysates revealed reduced GALE protein levels, a significant decrease in N-acetyl-lactosamine (LacNAc), showing a hypoglycosylation pattern, reduced surface expression of gylcoprotein Ibα-IX-V (GPIbα-IX-V) complex and mature ß1 integrin, and increased apoptosis. In vitro studies performed with patients-derived megakaryocytes showed normal ploidy and maturation but decreased proplatelet formation because of the impaired glycosylation of the GPIbα and ß1 integrin, and reduced externalization to megakaryocyte and platelet membranes. Altered distribution of filamin A and actin and delocalization of the von Willebrand factor were also shown. Overall, this study expands our knowledge of GALE-related thrombocytopenia and emphasizes the critical role of GALE in the physiological glycosylation of key proteins involved in platelet production and function.

The importance of genetic variant cleaners: From patient to wet lab and back to clinical practice.

Laureano J. Kamiya and colleagues have identified two novel missense variants (Gly168Arg and Gly168Glu) in the RUNX1 gene. These variants, identified in two unrelated families with familial platelet disorder with a predisposition to acute myeloid leukaemia (FPD/AML) phenotype, were initially classified as variants of uncertain significance (VUS). However, functional studies of RUNX1 target genes enabled their reclassification as likely pathogenic. The findings highlight Gly168 as a new mutation hotspot in RUNX1, providing important insights for genetic counselling and leukaemia monitoring. The study emphasizes the necessity for continuous updates to diagnostic guidelines and data sharing among laboratories to improve the classification and interpretation of genetic variants. Commentary on: Kamiya et al. Two novel families with RUNX1 variants indicate glycine 168 as a new mutational hotspot: Implications for FPD/AML diagnosis. Br J Haematol 2024 (Online ahead of print). doi: 10.1111/bjh.19776.

Mast cells promote choroidal neovascularization in a model of age-related macular degeneration.

'Wet' age-related macular degeneration (AMD) is characterized by pathologic choroidal neovascularization (CNV) that destroys central vision. Abundant evidence points to inflammation and immune cell dysfunction in the progression of CNV in AMD. Mast cells are resident immune cells that control the inflammatory response. Mast cells accumulate and degranulate in the choroid of patients with AMD, suggesting they play a role in CNV. Activated mast cells secrete various biologically active mediators, including inflammatory cytokines and proteolytic enzymes such as tryptase. We investigated the role of mast cells in AMD using a model of CNV. Conditioned media from activated mast cells exerts proangiogenic effects on choroidal endothelial cells and choroidal explants. Laser-induced CNV in vivo was markedly attenuated in mice genetically depleted of mast cells (KitW-sh/W-sh) and in wild-type mice treated with mast cell stabilizer, ketotifen fumarate. Tryptase was found to elicit pronounced choroidal endothelial cell sprouting, migration and tubulogenesis; while tryptase inhibition diminished CNV. Transcriptomic analysis of laser-treated RPE/choroid complex revealed collagen catabolism and extracellular matrix (ECM) reorganization as significant events correlated in clusters of mast cell activation. Consistent with these analyses, compared to wildtype mice choroids of laser-treated mast cell-deficient mice displayed less ECM remodelling evaluated using collagen hybridizing peptide tissue binding. Findings herein provide strong support for mast cells as key players in the progression of pathologic choroidal angiogenesis and as potential therapeutic targets to prevent pathological neovascularization in 'wet' AMD.

An adaptive optics module for deep tissue multiphoton imaging in vivo.

Understanding complex biological systems requires visualizing structures and processes deep within living organisms. We developed a compact adaptive optics module and incorporated it into two- and three-photon fluorescence microscopes, to measure and correct tissue-induced aberrations. We resolved synaptic structures in deep cortical and subcortical areas of the mouse brain, and demonstrated high-resolution imaging of neuronal structures and somatosensory-evoked calcium responses in the mouse spinal cord at great depths in vivo.

Health Care Access and COVID-19 Vaccination in the United States: A Cross-Sectional Analysis.

BACKGROUND: Although federal legislation made COVID-19 vaccines free, inequities in access to medical care may affect vaccine uptake. OBJECTIVE: To assess whether health care access was associated with uptake and timeliness of COVID-19 vaccination in the United States. DESIGN: A cross-sectional study. SETTING: 2021 National Health Interview Survey (Q2-Q4). SUBJECTS: In all, 21,532 adults aged≥18 were included in the study. MEASURES: Exposures included 4 metrics of health care access: health insurance, having an established place for medical care, having a physician visit within the past year, and medical care affordability. Outcomes included receipt of 1 or more COVID-19 vaccines and receipt of a first vaccine within 6 months of vaccine availability. We examined the association between each health care access metric and outcome using logistic regression, unadjusted and adjusted for demographic, geographic, and socioeconomic covariates. RESULTS: In unadjusted analyses, each metric of health care access was associated with the uptake of COVID-19 vaccination and (among those vaccinated) early vaccination. In adjusted analyses, having health coverage (adjusted odds ratio [AOR] 1.60; 95% CI: 1.39, 1.84), a usual place of care (AOR 1.58; 95% CI: 1.42, 1.75), and a doctor visit within the past year (AOR 1.45, 95% CI: 1.31, 1.62) remained associated with higher rates of COVID-19 vaccination. Only having a usual place of care was associated with early vaccine uptake in adjusted analyses. LIMITATIONS: Receipt of COVID-19 vaccination was self-reported. CONCLUSIONS: Several metrics of health care access are associated with the uptake of COVID-19 vaccines. Policies that achieve universal coverage, and facilitate long-term relationships with trusted providers, may be an important component of pandemic responses.

Epstein Barr virus-directed T-cell therapy for refractory EBV-PTLD in a toddler post Orthotopic heart transplantation.

Epstein-Barr Virus (EBV) is a ubiquitous herpes type virus that is associated with post-transplant lymphoproliferative disorder (PTLD). Usual management includes reduction or cessation of immunosuppression and in some cases chemotherapy including rituximab. However, limited therapies are available if PTLD is refractory to rituximab. Several clinical trials have investigated the use of EBV-directed T cells in rituximab-refractory patients; however, data regarding response is scarce and inconclusive. Herein, we describe a patient with EBV-PTLD refractory to rituximab after orthotopic heart transplantation (OHT) requiring EBV-directed T-cell therapy. This article aims to highlight the unique and aggressive clinical presentation and progression of PTLD with utilization of EBV-directed T-cell therapy for management and associated pitfalls.

Impact of Frailty on Heart Failure Prognosis: Is Sex Relevant?

PURPOSE OF REVIEW: Heart failure (HF) is one of the most frequent causes of hospital admission in elderly patients, especially in women, who present a high prevalence of geriatric syndromes like frailty. Studies have suggested that frailty and its impact may also differ between males and females. Understanding how frailty may differently affect HF patients depending on sex is therefore imperative for providing personalized care. The aim of this review is to summarize the role of sex in the prognostic impact of frailty in HF patients. RECENT FINDINGS: Numerous studies have identified frailty as a significant predictor of all-cause mortality and hospital readmissions. A recent study of elderly HF out-patients demonstrated that while women had a higher prevalence of frailty, it was an independent predictor of mortality and readmission only in men. Moreover, another study revealed that physical frailty was associated with time to first clinical event among men but not among women. These results raise the question about why frailty affects differently HF prognosis in men and women. Women with HF present a higher prevalence of frailty, especially when it is considered as physical decline. Nevertheless, frailty affects differently HF prognosis in men and women. Women with HF present lower mortality than men and frailty is related with prognosis only in men. The different severity of HF between men and women and other hormonal, psychosocial, and clinical factors might be involved in this fact.

The Radical Welcome Engagement Restoration Model and Assessment Tool for Community-Engaged Partnerships.

People experiencing addiction, houselessness, or who have a history of incarceration have worse health outcomes compared with the general population. This is due, in part, to practices and policies of historically White institutions that exclude the voices, perspectives, and contributions of communities of color in leadership, socio-economic development, and decision-making that matters for their wellbeing. Community-based participatory research (CBPR) approaches hold promise for addressing health inequities. However, full engagement of people harmed by systemic injustices in CBPR partnerships is challenging due to inequities in power and access to resources. We describe how an Allentown-based CBPR partnership-the Health Equity Activation Research Team of clinicians, researchers, and persons with histories of incarceration, addiction, and houselessness-uses the Radical Welcome Engagement Restoration Model (RWERM) to facilitate full engagement by all partners. Data were collected through participatory ethnography, focus groups, and individual interviews. Analyses were performed using deductive coding in a series of iterative meaning-making processes that involved all partners. Findings highlighted six defining phases of the radical welcome framework: (a) passionate invitation, (b) radical welcome, (c) authentic sense of belonging, (d) co-creation of roles, (e) prioritization of issues, and (f) individual and collective action. A guide to assessing progression across these phases, as well as a 32-item radical welcome instrument to help CBPR partners anticipate and overcome challenges to engagement are introduced and discussed.

Enhancing Dynamic Spectral Diffusion in Metal-Organic Frameworks through Defect Engineering.

The crystal packing of organic chromophores has a profound impact on their photophysical properties. Molecular crystal engineering is generally incapable of producing precisely spaced arrays of molecules for use in photovoltaics, light-emitting diodes, and sensors. A promising alternative strategy is the incorporation of chromophores into crystalline metal-organic frameworks (MOFs), leading to matrix coordination-induced emission (MCIE) upon confinement. However, it remains unclear how the precise arrangement of chromophores and defects dictates photophysical properties in these systems, limiting the rational design of well-defined photoluminescent materials. Herein, we report new, robust Zr-based MOFs constructed from the linker tetrakis(4-carboxyphenyl)ethylene (TCPE4-) that exhibit an unexpected structural transition in combination with a prominent shift from green to blue photoluminescence (PL) as a function of the amount of acid modulator (benzoic, formic, or acetic acid) used during synthesis. Time-resolved PL (TRPL) measurements provide full spectral information and reveal that the observed hypsochromic shift arises due to a higher concentration of linker substitution defects at higher modulator concentrations, leading to broader excitation transfer-induced spectral diffusion. Spectral diffusion of this type has not been reported in a MOF to date, and its observation provides structural information that is otherwise unobtainable using traditional crystallographic techniques. Our findings suggest that defects have a profound impact on the photophysical properties of MOFs and that their presence can be readily tuned to modify energy transfer processes within these materials.

Quantifying and Modulating Protein Encapsulation in Guanosine-Based Supramolecular Particles.

The encapsulation of proteins is an effective way to preserve their structure and enhance their function. One exciting possibility is adjusting the protective agent to match the specific protein's characteristics to influence its properties. In a recent study, we developed a flow cytometry-based method to quantify the encapsulation of small-molecule dyes in colloidal particles made from guanosine derivatives (supramolecular hacky sacks (SHS) particles). We aimed to determine whether this method could quantify protein encapsulation and track changes and if the particles could be tuned to bind to specific proteins. Our results showed that fluorescein isothiocyanate (FITC)-labeled proteins had apparent association constants in the micromolar range with hydrophobicity as the dominant factor enhancing the affinities. Confocal laser scanning microscopy (CLSM) imaging supported these results and provided additional information about the protein distribution within the particles. We also tested the feasibility of tuning the avidin affinity (AVI) for SHS particles with a biotin ligand. We found that increasing the amount of biotin initially enhanced AVI binding, but then reached saturation, which we hypothesize results from noncovalent cross-linking caused by strong biotin/AVI interactions. CLSM images showed that the linker also impacted the AVI distribution within the particles. Our strategy provides an advantage over other methods for quantifying protein encapsulation by being suitable for high-throughput analysis with high reproducibility. We anticipate that future efforts to use lower-affinity ligands would result in better strategies for modulating protein affinity for drug delivery applications.

Faecal Microbiota Divergence in Allopatric Populations of Podarcis lilfordi and P. pityusensis, Two Lizard Species Endemic to the Balearic Islands.

Gut microbial communities provide essential functions to their hosts and are known to influence both their ecology and evolution. However, our knowledge of these complex associations is still very limited in reptiles. Here we report the 16S rRNA gene faecal microbiota profiles of two lizard species endemic to the Balearic archipelago (Podarcis lilfordi and P. pityusensis), encompassing their allopatric range of distribution through a noninvasive sampling, as an alternative to previous studies that implied killing specimens of these IUCN endangered and near-threatened species, respectively. Both lizard species showed a faecal microbiome composition consistent with their omnivorous trophic ecology, with a high representation of cellulolytic bacteria taxa. We also identified species-specific core microbiota signatures and retrieved lizard species, islet ascription, and seasonality as the main factors in explaining bacterial community composition. The different Balearic Podarcis populations are characterised by harbouring a high proportion of unique bacterial taxa, thus reinforcing their view as unique and divergent evolutionary entities.

GALE variants associated with syndromic manifestations, macrothrombocytopenia, bleeding, and platelet dysfunction.

GALE gene encodes the uridine diphosphate [UDP]-galactose-4-epimerase, which catalyzes the bidirectional interconversion of UDP-glucose to UDP-galactose, and UDP-N-acetyl-glucosamine to UDP-N-acetyl-galactosamine. In that way, GALE balances, through reversible epimerization, the pool of four sugars that are essential during the biosynthesis of glycoproteins and glycolipids. GALE-related disorder presents an autosomal recessive inheritance pattern, and it is commonly associated with galactosemia. Peripheral galactosemia generally associates with non-generalized forms or even asymptomatic presentations, while classical galactosemia may be related to complications such as learning difficulties, developmental delay, cardiac failure, or dysmorphic features. Recently, GALE variants have been related to severe thrombocytopenia, pancytopenia, and in one patient, to myelodysplastic syndrome.

What is the context? GALE gene encodes for the UDP-Galactose 4-Epimerase, an enzyme involved in the Leloir pathway of galactose catabolism and protein glycosylation.Homozygous or compound heterozygous GALE variants associate with the disorder known as galactosemia type III.Three types of galactosemia can be distinguished: the peripheral, the intermediate, and the generalized form, which associate with different clinical symptoms and GALE genetic variants.Peripheral form is considered benign, while the intermediate and the generalized form is associated with severe and syndromic manifestations, including learning difficulties, delayed growth, sensorineural hearing loss, and early-onset cataracts, among others.What is new? In the last few years, GALE variants have been linked to hematological manifestations, such as anemia, febrile neutropenia, and severe thrombocytopenia.To date, the only GALE variants described in patients presenting hematological disorders are GALE p.Arg51Trp, p.Lys78ValfsX32, p.Val128Met, p.Thr150Met, p.Leu223Pro, and p.Gly237Asp.The thrombocytopenia observed in GALE patients is associated with reduced GPIbα and ß1 integrin glycosylation and externalization to the megakaryocyte and platelet surface, disrupting the actin cytoskeleton remodeling.What is the impact? GALE is an essential protein for the correct megakaryocyte and platelet glycosylation.

Palbociclib (PD 0332991) Interaction with Kinases. Theoretical and Comparative Molecular Docking Study.

The optimized geometry of palbociclib, (PD 0332991) (8-cyclopentyl-6-ethanoyl-5-methyl-2-(5-(piperazin-1-yl)pyridin-2-ylamino)pyrido[2,3-d]pyrimidin-7(8H)-one), electrostatic potential map, molecular orbitals were calculated using the density functional theory. The geometry was used in a molecular docking study of palbociclib-kinase complexes, results could be explained by the charge of the nitrogen and oxygen atoms within the palbociclib. Energy gap of HOMO-LUMO surfaces, could help to explain the reactivity of the ligand and the hydrogen bonding with three different kinases, two of CDK6 and one of CDK4 type. Docking results are similar and complementary with literature reports using molecular dynamics, were hydrogen bonding was obtained and analyzed. The promiscuity of three kinases with palbociclib was detected by the docking results, thus, palbociclib could be used in other types of cancer besides myeloid leukemia. Some similarities are found with CDK4/CDK6 kinases which allow us to determine that palbociclib could be used to control other resistant inhibitor types of cancer.

Inherited Thrombocytopenia Caused by Variants in Crucial Genes for Glycosylation.

Protein glycosylation, including sialylation, involves complex and frequent post-translational modifications, which play a critical role in different biological processes. The conjugation of carbohydrate residues to specific molecules and receptors is critical for normal hematopoiesis, as it favors the proliferation and clearance of hematopoietic precursors. Through this mechanism, the circulating platelet count is controlled by the appropriate platelet production by megakaryocytes, and the kinetics of platelet clearance. Platelets have a half-life in blood ranging from 8 to 11 days, after which they lose the final sialic acid and are recognized by receptors in the liver and eliminated from the bloodstream. This favors the transduction of thrombopoietin, which induces megakaryopoiesis to produce new platelets. More than two hundred enzymes are responsible for proper glycosylation and sialylation. In recent years, novel disorders of glycosylation caused by molecular variants in multiple genes have been described. The phenotype of the patients with genetic alterations in GNE, SLC35A1, GALE and B4GALT is consistent with syndromic manifestations, severe inherited thrombocytopenia, and hemorrhagic complications.

Advances in two-dimensional engineered nanomaterials applications for the agro- and food-industries.

Two-dimensional nanomaterials, such as graphene, transition metal dichalcogenides, MXenes, and other layered compounds, are the subject of intense theoretical and experimental research for applications in a wide range of advanced technological solutions, given their outstanding physical, chemical, and mechanical properties. In the context of food science and technology, their contributions are starting to appear, based on the advantages that two-dimensional nanostructures offer to agricultural- and food-related key topics, such as sustainable water use, nano-agrochemicals, novel nanosensing devices, and smart packaging technologies. These application categories facilitate the grasping of the current and potential uses of such advanced nanomaterials in the field, backed by their advantageous physical, chemical, and structural properties. Developments for water cleaning and reuse, efficient nanofertilizers and pesticides, ultrasensitive sensors for food contamination, and intelligent nanoelectronic disposable food packages are among the most promising application examples reviewed here and demonstrate the tremendous impact that further developments would have in the area as the fundamental and applied research of two-dimensional nanostructures continues. We expect this work will contribute to a better understanding of the promising characteristics of two-dimensional nanomaterials that could be used for the design of novel and feasible solutions in the agriculture and food areas. © 2023 Society of Chemical Industry.

Weyl-mediated helical magnetism in NdAlSi.

Emergent relativistic quasiparticles in Weyl semimetals are the source of exotic electronic properties such as surface Fermi arcs, the anomalous Hall effect and negative magnetoresistance, all observed in real materials. Whereas these phenomena highlight the effect of Weyl fermions on the electronic transport properties, less is known about what collective phenomena they may support. Here, we report a Weyl semimetal, NdAlSi, that offers an example. Using neutron diffraction, we found a long-wavelength helical magnetic order in NdAlSi, the periodicity of which is linked to the nesting vector between two topologically non-trivial Fermi pockets, which we characterize using density functional theory and quantum oscillation measurements. We further show the chiral transverse component of the spin structure is promoted by bond-oriented Dzyaloshinskii-Moriya interactions associated with Weyl exchange processes. Our work provides a rare example of Weyl fermions driving collective magnetism.

Chiasmatic lesions on conventional magnetic resonance imaging during the first event of optic neuritis in patients with neuromyelitis optica spectrum disorder and myelin oligodendrocyte glycoprotein-associated disease in a Latin American cohort.

BACKGROUND AND PURPOSE: Optic neuritis (ON) is often the initial symptom of neuromyelitis optica spectrum disorder (NMOSD) and myelin oligodendrocyte glycoprotein-associated disease (MOGAD). We aimed to compare the frequency and pattern of chiasmatic lesions in MOGAD-related ON (MOGAD-ON) and NMOSD-related ON (NMOSD-ON) using conventional brain imaging (magnetic resonance imaging [MRI]) in Latin America (LATAM). METHODS: We reviewed the medical records and brain MRI (≤30 days from ON onset) of patients with a first event of MOGAD-ON and NMOSD-ON. Patients from Argentina (n = 72), Chile (n = 21), Ecuador (n = 31), Brazil (n = 30), Venezuela (n = 10) and Mexico (n = 82) were included. Antibody status was tested using a cell-based assay. Demographic, clinical, imaging and prognostic (as measured by the Visual Functional System Score [VFSS] of the Expanded Disability Status Scale) data were compared. RESULTS: A total of 246 patients (208 NMOSD and 38 MOGAD) were included. No differences were found in gender and ethnicity between the groups. We observed chiasmatic lesions in 66/208 (31.7%) NMOSD-ON and in 5/38 (13.1%) MOGAD-ON patients (p = 0.01). Of these patients with chiasmatic lesions, 54/66 (81.8%) and 4/5 had associated longitudinally extensive optic nerve lesions, 45/66 (68%) and 4/5 had bilateral lesions, and 31/66 (47%) and 4/5 showed gadolinium-enhancing chiasmatic lesions, respectively. A positive correlation was observed between VFSS and presence of bilateral (r = 0,28, p < 0.0001), chiasmatic (r = 0.27, p = 0.0001) and longitudinally extensive lesions (r = 0,25, p = 0.0009) in the NMOSD-ON group, but no correlations were observed in the MOGAD-ON group. CONCLUSIONS: Chiasmatic lesions were significantly more common in NMOSD than in MOGAD during an ON attack in this LATAM cohort. Further studies are needed to assess the generalizability of these results.

Conversion of oxadiazolo[3,4-b]pyrazines to imidazo[4,5-b]pyrazines via a tandem reduction-cyclization sequence generates new mitochondrial uncouplers.

We report new mitochondrial uncouplers derived from the conversion of [1,2,5]oxadiazolo[3,4-b]pyrazines to 1H-imidazo[4,5-b]pyrazines. The in situ Fe-mediated reduction of the oxadiazole fragment followed by cyclization gave access to imidazopyrazines in moderate to good yields. A selection of orthoesters also allowed functionalization on the 2-position of the imidazole ring. This method afforded a variety of imidazopyrazine derivatives with varying substitution on the 2, 5 and 6 positions. Our studies suggest that both a 2-trifluoromethyl group and N-methylation are crucial for mitochondrial uncoupling capacity.

Clinical and epidemiologic evaluation of a 2020 chikungunya outbreak in Cambodia.

BACKGROUND: In 2020, the Kingdom of Cambodia experienced a nationwide outbreak of chikungunya virus (CHIKV). Despite an increase in the frequency of outbreaks and expanding geographic range of CHIKV, diagnostic challenges remain, and limited surveillance data of sufficient granularity are available to characterize epidemiological profiles and disease dynamics of the virus. METHODS: An ongoing and long-standing cross-sectional study of acute undifferentiated febrile illness (AUFI) in Cambodia was leveraged to describe the disease epidemiology and characterize the clinical presentation of patients diagnosed with CHIKV during the 2020 outbreak. Participants presenting with AUFI symptoms at ten study locations provided acute and convalescent blood samples and were tested for CHIKV using a reverse transcription-polymerase chain reaction (RT-PCR) and serological diagnostic methods including IgM and IgG. Acute and follow-up clinical data were also collected. RESULTS: From 1194 participant blood samples tested, 331 (27.7%) positive CHIKV cases were detected. Most CHIKV positive individuals (280, 84.6%) reported having a fever 3 to 4 days prior to visiting a health facility. Symptoms including chills, joint pain, nausea, vomiting, and lesions were all statistically significant among CHIKV positive participants compared to CHIKV negative AUFI participants. Cough was negatively associated with CHIKV positive participants. Positivity proportions were significantly higher among adults compared to children. No significant difference was found in positivity proportion between rainy and dry seasons during the outbreak. Positive CHIKV cases were detected in all study site provinces, with the highest test positivity proportion recorded in the rural northeast province of Kratie. CONCLUSIONS: Surveillance data captured in this study provided a clinical and epidemiological characterization of positive CHIKV patients presenting at selected health facilities in Cambodia in 2020, and highlighted the widespread distribution of the outbreak, impacting both urban and rural locations. Findings also illustrated the importance of utilizing both RT-PCR and serological testing for effective CHIKV surveillance.