Porcine epidemic diarrhea virus (PEDV) poses a significant threat to the swine industry, and distinguishing circulating field strains from vaccine strains under field conditions remains challenging. In this study, we established a one-tube visual detection platform based on recombinase‑aided amplification (RAA) and PfAgo nuclease for simultaneous detection of the highly conserved N gene and the virulence‑associated ORF3 gene of PEDV. The assay completes within 60min, with a sensitivity of 100 cDNA copies/μL. Specificity tests showed no cross‑reactivity with other porcine viruses tested. Using FAM‑ and ROX‑labeled probes, the N gene produces green fluorescence, ORF3 generated red fluorescence, and dual positivity appears yellow, which all visible to the naked eye under blue light. In validation with 36 clinical samples, 12 were identified as PEDV‑positive, including 3 field strains. Using RT‑PCR sequencing as the reference, the method achieved 100% specificity. Overall, this RAA‑PfAgo approach integrates rapidity, high sensitivity, specificity, and visualization without complex instrumentation, offering a powerful tool for on‑site clinical diagnosis, strain differentiation, and epidemiological monitoring in pig farms.
The Capripoxviruses (CaPVs) are highly contagious viruses that includes sheeppox virus, goatpox virus, and lumpy skin disease virus infecting sheep, goats, and cattle respectively resulting in significant morbidity and mortality. This study presents a diagnostic tool based on recombinase polymerase amplification (RPA) assisted with CRISPR-Cas12a platform coupled with a lateral flow assay (LFA) that facilitate timely detection and differentiation of Capripox viruses, and support effective surveillance programs. The assay operates through the cleavage of a dual labelled oligonucleotide reporter molecule (Biotin-Fam), generating a detectable signal. The results can be interpreted either using a fluorescence-based detection system or through a lateral flow assay (LFA), where gold nanoparticles conjugated with anti-FAM antibodies enable visual signal detection. Under optimized assay conditions (50nM LbCas12a and 1µM reporter probe with a reaction time of 25min), the CRISPR-Cas12a platform along with RPA (39°C for 20min) substantially enhanced analytical sensitivity, enabling reliable detection of as few as 10 copies/µL of viral DNA. The specificity of the assay was rigorously evaluated using individual targets, mixed-template samples, and 90 clinical specimens, demonstrating complete species-specific discrimination without any detectable cross-reactivity. Collectively, the developed single-gene RPA-CRISPR-Cas12a lateral flow assay (LFA) represents a rapid, highly sensitive, and specific point-of-care diagnostic platform, providing visual readout by the naked eye and offering significant potential for decentralized veterinary diagnostics and field-based disease surveillance.
Background:Acute myocardial infarction (AMI) remains a leading cause of premature death (<65 years) in the United States, with a significant association with psychoactive substance use (PSU). However, long-term trends and future projections of this combined burden remain unclear. Methods:We conducted a retrospective observational study using the Centers for Disease Control and Prevention Wide-Ranging Online Data for Epidemiologic Research Multiple Cause of Death database to assess premature mortality (ages 25-65 years) related to AMI and PSU from 1999 to 2023. Age-adjusted mortality rates (AAMRs) were derived, and annual percentage changes (APCs) were calculated using joinpoint regression. An autoregressive integrated moving average (ARIMA) model was used to forecast mortality through 2035. Results:From 1999 to 2023, 176,641 premature deaths were attributed to AMI and PSU. The AAMR rose from 1.4 per 100,000 in 1999 to 4.16 in 2023. Mortality increased sharply from 1999 to 2005 (APC 19.04), continued rising through 2021 (APC 2.63), and declined between 2021 and 2023 (APC -9.13). Middle-aged adults (45-65 years) had substantially higher mortality than younger adults (25-45 years) (AAMR 9.63 vs 0.91, 2018-2023). Men had higher mortality than women (6.81 vs 2.53). American Indian/Alaska Native and White populations showed the higher racial burden. Nonmetropolitan areas had markedly higher mortality than metropolitan regions. South Dakota, Kentucky, and Arkansas had the highest state-level mortality. Forecasting predicts only a modest decline to 3.80 per 100,000 by 2035. Conclusion:Despite recent declines, premature mortality from AMI and PSU is projected to remain high through 2035, underscoring the need for targeted, evidence-based prevention strategies.
Toscana virus (TOSV) is a mosquito-borne phlebovirus endemic to the Mediterranean region and an important cause of arboviral infections in humans, particularly during the summer months. Although TOSV infections are often asymptomatic or mild, the virus can cause neuroinvasive disease, including meningitis and encephalitis, highlighting the need for rapid and reliable tools for its detection and characterization. N protein was identified as a promising diagnostic target for TOSV due to its accumulation in infected cells and its role as primary immune target. The aim of this study was to establish a rapid and easily adaptable workflow for the recombinant production of the nucleoprotein (N) of Toscana virus (TOSV). We introduced a Gibson® Assembly-based cloning strategy using the pT7CFE1-CHis plasmid, specifically selected for its compatibility with cell free protein expression systems. The Gibson® approach enabled fast and efficient construct generation, overcoming the constraints typically associated with classical restriction-based cloning. Subsequently, cell free in vitro synthesis produced the recombinant protein within 16 h, enabling an efficient production. The yield was sufficient to perform downstream assays, including Western blot, thereby demonstrating the suitability of this combined strategy for rapid antigen prototyping and preliminary characterization. Our results highlight the recombinant TOSV antigen for diagnostics to support timely and disease detection and control.
Rapid and accurate identification of respiratory viruses is essential for effective clinical management and infection control. However, conventional RT-qPCR methods are time-consuming and require complex workflows. To address these limitations, we developed a novel Switching RT-qPCR assay designed for multiplex detection of respiratory viruses with enhanced efficiency and sensitivity. This study aimed to evaluate the analytical and clinical performance of the switching RT-qPCR assay in the detection of seven major respiratory viruses: Influenza A and B, respiratory syncytial virus (RSV) A and B, adenovirus (AdV), human metapneumovirus (hMPV), and SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2). Nasopharyngeal swab specimens (n = 808) were collected from patients with respiratory symptoms. RNA was extracted and tested using both a Switching RT-qPCR assay and commercial multiplex RT-qPCR method (Allplex Respiratory Panels and Allplex SARS-CoV-2 Assay). The concordance, sensitivity, specificity, and analytical performances of the two methods were compared. The Switching RT-qPCR assay demonstrated a clinical sensitivity of 93.6% and a specificity of 100%, with an overall concordance rate exceeding 96%, compared with the conventional assay. Analytical sensitivity was comparable or superior across most viral targets, particularly RSV A, AdV, and hMPV. The duplex assay format enabled the efficient simultaneous detection of two targets per reaction, reducing the overall testing time while maintaining analytical accuracy. This Switching RT-qPCR assay offers a reliable, rapid, and efficient alternative for respiratory virus detection. Its simplified duplex format and high concordance with standard molecular diagnostics indicate its potential utility in routine clinical laboratories.
Persistent infection with high-risk human papillomavirus (HPV) types 16 and 18 is the primary cause of cervical cancer, and vaccination is an effective prevention method. The assessment of vaccine efficacy relies on measuring neutralizing antibodies (nAbs). However, the pseudovirion-based neutralization assay (PBNA), which is widely used as a reference method has limitations such as low throughput and technical complexity. To address this issue, we have developed a rapid and accurate magnetic bead-based competitive chemiluminescence immunoassay (CLIA) for the separate quantitative assessment of HPV-16 and HPV-18 nAbs. This platform uses two assay configurations: HPV-16 detection involves monoclonal antibody (mAb)-coated beads and acridinium ester (AE)-labeled antigens (Ags), while HPV-18 detection involves Ag-coated beads and AE-labeled mAbs. This assay works by measuring the competitive inhibition of tracer binding by sample Abs, resulting in a chemiluminescence signal that is inversely proportional to nAbs concentration. The method has shown acceptable analytical performance, with strong linearity (R² > 0.999) and recovery values within the predefined acceptance range for both assays. A preliminary comparison using 40 serum samples from vaccinated participants showed 97.5% agreement and a strong correlation (R² > 0.95) with PBNA. The selected analytical parameters remained generally comparable after short-term thermal stress at 37 °C for 7 days. These findings support the analytical feasibility of the platform and its further evaluation in HPV vaccine immunogenicity studies and serological monitoring.
Background:Cardioplegia remains a cornerstone of myocardial protection during cardiac surgery; however, the configuration of its delivery circuit, particularly priming volume and blood-synthetic surface area, can exert a measurable influence on patient outcomes. Oversized or non-tailored circuits inherently increase hemodilution, inflammatory activation, and coagulation disturbances, effects that are especially pronounced in neonates and small children, whose circulating blood volume is limited. Advances in modular heart-lung machine (HLM) technology have introduced the possibility of tailoring cardioplegia circuits to patient size and procedural requirements, aligning mechanical design with physiological principles. Materials and Methods:A narrative review was conducted using PubMed, Scopus, and Google Scholar for studies published between January 1976 and July 2025. Search terms included "cardioplegia," "myocardial protection," "cardioplegia circuit," "priming volume," "modular heart-lung machine," "microplegia," "del Nido cardioplegia," and related keywords. The search identified 286 records; after duplicate removal and eligibility screening, 22 studies were included in the final qualitative synthesis. Evidence was analyzed according to four domains: priming volume and blood conservation, contact surface area and inflammation, modularity and circuit customization, and physiological implications of delivery strategy. Results:Cardioplegia delivery circuit design appears to be a clinically relevant but underrecognized component of myocardial protection. Modular HLM platforms provide a practical approach to reduce circuit complexity and adapt extracorporeal circulation to individual patient requirements. Further prospective multicenter studies are needed to define the impact of modular cardioplegia circuits on clinical outcomes. Conclusions:Modular HLM technology provides a flexible and physiologically grounded platform for tailoring cardioplegia delivery to individual patient profiles. Standardizing weight-banded modular configurations and implementing routine monitoring of priming volume-to-weight ratios could improve consistency in practice and serve as a foundation for prospective studies. The cumulative evidence suggests that extending modular principles to the cardioplegia circuit represents an underutilized but promising opportunity to enhance both pediatric and adult cardiac surgical outcomes.
Avian reovirus (ARV) causes viral arthritis in poultry, and emerging variants in China demand improved serosurveillance. We developed an indirect competitive ELISA (ic-ELISA) using a monoclonal antibody against recombinant σC protein. After optimization (coating antigen 6.0 μg/mL, mAb 1:4000, blocking 90 min, serum 60 min, mAb 90 min), the cut-off percent inhibition was set at 44.5% based on 40 negative sera. The assay showed no cross-reactivity with six common avian pathogens, a detection limit of 1:400 serum dilution, and excellent reproducibility (intra-CV ≤0.70%, inter-CV ≤7.70%). Testing 87 field sera from various regions of China gave an overall seropositivity of 59.8% (regional range 50.0-75.0%). Compared with a commercial IDEXX kit, our ic-ELISA detected 52 positives vs. 48 (χ²=4.32, P = 0.038), demonstrating superior sensitivity. This reliable, sensitive, and specific serological tool is suitable for large-scale ARV surveillance and vaccine monitoring.
Background:Pacemakers (PMs) are crucial for treating symptomatic bradyarrhythmias and improving patient health-related quality of life (HRQoL). While Chagas disease (ChD) is known to impair HRQoL, its specific impact on PM recipients, as measured by the AQUAREL questionnaire, is not well understood. This study aimed to compare HRQoL between pacemaker patients with and without ChD. Methods:This cross-sectional study included 57 adult patients with PM (29 with ChD and 28 without). HRQoL was evaluated using the AQUAREL questionnaire, which assesses three domains: Chest Discomfort, Arrhythmia, and Dyspnea. We used multivariate inflated beta regression models, adjusting for clinical and demographic covariates, to analyze the data. Results:Participants with ChD (median age 64 years, interquartile range [IQR], 56-72 years) reported significantly worse HRQoL than those without ChD (median age 57 years, IQR, 53-69 years). After multivariate adjustment, ChD remained significantly associated with poorer HRQoL scores in all domains: Chest discomfort (adjusted odds ratio [OR] 0.59; p = .044), Arrhythmia (adj. OR 0.38; p = .004), and Dyspnea (adj. OR 0.61; p = .049). An OR < 1 indicates worse outcomes for the ChD group. Conclusions:ChD is independently associated with a significantly poorer perception of HRQoL in pacemaker patients compared to those without ChD. This impairment spans the critical domains of chest discomfort, arrhythmia, and dyspnea, highlighting the distinct health burden imposed by ChD in this patient population.
Background:Ultrathin bioresorbable-polymer sirolimus-eluting stents (BP-SES), including bioresorbable scaffolds (BRS), were developed to mitigate adverse events associated with durable-polymer everolimus-eluting stents (DP-EES). This study aims to compare the efficacy and safety of BP-SES versus DP-EES in patients undergoing percutaneous coronary intervention for de novo coronary artery lesions. Methods:We searched PubMed, Embase, ScienceDirect and ClinicalTrials.gov from inception to May 2026 for randomised controlled trials (RCTs) comparing BP-SES or BRS with DP-EES. Random-effects models were used to pool risk ratios (RRs) or mean differences with 95% confidence intervals (CIs). Results:Nineteen RCTs involving 18,501 patients were included. No significant differences were observed between BP-SES and DP-EES for target lesion failure (RR 1.15, 95% CI 0.74-1.79; I2 = 94%), target vessel failure (RR 0.93, 95% CI 0.84-1.03; I2 = 10%), cardiac death (RR 0.96, 95% CI 0.78-1.18), target vessel myocardial infarction (RR 0.89, 95% CI 0.77-1.03), clinically indicated target lesion revascularisation (RR 0.99, 95% CI 0.80-1.22). Binary restenosis was significantly higher in the BP-SES arm overall (RR 2.46, 95% CI 1.41-4.29; p = 0.001). Conclusions:Metallic BP-SES and DP-EES showed similar rates for most clinical endpoints. However, extreme heterogeneity and accrual of only 5.7% of the required information size render the evidence for target lesion failure inconclusive. Higher binary restenosis increased target lesion revascularisation with fully BRS indicate that these platforms are not interchangeable with contemporary metallic stents. Further large, long-term trials are required.
Since the first implant over two decades ago, transcatheter aortic valve implantation (TAVI) has become the standard treatment for patients with severe aortic stenosis at high and intermediate risk, with mounting evidence to support its use in the younger lower-risk patient. Increasing operator experience and ongoing technological advances have both refined the procedure and reduced overall risk. However, important challenges remain, including coronary access after TAVI, conduction disturbance following implantation, the long-term consequences of paravalvular leak, cost-effectiveness and broader implications on healthcare systems. This review is timely following publication of the latest European Society of Cardiology guidelines on the management of valvular heart disease and recent landmark trials. We aim to provide an update on key areas of development including patient selection, imaging, management of coronary disease, valve durability and lifetime management.
The CRISPR/Cas (clustered regularly interspaced short palindromic repeats) system is a versatile technology for developing antiviral medicines and editing viral genomes in both diagnostics and vaccine synthesis. Emerging insights into class 2 effectors, such as Cas9, Cas12, and Cas13, which target viral DNA and RNA, have revolutionized vaccines against viruses such as HIV, HPV, HBV, and EBV. Innovative diagnostic techniques such as SHERLOCK, DETECTR, and FELUDA have demonstrated system's diversity and accuracy in detecting the virus markers, supporting clinical decision-making, indicating adaptability and precision of CRISPR. This review critically evaluates CRISPR's role in RNA editing, emphasizing its importance for functional genomics and development of recombinant vaccines. Translational challenges are critically discussed, including off-target effects, delivery limitations, and ethical issues, for which unique approaches such as high-fidelity Cas variants, non-viral delivery systems, and bioethical frameworks are evaluated to address these limitations. This review also covers other social implications, such as accessibility and biosecurity risks, associated with CRISPR technologies Collectively, these advances underscore the transformative potential of CRISPR technologies in shaping next-generation antiviral diagnostics and therapeutics.
BACKGROUND:Viral gastroenteritis is a major global health concern, causing billions of infections annually. Multiplex real-time PCR assays for gastro-intestinal viruses (GIV) ensure timely diagnosis, enabling clinical management, improved antimicrobial stewardship and infection prevention actions and supports surveillance. To improve GIV diagnostics used in our tertiary hospital, we adapted and validated our current manual in-house in vitro diagnostic (IH-IVD) PCR panel for GIV on the Hologic Panther Fusion® system (a high-throughput, fully automated sample-to-result IVD system with Open Access™ functionality). STUDY-DESIGN:We compared our current manual IH-IVD method on Roche Flow with the automated Hologic Panther Fusion® system using identical primers and probes targeting rota-, noro- (genotype GI and GII), adeno- and adeno- F (type 40-41), astro-, sapo-, entero- and parechovirus. For clinical validation, 301 fecal samples collected between 2015 and 2024 were analyzed with the Hologic Panther Fusion® system and compared with prior IH-IVD assay results. Analytical sensitivity was assessed using serial dilutions of viral stocks and an external quality assessment (EQA) panel by Quality Control for Molecular Diagnostics (QCMD). RESULTS:Clinical sensitivity and specificity ranged from 91.3% to 100% and 99.3-100%, respectively. Analytical sensitivity was slightly lower for Panther Fusion® (10-fold), except for adeno-F- and norovirus genotype GII which showed higher sensitivity (5-fold). EQA panels were concordant with both systems and the expected QCMD outcomes. CONCLUSIONS:The Panther Fusion® is a fully automated system with minimal hands-on time and rapid sample-to-result turnaround, which offers a reliable outcome despite slightly lower analytical sensitivity compared to IH-IVD.
Genomic surveillance proved invaluable during the COVID-19 pandemic for tracking SARS-CoV-2 variants and guiding outbreak responses, underscoring the ongoing need to reduce whole-genome sequencing (WGS) costs and improve workflow efficiency to ensure accessibility in resource limited settings. Here, we evaluated a one-step reverse transcription polymerase chain reaction (RT-PCR) approach using the Midnight V2 primer scheme for targeted amplification of the SARS-CoV-2 genome, assessed its compatibility with Illumina sequencing, and compared its performance to a well-established two-step method. Initially, we determined optimal RT-PCR reaction conditions using the Midnight V2 primer panel for the one-step RT-PCR kit and scaled reaction volumes for both RT-PCR and library preparation. Clinical specimens (n = 53) that had undergone routine WGS for surveillance purposes using the established two-step RT-PCR method were compared using the one-step RT-PCR assay. For samples with genome completeness greater than 70%, both methods gave comparable results with similar sequence coverage and 100% concordance for lineage assignment. Further investigation revealed a higher percentage of reads aligning to the SARS-CoV-2 genome with a greater depth of coverage using the one-step method compared to the two-step method. Finally, analysis of scaled one-step and library reaction volumes revealed significant cost savings for samples undergoing WGS. Overall, the results presented here verify the accuracy and reproducibility of one-step targeted amplification and offer an efficient and cost-effective workflow for routine SARS-CoV-2 genomic surveillance.
Tomato leaf curl diseases caused by begomoviruses result in significant yield losses in solanaceous and cucurbit crops across East Africa. To effectively manage and monitor these diseases, rapid and field-deployable diagnostic tools are essential. However, traditional Polymerase Chain Reaction (PCR)-based detection methods require laboratory facilities, making them unsuitable for on-site diagnosis. In this study, we developed epitope-targeted peptide antibodies for the serological detection of two begomoviruses in Kenya: tomato leaf curl Uganda virus (ToLCUV) and tomato leaf curl Kunene virus (ToLCKunV). We identified conserved coat protein epitopes on the basis of bioinformatics-based predictions and structural modeling and then used them to generate two peptide-specific antibodies (ToL-asA and ToL-asB). Both antibodies showed strong reactivity with extracts from plants infected with ToLCUV and ToLCKunV in various assays, including indirect ELISA, dot immunobinding assay, and western blotting. Notably, cross-reactivity with unrelated plant viruses was undetectable. In a double-antibody sandwich ELISA, specific antibody pairs generated strong detection signals, indicating that the targeted epitopes were accessible and suitable for immunochromatographic assays. Sequence alignment and phylogenetic analyses confirmed that the targeted epitopes are conserved in several begomoviruses prevalent in East Africa. Furthermore, the use of these antibodies improved viral capture in immunocapture PCR, significantly increasing detection sensitivity for analyses of crude plant extracts. Finally, the peptide antibodies were incorporated into a lateral flow immunoassay platform, thereby enabling the rapid visual detection of begomovirus infections. Overall, these findings demonstrate that epitope-based peptide antibodies are effective reagents for developing rapid and field-deployable diagnostic tools for tomato leaf curl diseases.
Tricuspid regurgitation (TR) has a community prevalence of 3%, which increases with age and affects up to 7% of elderly patients. TR is secondary (functional) in 90% of cases (usually resulting from left heart disease and/or pulmonary hypertension), although atrial dilatation is an increasingly recognised cause. Awareness of the poor prognosis associated with increasing TR severity has refocused the quest for effective treatments. Surgical intervention has been hindered by high in-hospital mortality (up to 10%), even in carefully selected cases, and transcatheter tricuspid valve interventions (TTVIs) have emerged as a low-risk alternative for this high-risk cohort. Increasing data demonstrate the clinical safety and efficacy of TTVIs, but uncertainties remain concerning optimal case selection and procedural timing. Herein, we review newly established techniques alongside emerging clinical and procedural outcome data.
Rabies, a fatal zoonosis, remains a burden in developing countries. Concerns about indirect environmental transmission and appropriate post‑exposure prophylaxis (PEP) are increasing, yet data on RABV stability under various conditions are limited. In this study, we systematically evaluated the stability of RABV (CVS‑11 strain) on six surface (glass, rubber, plastic, polypropylene surgical mask, fabric, and paper) at 25℃, and its stability in brain tissue, muscle, and simulated saliva under three simulated seasonal conditions: summer (35℃, 75% RH), spring/autumn (15℃, 43% RH), and winter (4℃, 33% RH). Viral titers were determined by fluorescent focus assay, complemented by direct fluorescent antibody staining and qPCR for nucleic acid detection. On surfaces, RABV titers declined from about 107 FFU/mL to 103 FFU/mL over 72 h, with inactivation occurring significantly faster on fabric than on other materials. Under simulated summer conditions, no infectious virus could be recovered from saliva after 12 h or from brain and muscle tissues after 24 h, but viral RNA remained detectable by qPCR. However, under spring/autumn and winter conditions, the virus still exhibited relatively high titers at 24 h, with enhanced stability at 4℃. These findings provide an evidence‑based framework for diagnostic laboratories to interpret nucleic acid tests in degraded specimens and to establish sample acceptance criteria. By clarifying the limited time window of environmental infectivity, they also help alleviate rabies‑related anxiety and support more nuanced post‑exposure prophylaxis decisions.
Accurate and timely sequencing of poliovirus is critical for global eradication efforts, particularly for molecular epidemiology based on the typing region of the genome, viral protein 1 (VP1). While Oxford Nanopore Technologies (ONT) sequencing has expanded capabilities for poliovirus surveillance, the relative performance of different ONT library preparation methods, including ligation-based (Native Barcoding) and transposase-based (Rapid Barcoding) approaches, has not been systematically evaluated. In this study, we compared rapid barcoding and native barcoding workflows for sequencing VP1 amplicons from 17 type 2 poliovirus-positive samples, each processed in triplicate. Native barcoding generated significantly more sequencing output, producing approximately 2.3-fold greater total read yield than rapid barcoding, and demonstrated higher run-to-run reproducibility (R2 = 0.979-0.998 vs. 0.847-0.929, respectively; p < 0.001). In addition, native barcoding generated 80% of the total yield achieved by rapid barcoding within approximately 7 h, whereas rapid barcoding required approximately 40 h to reach the same output. Despite these differences, both methods produced identical VP1 consensus sequences across all samples, with comparable read quality (median per-base Q-scores of approximately Q17-Q18). Rapid barcoding provided substantial practical advantages, reducing hands-on library preparation time (55 vs. 200 min) and per-sample cost ($12.82 vs. $16.54), while simplifying workflow and reducing technical complexity. These findings indicate that sequencing yield may not be a determinant of downstream analytical outcomes for poliovirus VP1 ONT sequencing. Rapid barcoding therefore represents a cost-effective and efficient approach for routine poliovirus surveillance, whereas native barcoding remains advantageous in applications requiring rapid data generation or maximal sequencing depth.
Arboviruses such as Usutu virus (USUV) and West Nile virus (WNV) cycle enzootically between competent mosquito vectors and avian reservoir hosts. With USUV established and WNV incidence rising in the Netherlands, detecting antibodies in wild birds captured at ringing sites is critical for surveillance. Currently, no multi-analyte, species-independent serological assays exist for on-site field analysis. Using a recently developed species-independent Lateral flow Microarray ImmunoAssay (LMIA) capable of detecting both USUV non-structural protein 1 (NS1)- and WNV NS1-specific antibodies, a total of 82 wild birds were captured and tested in the field. Of these, 8 birds were USUV LMIA-reactive, all of which were included in a subset of 18 birds that were additionally analysed using gold-standard USUV and WNV 90% Focus Reduction Neutralization Test (FRNT90). Compared to FRNT90 results, the LMIA showed a sensitivity and specificity for USUV of 100% and 89%, respectively. The LMIA also detected seroconversion to USUV in a Eurasian blackbird, which was confirmed by FRNT90. A scoring system based on the WHO's REASSURED criteria was also used as a framework to evaluate the LMIA field performance, receiving 81% of the highest possible score. While minor optimization and broader validation remain, this study demonstrates LMIA feasibility for on-site USUV serological screening. Its rapid, multi-analyte, and equipment-light format supports point-of-capture testing and targeted surveillance in Dutch bird-ringing sites and resource-limited settings.
