Serological progression and time to seroconversion in serodiscordant and seronegative dogs tested for visceral leishmaniasis in an endemic area of Brazil.

Development of a time-resolved fluorescent lateral flow immunochromatographic strip for detection of Mycoplasma bovis antibodies.

Engineered metal-organic frameworks for advanced lateral flow immunoassays: structural design, functional hybridization, and biosensing applications

Orthoebolaviruses, like Ebola, Sudan, and Bundibugyo viruses, are responsible for causing sporadic and unpredictable outbreaks of severe disease throughout Sub-Saharan Africa. Accurate and reliable diagnostic tests are key to identifying cases of disease and restricting its spread, yet options are mostly limited to molecular assays, such as reverse transcriptase quantitative PCR, that require time, training, and complex equipment to run. To complement existing diagnostic modalities, and to address some of their shortcomings, we developed a prototype immunochromatographic lateral flow rapid diagnostic test (ILF-RDT) that detects the glycoproteins (GPs) from all known orthoebolaviruses, including Bundibugyo virus, which is currently causing a large outbreak in the Democratic Republic of the Congo and Uganda. This test depends on a pair of novel, broadly cross-reactive monoclonal antibodies capable of binding the viral GP-including the secreted GP-in a non-competitive manner. Here, we show that this ILF-RDT detects both recombinant GP and authentic virus in a variety of substrates, including samples spiked with human blood and blood samples taken from experimentally infected animals. Indeed, the ILF-RDT was able to detect Ebola virus in nonhuman primate samples at 4 days post-infection, before the animals began exhibiting signs of severe disease. Given the uniquely broad cross-reactivity of this diagnostic assay, its ability to detect low levels of virus early during infection, and its suitability for use at the point-of-care in resource-limited settings, this ILF-RDT could have a significant impact on the management and mitigation of future orthoebolavirus outbreaks.

Fluoroquinolones (FQs) are widely used in animal husbandry for the prevention of a variety of animal diseases. However, residues of FQs in animal products that exceed safe limits may pose a serious threat to human health. Therefore, the development of colloidal gold immunochromatographic test strips with high specificity and high sensitivity is crucial for the accurate detection and management of such antibiotic residues. In this study, a monoclonal antibody (mAb) with high specific reactivity to FQs was prepared, which exhibited cross-reactivity with more than 10 fluoroquinolones. The mAb showed that the half-maximal inhibitory concentration (IC50) for PEF was 0.023 ng mL-1, with a linear range from 0.0103 to 0.069 ng mL-1. A competitive enzyme-linked immunosorbent assay (ic-ELISA) was established. The recoveries of PEF ranged from 80% to 120%, and the average coefficient of variation was below 10%. Based on this mAb, an immunochromatographic test strip for the detection of FQ residues in actual milk samples was developed. The results demonstrated that the test strip could simultaneously detect 12 fluoroquinolones, with a LOD of 1-10 ng mL-1. This study highlights that establishing a method capable of simultaneous broad-spectrum screening and high-sensitivity detection is essential for effectively monitoring fluoroquinolone residues and ensuring consumer safety.

While high-performance liquid chromatography (HPLC) is well-established for β-thalassaemia and haemoglobinopathies, phenotypic screening for α0-thalassaemia has been limited. To address this limitation, we aimed to translate the discovery of the α-thalassemia early eluting peak (αEEP) in HPLC into clinical practice by comparing its diagnostic performance with other existing methods (haemoglobin H inclusion test [HbHi] and immunochromatographic strip test [ICT]) in a multicentre setting, and elucidating the nature of the αEEP by liquid chromatography-tandem mass spectrometry (LC-MS/MS). With a cohort of 820 genotyped patients, the αEEP showed superior diagnostic performance in detecting --SEA (sensitivity 99.6%, specificity 100%) compared with HbHi (sensitivity 95.8%, P = 0.006; specificity 97.3%, P < 0.001) and ICT (sensitivity 95.8%, P = 0.006; specificity 75.4%, P < 0.001). Both HbHi and ICT showed reduced sensitivity in β-thalassaemia carriers versus non-carriers. ICT showed reduced specificity when Hb F ≥ 1% compared with < 1%. The αEEP remained robust across all subgroups. LC-MS/MS revealed a strong association between the αEEP and embryonic ζ-globin chains (P < 0.001). The αEEP offered cost reductions of 98.6% over HbHi and 97.3% over ICT. Collectively, the αEEP is a highly reliable and cost-effective marker for detecting --SEA carriers, enabling a novel "all-in-one" HPLC screening strategy for --SEA, β-thalassaemia and haemoglobinopathies. Trial registration number: not applicable.

Feline leishmaniosis (FeL) caused by Leishmania infantum is increasingly recognized in endemic areas, but factors influencing susceptibility in cats remain incompletely understood. Because blood group antigens may modulate host-pathogen interactions, this study evaluated whether feline AB blood system phenotypes are associated with L. infantum seropositivity and/or molecular positivity in cats from Italy. Exploratory analyses further assessed whether blood phenotype was associated with the magnitude of indirect fluorescent antibody test (IFAT) antibody titres or with real-time PCR (qPCR) parasite load. In this retrospective cross-sectional study, cats were classified as L. infantum-positive when they had an IFAT titre ≥1:80 and/or a positive qPCR on blood or lymph node aspirates. Feline AB blood typing was performed by tube agglutination, with type B and AB samples confirmed by immunochromatographic testing and back typing. A total of 706 cats were included. Overall, 67/706 cats (9.5%) were classified as L. infantum-positive. Blood phenotype distribution was 83.1% type A, 10.1% type B, and 6.8% type AB. L. infantum positivity was detected in all three phenotypes, and no evidence of association was found between blood phenotype and L. infantum positivity, IFAT seropositivity, qPCR positivity, IFAT titre, or qPCR parasite load. After adjustment for region, blood phenotype remained not significantly associated with L. infantum positivity. These findings suggest that feline AB blood system phenotypes were not associated with L. infantum infection in this feline cohort. Future studies should investigate whether blood phenotype may influence other aspects of FeL, such as clinical expression or disease outcome.

BackgroundContemporary multicentre data on carbapenemase distribution among carbapenem-resistant Enterobacterales (CRE) in Lebanon remain limited. This study aimed to characterize the distribution of carbapenemase enzyme families among clinical CRE isolates collected from Lebanese hospitals.MethodsA multicentre study was conducted across 14 hospitals representing different Lebanese regions between March and October 2025. CRE isolates were centrally processed using standardized phenotypic microbiological methods and interpreted according to the 2025 European Committee on Antimicrobial Susceptibility Testing breakpoints. Carbapenemase identification was performed using NG-Test® CARBA-5. Agreement between phenotypic prediction and rapid immunochromatographic testing was evaluated.ResultsA total of 59 non-duplicate CRE isolates from individual patients were included. New Delhi metallo-β-lactamase (NDM) was the predominant carbapenemase enzyme family (64.4%), followed by OXA-48–like enzymes (23.7%), with co-production observed in 10.2% of isolates. Among carbapenem-resistant Escherichia coli (34 isolates), NDM predominated (73.5%). In contrast, carbapenem-resistant Klebsiella pneumoniae (15 isolates) more frequently harboured OXA-48–like enzymes (53.3%), although NDM-producing isolates were also identified (33.3%). No NDM-producing isolate was susceptible to ceftazidime–avibactam, whereas OXA-48–like producers retained higher susceptibility (71.4%). Phenotypic carbapenemase prediction showed high agreement with NG-Test® CARBA-5 results (κ = 0.89), including 100% sensitivity for metallo-β-lactamase detection.ConclusionsNDM was the predominant carbapenemase among CRE isolates recovered from participating Lebanese hospitals, while Oxacillinase-48 carbapenemase (OXA-48)–like enzymes remained common. These findings highlight the importance of carbapenemase-specific characterization, as therapeutic options differ according to the underlying resistance mechanism. Rapid carbapenemase detection using NG-Test® CARBA-5, coupled with phenotypic prediction, may support resistance mechanism-guided therapy, antimicrobial stewardship and surveillance efforts.

BEACON: AIE-based point-of-care test for cross-species Brucella antibody detection.

Severe Fever with Thrombocytopenia Syndrome (SFTS), caused by SFTS virus (SFTSV), is a widely distributed infection with significant mortality. Diagnosis in resource-limited settings remains challenging. For rapid and convenient diagnosis, we developed a portable rapid diagnosis method that combines Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas12a technology with immunochromatographic test strips. The SFTSV RNA was amplified by reverse transcription loop-mediated isothermal amplification (RT-LAMP). The homologous target sequence and single stranded DNA (ssDNA) reporter gene were cleaved by CRISPR/Cas12a in parallel, and ssDNA probes labeled with FAM fluorescein and biotin were captured by an immunochromatographic strip. Finally, the signal on the immunochromatographic strips became visible to the naked eye. Based on CRISPR/Cas12a, a rapid SFTSV detection method was developed, featuring simplicity, rapidity, low cost, and ease of use. The method was applied for the nucleic acid detection of SFTSV in 40 clinical serum samples and compared with RT-polymerase chain reaction (PCR). The new method showed 100% sensitivity and 100% specificity with a detection agreement rate of 100%. The minimum detection limit of the method was 2.5 copies/μL, and no cross-reactivity with nucleic acids from other common pathogens was observed. Detection can be completed within 80 min, and results are observable with the naked eye. For the analysis of clinical samples, the method exhibits good detection performance and thus provides an attractive option for the nucleic acid detection of SFTSV in point-of-care and resource-limited medical settings.

Francisella tularensis is a bacterial pathogen that can contaminate drinking water and food products, causing tularemia - a severe zoonotic disease that affects both humans and animals. This infection is dangerous to human health and may be fatal without diagnosis and treatment. Therefore, rapid, sensitive, and selective detection of this microorganism is in high demand for clinical diagnostics, environmental monitoring, and ensuring food safety. This study presents the development of immunochromatographic (lateral flow) tests for revealing F. tularensis cells in natural and drinking water samples. A special feature of the test system is catalytically active Au@Pt nanoparticles (a peroxidase-like nanozyme) used as a label for specific antibodies. Au@Pt nanozyme can catalyze the oxidation of the peroxidase substrate, followed by the formation of a colored product, which amplifies the colorimetric signal on the test strip and significantly improves detection sensitivity. The tests allow for the detection of F. tularensis cells in concentrations down to 102-103 cells per mL, depending on the strain, with visual result assessment. The application of Au@Pt nanozyme decreased the detection limits by 321-9600 times compared to gold nanoparticles commonly used in immunochromatography. The assay duration is 14 min, including the catalytic enhancement step. Monoclonal antibodies against bacterial lipopolysaccharide provide selective detection of virulent strains, excluding cross-reactions with non-pathogenic strains of F. tularensis and other microorganisms that may also contaminate water sources. The effective testing of natural and tap water samples conducted with no preliminary sample preparation has proven the relevance of the developed approach.

Development of a quantum dot-based immunochromatographic test strip for rapid detection of infectious bursal disease virus.

Onychomycosis is a common nail infection predominantly caused by dermatophytes. Accurate diagnosis is essential to guide antifungal therapy, yet conventional methods such as direct examination (DE) and fungal culture have important limitations, including operator dependence and long turnaround times. Rapid antigen-based assays, such as immunochromatographic tests (ICTs), have emerged as potential alternatives, but their performance under routine laboratory conditions remains insufficiently defined. To evaluate the diagnostic performance of the PreventID Dermatophyte immunochromatographic test under controlled conditions and during routine clinical implementation, using fungal culture as the reference standard. This study was conducted in two phases at a tertiary care hospital. During the pilot phase (November 2023-February 2024), nail samples from patients with suspected onychomycosis were analyzed in parallel using ICT, DE, and fungal culture. During the implementation phase (September 2024-September 2025), ICT was introduced as a first-line screening tool. A subset of samples processed in October 2024 and January 2025 underwent parallel testing with ICT, DE, and culture for direct comparison. Diagnostic performance metrics were calculated using dermatophyte culture as the reference standard. In the pilot phase, 49 samples were analyzed, with dermatophytes isolated in 18 cases. ICT showed a sensitivity of 88.9%, specificity of 90.3%, and substantial agreement with culture (κ = 0.78), outperforming DE in sensitivity. During routine implementation, 904 ICTs were performed; among ICT-negative samples undergoing culture, dermatophytes were isolated in 52 cases, yielding a negative predictive value of 87.5%. During the systematic comparison period, ICT sensitivity decreased to 37.5% with poor agreement (κ = 0.08), whereas DE showed higher sensitivity (63.9%) and moderate agreement (κ = 0.53). PreventID Dermatophyte performs well under controlled conditions but shows reduced sensitivity in routine practice. Although its rapid turnaround makes it a useful screening tool, the observed false-negative rate limits its reliability as a stand-alone test to exclude infection. Therefore, ICT results should be interpreted in conjunction with clinical findings and confirmed by conventional methods, supporting its role within a multimodal diagnostic strategy.

Identifying risk factors for gastrointestinal carbapenem-resistant Enterobacterales (CRE) colonization and acquisition is essential for guiding infection prevention and control strategies; however, most studies focus exclusively on colonization at hospital admission. We aimed to distinguish determinants of CRE colonization present at intensive care unit (ICU) admission from those driving acquisition during ICU stay using serial surveillance screening. A prospective cohort study was conducted in two ICUs in Brazil. Screening relied on rectal swab cultures from April 2022 to May 2024, supplemented with immunochromatographic testing in one ICU and polymerase chain reaction in the other during October 2023-May 2024. Colonization at admission and incident acquisition during stay were outcomes of interest. Pre-ICU predictors were evaluated using logistic regression with least absolute shrinkage and selection operator selection, whereas post-ICU factors were assessed through generalized estimating equations. Among 751 patients, 39 (5%) were colonized with CRE at ICU admission. Previous acute care hospitalization (odds ratio [OR] = 2.70; P = 0.006) and transfer from another hospital (OR = 2.62; P = 0.046) were independent risk factors. Post-ICU-admission incident CRE colonization was associated with ICU B stay (OR = 2.20; P = 0.013), broad-spectrum antibiotics (OR = 1.90; P = 0.018), urinary catheter use (OR = 2.33; P = 0.024), and shock (OR = 1.91; P = 0.043). Hand hygiene compliance and contact precautions adherence were >80% across both units. CRE acquisition in ICU appears related to colonization pressure, antibiotic disruption, and specific device use. Prevention of CRE acquisition in high-prevalence ICUs likely requires interventions beyond standard infection prevention and control measures.

Pt single-atom catalyst-Enhanced aerogenous reaction for pressure signal-based immunochromatographic test strip.

Traumatic brain injury (TBI) represents a significant global health challenge requiring rapid and accurate diagnostic tools. S100 calcium-binding protein B (S100B) serves as a consensus-endorsed serum biomarker for TBI, but current detection methods lack the speed and simplicity needed for on-site clinical applications. This study reports the development of a time-resolved fluorescence immunochromatographic test strip (TRFIS) for rapid and quantitative serum S100B detection. High-affinity monoclonal antibodies against S100B were generated via hybridoma technology, and time-resolved fluorescent microspheres (TRFMs) were integrated with lateral flow immunoassay. The TRFIS demonstrated excellent analytical performance with a linear range of 15.6-1000 pg/mL (R2 = 0.9885), a detection limit of 12.6 pg/mL, and a test time of approximately 13 min. Intra- and inter-batch coefficients of variation were both < 10.0%, confirming high reproducibility. This platform offers high sensitivity, accuracy, and simplicity, making it potentially suitable for on-site TBI diagnosis in resource-limited healthcare settings.

Helminth infections remain a major public health problem in the Lao People's Democratic Republic (Lao PDR), particularly in rural communities where foodborne trematodes and soil-transmitted helminths are co-endemic. Opisthorchis viverrini is a well-established risk factor for cholangiocarcinoma, while Strongyloides stercoralis can cause severe and potentially fatal disease. This study aimed to determine the prevalence and patterns of helminth infections in southern Lao PDR using combined parasitological and serological diagnostic approaches. A community-based cross-sectional study was conducted among 242 adults residing in three rural villages in Champasak Province, southern Lao PDR. Stool samples were examined using the formalin-ethyl acetate concentration technique (FECT), and point-of-care immunochromatographic tests (ICTs) were applied to detect IgG antibodies against O. viverrini and S. stercoralis. Overall, 69.0% of participants were infected with at least one helminth species. O. viverrini was the most prevalent parasite, detected in 51.7% by FECT and 65.3% by ICT. Other common infections included minute intestinal flukes (20.2%), hookworms (14.9%), S. stercoralis (9.9% by FECT; 14.5% by ICT), Trichostrongylus spp. (9.1%), and Taenia spp. (6.2%). Polyparasitism was frequent, with 9.1% harboring three or more helminth species. The estimated true prevalences, based on combined results from stool examination and serological testing, were 66.6% for opisthorchiasis and 13.5% for strongyloidiasis. ICT identified substantially more infections than stool examination alone for both O. viverrini and S. stercoralis. Serological ICT screening offers a sensitive, practical complement to parasitological methods. Integrated diagnostics are essential for improved surveillance and targeted interventions in resource-limited endemic areas. Clinical trial number: not applicable.

With the launch of Sri Lanka's National Strategic Plan for leishmaniasis control, case diagnosis has been identified as a key strategic intervention. However, the routine slit-skin smear (SSS) demonstrates variable sensitivity (33-78%), underscoring the need for rapid and sensitive diagnostic tools. This study evaluated the diagnostic performance of a newly developed nanoparticle-based anti-rKRP42 (recombinant kinesin-related protein antigen-42) IgG immunochromatographic test (rKRP42-ICT) for detecting cutaneous leishmaniasis caused by Leishmania donovani. A cross-sectional pilot study was conducted among 58 adults with clinical CL at Base Hospital Tangalle. CL was confirmed by SSS (reference test). Serum from CL patients and Japanese negative controls was tested using rKRP42-ICT, rKRP42 IgG ELISA, and rK39-ICT. Diagnostic performance of the ICT (sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV)) was calculated against SSS. Clinical associations were analysed by Chi-square, and ELISA OD means were compared using non-parametric tests (with 95% confidence interval). The majority of the CL cohort (62%) were male. The mean duration at presentation was 5.1months. Most had single, small (< 2cm) lesions, with ulcers being the commonest type (41%). rKRP42-ICT demonstrated a positive rate of 43.1% among the clinical CL cohort while SSS and rK39-ICT positivity was 55.2% and 5.2%, respectively. The rKRP42-ICT showed 43.8% sensitivity, 57.7% specificity, and 56% PPV and 45.5% NPV against the reference test. The rKRP42-ICT could detect positive samples across a wide range of ELISA OD values, from 0.017 to 2.535. rKRP42-ICT positivity correlated with higher ELISA OD values (p < 0.05), but SSS positivity did not. The rKRP42-ICT and SSS agreement was slight (Kappa = 0.014). Negative controls (n = 43) were negative by both ICTs. Male gender, ulcerated, large (> 2cm), and multiple lesions were associated with higher rKRP42-ICT positivity (p < 0.05) and higher mean ELISA OD values. Defining confirmed cases as clinical CL with either SSS or rKRP42-ICT positivity increased case detection from 55.2 to 74.1% (20%). The rKRP42 IgG-based ICT showed promise as a supplementary diagnostic tool for CL, potentially improving overall case detection beyond routine microscopy and providing rapid results for clinical decision-making. With further optimization and validation, it may support national diagnostic and surveillance efforts.

Point-of-care, immunochromatographic tests have not reliably detected the rabies virus and have not utilized a consistent diagnostic protocol. International diagnostic standards established by the World Organization for Animal Health require strategic and large-scale validation studies prior to widespread use. The United States (US) Centers for Disease Control and Prevention, National Rabies Reference Laboratory, undertook a large-scale validation of the BioNote Anigen Rapid Rabies Antigen Test in collaboration with eight US public health laboratories and three international laboratories. Modifications were made according to the manufacturer's instructions to maximize antigen concentration in the test diluent. A total of 1,399 samples underwent paired testing with one of three US gold standard tests, consisting of 31 types of animals and 9 rabies virus variants (RVVs). Test sensitivity and specificity were 97.11% (CI: 95.21%-98.27%) and 99.89% (CI: 99.36%-99.98%). Fourteen samples resulted in false-negative results, primarily impacting dogs that were euthanized early or shown to have a low viral load. Limit-of-detection studies found that false-negative results often occurred when the sample had a PCR Ct value >23. The BioNote Anigen Test performed well across diverse RVVs found in North America. Sensitivity of the test was slightly lower than, but not statistically inferior to, the minimum 98% value established for the current gold standard tests. This is the largest systematic evaluation of a rabies point-of-care test that includes diverse RVV and animal types, and results suggest that the BioNote Anigen Test with the procedural changes would have broad benefits for rapid diagnosis in animals.IMPORTANCEDespite more than half a century of highly accurate rabies diagnostic methods, a global landscape analysis conducted in 2021 found that nearly all countries in Africa and Asia have inadequate rabies surveillance and testing programs. This dearth of surveillance and testing has been identified as a leading factor for rabies' longstanding status as a neglected disease. Studies have consistently identified barriers with current diagnostic approaches, which render them unlikely to be utilized effectively in low- and middle-income countries, where an estimated 70,000 people die from the disease each year. In 2023, the World Organization for Animal Health cautioned against the use of unvalidated lateral flow assay tests outside of research or evaluation programs. Despite this statement of caution, numerous field deployments of these tests have been published. This evaluation found a high sensitivity and specificity for the BN-LFA, with values similar to those reported for other gold standard rabies tests.

Rapid detection of syphilis infection enables timely antibiotic treatment to prevent long-term complications and further transmission. We implemented a treponemal rapid lateral flow immunochromatographic test that was performed by trained clinical laboratory personnel on a 24/7 basis in an urban county hospital. From March 2023 to February 2024, 461 rapid syphilis tests (RSTs) were performed on serum from 449 patients. Specimens were also tested by a conventional reverse algorithm beginning with an enzyme immunoassay (EIA), followed by a rapid plasma reagin (RPR) test, and when necessary, an additional treponemal test to resolve discordant results. Most patients (349, 77.7%) initially presented to the emergency department. The most common patient risk factors for syphilis were substance use disorder (51.9%) and unstable housing (42.8%). Compared to the reverse algorithm, RST sensitivity was 52.2%, specificity 98.2%, positive predictive value 83.7%, and negative predictive value 92.1%. After excluding 45 patients who had a documented prior history of syphilis, RST sensitivity was 83.3%, specificity 98.2%, positive predictive value 74.1%, and negative predictive value 99.0%. Nearly all of the 33 patients testing negative by the RST but positive by EIA were more likely to have been treated for a previous infection: 27 had a documented history of treatment and 30 had undetectable or low RPR titers (<1:2). The median RST turnaround time was 1.6 hours compared to 197.8 hours for reverse algorithm screening. RST is a useful option for high-risk patients in urban settings. Other testing methods are preferable for previously treated syphilis, and conventional follow-up testing is required to confirm results.