Search Results (185)

Antibiotic stereoisomers as components of medicines are typically characterized by different biological activities. Because pharmaceuticals can include a racemic mixture of stereoisomers, monitoring of all forms is required. One contaminant of food products, antibiotic ofloxacin (OFL), as a chiral compound, has two enantiomers—the biologically active S-isomer and less active R-isomer. In this study, a sensitive immunochromatographic test system for simultaneous enantiospeсific detection of the two OFL isomers was developed for the first time. For this, polyclonal antibodies were produced, and conditions for a double lateral flow immunoassay (LFIA) were selected and optimized so that the cross-reactivity with another enantiomer was negligible. The LFIA was performed in a competitive format with gold nanoparticles as a label for secondary antibodies. The achieved LODs/cutoffs were 0.001/10 and 0.007/30 ng/mL for S-OFL and R-OFL detection, respectively; the assay procedure took only 15 min. A double LFIA was performed to detect S-OFL and R-OFL in milk with minimal sample pretreatment; the recoveries were 85–95%. The developed test system is an effective tool for the selective detection of both isomers of OFL, allowing for the avoidance of false negative results. This immunochromatographic approach can be promising for the control of other optically active food toxicants. Full article

Pseudomonas aeruginosa (PA) is a significant pathogen of clinical concern that is frequently associated with multidrug resistance, leading to respiratory tract, wound, and hospital-acquired infections. To enable rapid and accurate detection, we developed a fluorescence-based loop-mediated isothermal amplification (LAMP) method, targeting the PA-specific ecfX gene. Among ten primer sets designed, the optimal set (EC2) was identified, and reaction conditions were optimized (Bst polymerase 320 U/mL, Mg²⁺ 8 mM, dNTP 1.4 mM, inner/outer primer ratio 1:8, 64 °C, 20 min). The assay demonstrated a detection limit that was comparable to a real-time polymerase chain reaction and immunochromatographic assays, but with a markedly reduced turnaround time. No cross-reactivity was observed with non-PA pathogens, and reproducibility tests confirmed high stability. In addition, the reliability of the results was further verified using 60 standard bacterial strains, and the feasibility of the assay was validated with 2 real soil samples and 1 water sample. This LAMP method offers a simple, rapid, and sensitive tool for on-site detection of PA, with potential applications in clinical diagnostics and public health surveillance. Full article

African Swine Fever (ASF) control is severely limited by a diagnostic gap, as laboratory-based quantitative polymerase chain reaction (qPCR) is highly sensitive but slow, whereas field-deployable immunochromatographic assays (ICAs) are rapid but unreliable. To address this limitation, this study evaluated a novel, rapid isothermal assay, RNase hybridization-assisted amplification (RHAM), as a high-sensitivity, point-of-need diagnostic solution. This study compared the performance of RHAM and a conventional p72-based ICA against the qPCR reference standard using 106 diverse clinical field samples, including oral swabs, blood, serum, and organs, collected from suspected ASF cases in Thailand. The ICA exhibited markedly low diagnostic performance, achieving only 56.76% sensitivity and showing moderate agreement (κ = 0.421) with qPCR, highlighting the need for a more reliable alternative. In contrast, the RHAM assay achieved 94.59% sensitivity and 96.88% specificity, providing results rapidly within 35 min. This statistically superior performance (McNemar’s test, p < 0.0001) demonstrated almost perfect agreement (κ = 0.891) with the qPCR reference standard, missing only four samples with very high Ct values (>30). In conclusion, RHAM is a powerful, accurate, and field-deployable diagnostic tool that effectively bridges the diagnostic gap, offering qPCR-like sensitivity for the rapid containment of ASF outbreaks. Full article

Fumonisins are among the most prevalent mycotoxins in maize and maize-based products, posing significant food safety and public health risks due to their hepatotoxic, nephrotoxic, and potential carcinogenic effects. Given the strict regulatory limits set by the European Commission and Codex Alimentarius, the development of reliable, sensitive, and matrix–robust analytical methods remain a priority for routine monitoring in both food and feed systems. In this study, a reusable immuno-affinity purification methodology for the quantitative determination of fumonisin mycotoxins (FB1, FB2 and FB3) in foods and feeds (maize matrix) was developed. A single extraction protocol using 2% formic acid in water was employed, followed by cleanup with an immuno-affinity purification column and toxin elution by methanol/PBS (1:1, v/v). Detection and quantification of the mycotoxins was achieved by a normal phase ultra-high performance liquid chromatography coupled with electrospray ionisation triple quadrupole mass spectrometry (UHPLC/ESI-MS/MS). The chromatographic mobile phase utilised was a linear gradient of methanol/water containing 0.1% formic acid. The developed method has a limit of detection of 2.5 ng/g and a limit of quantification of 5 ng/g, all well below the European commission’s guidance values of 1000 ng/g for corn destined for human consumption and 800 ng/g for maize-based breakfast cereals and snacks. While the recovery rates of the method in this study ranged from 65–70% for the three fumonisin analogues in solutions, when tested in maize matrix, recoveries were markedly lower (~30%) due to pronounced matrix suppression. Good repeatability (standard deviation <10%) was achieved for all the fumonisin analogues. The developed method, although quick and effective in solvent systems, suffered limitations to its practical usage due to matrix suppression of the extracts derived from the immuno-affinity purification column, thus significantly reducing the application of the method in measuring fumonisin mycotoxins in food and feed samples. Overall, the method was effective in quantification of fumonisin mycotoxins in solvent solutions but not in food and feed matrices, thus necessitating further optimisation for practical usage. The performance of the developed method was compared to a commercial lateral flow immunochromatographic assay which proved to be better than the developed method in the quantification of toxins in food matrices, as the commercial lateral flow immunochromatographic assay outperformed the developed method in maize matrices. These findings highlight the need for matrix-based validation and further refinement of antibody stability to ensure robust application in regulatory monitoring of fumonisins using immunoaffinity purification methods. Full article

Background: Carbapenem-resistant Klebsiella pneumoniae (CRKp) is a critical global health threat due to its multidrug resistance, primarily driven by carbapenemase production. Rapid and accurate detection of carbapenemases is essential for effective treatment and infection control. This study evaluates the validity of the NG-Test CARBA 5, a rapid immunochromatographic assay, for detecting five major carbapenemases (KPC, NDM, VIM, IMP, OXA-48-like) in clinical CRKp isolates. Methods: Clinical isolates of CRKp were collected from various clinical specimens at the Military Medical Academy in Belgrade, Serbia, between January 2023 and October 2024. Detection of carbapenemases was performed using NG-Test CARBA 5, while PCR served as the reference method. Diagnostic performance was assessed by calculating sensitivity, specificity, and Cohen’s kappa coefficient. Results: Among 312 isolates, OXA-48-like was the most prevalent carbapenemase. NG-Test CARBA 5 showed high sensitivity (98.7%) and specificity (100%) overall, with excellent agreement for NDM (κ = 0.947), OXA-48-like (κ = 0.957), and KPC (κ = 0.978). However, it failed to detect VIM in five PCR-positive isolates, suggesting potential limitations. Conclusions: NG-Test CARBA 5 is a rapid and reliable tool for detecting major carbapenemases in CRKp, though its performance for VIM detection requires further investigation. This assay has the potential to improve clinical diagnostics and strengthen infection control in settings with high antimicrobial resistance. Full article

Brucella canis is a neglected zoonotic pathogen associated with canine reproductive disorders and emerging public health concerns. This study presents the first nationwide systematic review and meta-analysis of Brucella spp. in Portugal, integrated with a 13-year retrospective seroepidemiological investigation (2013–2025) of B. canis in dogs across mainland Portugal and Insular Autonomous Regions. Among 132 canine serum samples, a seropositivity of 23.48% was observed using an immunochromatographic assay confirmed by indirect immunofluorescence (IFAT). Significant associations were identified with seasonality (p < 0.001) and breed (p = 0.001), while sex and age were not statistically significant. Municipal-level analysis revealed marked heterogeneity, with Trofa showing the highest seropositivity (58.82%) and a pooled odds ratio of 11.28 (95% CI: 2.90–43.94; p < 0.001). In parallel, meta-analyses of published data estimated a pooled seroprevalence of 2.42% in animals (95% CI: 1.79–3.13) and 10.57% in humans (95% CI: 8.80–12.47), underscoring the broader burden of Brucella spp. exposure in Portugal. These findings suggest underdiagnosis of canine brucellosis and highlight the need for enhanced surveillance in high-risk breeds and regions. The study reinforces the importance of integrated One Health strategies to improve early detection, control, and prevention of B. canis infection in both veterinary and public health contexts. Full article

Gentamicin (GEN) is a broad-spectrum antibiotic widely used in livestock production, and its excessive residues in animal-derived foods pose potential health risks to consumers. However, conventional colloidal gold immunochromatographic assays (AuNPs-ICA) often suffer from low sensitivity and poor tolerance to sample matrices. Herein, a UiO-66-NH₂ framework decorated with gold nanoparticle (UiO-66-NH₂@Au)-based ICA was employed to construct an ICA platform for GEN detection, combining the optical advantages of AuNPs with the protective and stable octahedral framework of the Metal-organic framework (MOF) to enhance antibody stability under extreme conditions. The method achieved limits of detection for GEN of 0.1 µg/kg in four tested matrices, with recoveries of 80.1–112.0% and coefficients of variation below 11.7%. Compared to traditional AuNPs-ICA, the sensitivity was improved by up to 30-fold, the pH tolerance range was expanded from 6–8 to 4–10, and the organic solvent tolerance to organic solvents expanded up to 40%. Verification with 40 real samples demonstrated excellent correlation (R² > 0.99) with results from commercial ELISA kits. This UiO-66-NH₂@Au-ICA platform offers a new technical solution with high sensitivity, strong good anti-interference performance, and robustness for rapid field detection of GEN residues in products of animal origin and holds significant practical importance for advancing rapid food safety detection technologies. Full article

Ehrlichiosis and anaplasmosis are endemic to tropical and subtropical regions and pose significant zoonotic threats to both human and animal health. This study aimed to detect anti-Ehrlichia canis, anti-Borrelia burgdorferi, and anti-Anaplasma antibodies in dogs from the rural–urban area of Huamanga, Ayacucho. The cross-sectional survey was conducted at the Facultad de Ciencias Biológicas of the Universidad Nacional de San Cristóbal de Huamanga between May and August 2023. Samples were collected via venipuncture, and antibody detection was performed using the immunochromatographic assay Anigen Rapid CaniV-4 kit. Frequencies, percentages, and statistical analyses were conducted using the SPSS^® software package. A total of 107 samples from dogs in the Covadonga Human Settlement were analyzed, comprising 64 (59.8%) males and 43 (40.2%) females. The majority (78.5%) were from mixed-breed dogs, while other dogs breed included Schnauzers, Pekingese, and Pitbulls. Thirty positive samples were identified, with antibodies against Ehrlichia canis (15.9%), Anaplasma phagocytophilum/Anaplasma platys (3.7%), mixed infections of Ehrlichia canis and Anaplasma phagocytophilum/Anaplasma platys (6.5%), and Ehrlichia canis/Borrelia burgdorferi (1.9%) detected, as well as an association between vector exposure and the presence of Ehrlichia canis antibodies. These findings underscore the urgent need for the implementation of integrated control strategies and enhanced surveillance programs targeting tick-borne diseases in high-risk areas, along with targeted educational campaigns to promote responsible pet ownership and preventive measures. Full article

The spread of carbapenemase-producing Gram-negative bacteria poses a significant clinical challenge due to their association with severe Difficult-to-Treat nosocomial infections, as available therapies are drastically reduced. Rapid and accurate detection of carbapenemase-producing Gram-negative bacteria is critical for effective patient management, guiding appropriate antibiotic therapy, and implementing infection control measures to limit their dissemination within healthcare settings. Lateral flow immunoassays that detect the five main carbapenemases have become cornerstones in the fight against carbapenemase-producing Gram-negative bacteria. Carbapenemases evolve in response to antibiotic exposure, and therefore regular evaluation of these lateral flow immunoassays is crucial. Here, we have evaluated a novel assay, the KINVO assay (Medomics Medical Technology) and compared it to the Gold Standard of LFIAs for carbapenemase detection, the NG-TEST CARBA 5 assay (NG-Biotech) on a large panel of carbapenemase variants. The comparison between the two assays highlighted that both share key advantages such as rapidity and simplicity. However, NG-Test CARBA 5 demonstrated superior performance overall, particularly in accurately detecting IMP-type carbapenemases and the OXA-48 variant OXA-505. In contrast, the KINVO assay was more effective at detecting a broader range of KPC variants, including some that have lost carbapenem-hydrolyzing activity but gained resistance to ceftazidime/avibactam. If we consider these variants no longer as carbapenemases, and thus that they should not be detected, the NG-Test CARBA 5 performed better for KPC carbapenemase detection. Full article

Due to the severe hazard of aflatoxins (AFs) to humans, it is of great significance to detect the key aflatoxins, aflatoxin B₁ (AFB₁) and aflatoxin G₁ (AFG₁), in food and feed in simple, rapid, and semi-quantitative ways. The hybridoma clone 3A1 was prepared in this study, and anti-AFB₁ monoclonal antibody (mAb) with high specificity and affinity (9.38 × 10⁸ L/mol) from 3A1 was purified. The indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) demonstrated that the linear detection range for AFB₁ was 0.029–1.526 ng/mL with a limits of determination (LOD) of 0.023 ng/mL. A latex microsphere-based immunochromatographic test strip (LM-ICTS) was constructed based on 3A1, which showed that the strip could detect AFB₁ (LOD: lower than 1.79 ng/mL) and AFG₁ (LOD: lower than 8.08 ng/mL), and the linear detection ranges for AFB₁ and AFG₁ are 1.79–48.46 ng/mL and 8.08–107.40 ng/mL, respectively. The average recoveries of intra-assay and inter-assay for peanuts were (98.4 ± 4.7)% and (92.6 ± 7.6)%, and the average coefficient of variation (CVs) were 4.38% and 8.15%, respectively. For sunflower seeds, the intra-assay and inter-assay recoveries were (94.4 ± 7.2)% and (89.2 ± 4.3)%, and the average CVs were 6.6% and 4.9%, respectively. In summary, the developed LM-ICTS exhibited excellent sensitivity and specificity, which provided a rapidly stable on-site detection choice for AFB₁ and AFG₁ to contaminated agricultural samples, including grain and feed. Full article

The N-glycolylneuraminic acid (Neu5Gc), a major salivary acid molecule found on the cell surface of animals such as pigs, cows, and sheep, can be metabolically incorporated into the body through consumption of animal-derived foods like red meat. This leads to an immune response and chronic inflammation in individuals who do not naturally produce Neu5Gc, including humans and poultry, further increasing the risk of cancer. The trans-cleavage activity of Cas12a is activated by the recognition of the target aptamer by the crRNA, resulting in the cleavage of the dual-labeled probe. By combining this with immunochromatographic techniques, we established a chromatographic test strip assay that allows immediate on-site detection of Neu5Gc contamination in non-red meat samples devoid of Neu5Gc. Further optimization enabled specific detection within 25 min with a minimum detectable limit of 10 ng/mL. These analyses successfully detected the spiked samples and actual samples containing Neu5Gc. The developed lateral flow test strips based on aptamer-Cas12a can be utilized for detecting Neu5Gc contamination in non-red meat food products, animal bioproducts, and poultry feeds. Full article

Background: Carbapenem-resistant Enterobacterales infections are frequent in critically ill patients. Outbreaks caused by carbapenemase-producing Enterobacterales, in particular the New Delhi Metallo-beta-lactamase (NDM)-type carbapenemase-producing phenotype, are increasing in Italy. Unfortunately, the clinical impact of this new microorganism is still being defined, as well as the correlation between colonization and invasive infections. The aim of the study is to analyze factors related to the development of NDM infections in colonized patients and to evaluate their impact on patients’ outcome in high-complexity ICUs. Methods: All patients admitted to the General and Cardiac ICUs of ‘Città della Salute e della Scienza’ University Hospital in Turin (Italy) between January and August 2023 were enrolled. Microorganisms were examined by lateral flow immunochromatographic assays or molecular assays on weekly surveillance or clinically requested cultures. Antimicrobial susceptibility was determined by broth microdilution methods and interpreted according to EUCAST breakpoints. Results: Out of a total of 915 patients, 46 (5%) were positive for NDM-producing Enterobacterales and, among them, 13 (28%) developed an invasive infection. All patients were critical (SAPS II 40+/−13). The median times between ICU admission and colonization or infection were 6 and 16 days, respectively. Significant disparities emerged between colonized and infected patients regarding days of mechanical ventilation (1 vs. 28), ICU (7 vs. 39 days), and in-hospital (21 vs. 71 days) length of stay. Renal replacement treatment (OR 8.2461, p = 0.0173, 95% CI [1.3636–65.9114]) and surgery (OR 22.8747, p = 0.0149, CI95% [1.5986–1447.743]) seemed to impact the risk of developing infection. Six patients with invasive infection were treated with Cefiderocol and five with Ceftazidime/Avibactam and Aztreonam. In absence of early identification and appropriate treatment, patients may be at increased risk of colonization spread and potentially worse clinical outcomes. Conclusions: Early identification of the carbapenemase type is clinically relevant in critically ill patients with confirmed or suspected infection, as NDM production necessitates the use of specific agents for effective treatment. Full article

The Akabane virus (AKAV) is a significant member of the Orthobunyavirus genus, with its envelope glycoprotein Gc, focusing on its molecular structural features, immunoregulatory mechanisms, and application value in pathogen diagnosis and vaccine design. As a key structural protein of AKAV, Gc mediates virus adsorption and neutralizing antibody recognition through the N-terminal highly variable region (HVR), while the C-terminal conserved region (CR) dominates the membrane fusion process, and its glycosylation modification has a significant regulatory effect on protein function. In clinical diagnostics, serological assays based on Gc proteins (e.g., ELISA, immunochromatographic test strips) have been standardized; in vaccine development, the neutralizing epitope of Gc proteins has become a core target for subunit vaccine design. Follow-up studies were deeply needed to analyze the structure-function interaction mechanism of Gc proteins to provide theoretical support for the construction of a new type of AKAV prevention and control system. Full article

Nipah virus (NiV) and Hendra virus (HeV), which both belong to the genus henipavirus, are zoonotic pathogens that cause severe systemic, neurological, and/or respiratory disease in humans and a variety of mammals. Therefore, monitoring viral prevalence in natural reservoirs and rapidly diagnosing cases of henipavirus infection are critical to limiting the spread of these viruses. Current laboratory methods for detecting NiV and HeV include virus isolation, reverse transcription quantitative real-time PCR (RT-qPCR), and antigen detection via an enzyme-linked immunosorbent assay (ELISA), all of which require highly trained personnel and specialized equipment. Here, we describe the development of a point-of-care customized immunochromatographic lateral flow (ILF) assay that uses recombinant human ephrin B2 as a capture ligand on the test line and a NiV-specific monoclonal antibody (mAb) on the conjugate pad to detect NiV and HeV. The ILF assay detects NiV and HeV with a diagnostic specificity of 94.4% and has no cross-reactivity with other viruses. This rapid test may be suitable for field testing and in countries with limited laboratory resources. Full article

Background/Objectives: Since the World Health Organization (WHO) recommended HIV self-testing as an alternative to traditional facility-based testing in 2016, it has been increasingly adopted worldwide. This study aimed to evaluate the performance of the ConfiSign HIV Self-Test (GenBody Inc., Republic of Korea), a newly developed blood-based immunochromatographic assay for the qualitative detection of total antibodies (IgG and IgM) against HIV-1/HIV-2. Methods: The evaluation included four components: (1) retrospective analysis of 1400 archived serum samples (400 HIV-positive and 1000 HIV-negative samples), (2) prospective self-testing by 335 participants (112 HIV-positive participants and 223 individuals with an unknown HIV status, including healthy volunteers), (3) assessment using seroconversion panels and diverse HIV subtypes, and (4) analytical specificity testing for cross-reactivity and interference. The Elecsys HIV combi PT and Alinity I HIV Ag/Ab Combo assays were used as reference assays. Results: In retrospective testing, the ConfiSign HIV Self-Test achieved a positive percent agreement (PPA) of 100%, a negative percent agreement (NPA) of 99.2%, and a Cohen’s kappa value of 0.986, showing excellent agreement with the reference assays. In the prospective study, the test showed 100% sensitivity and specificity, with a low invalid result rate of 1.8%. All HIV-positive samples, including those with low signal-to-cutoff (S/Co) values in the Alinity I assay, were correctly identified. The test also reliably detected early seroconversion samples and accurately identified a broad range of HIV-1 subtypes (A, B, C, D, F, G, CRF01_AE, CRF02_AG, and group O) as well as HIV-2. No cross-reactivity or interference was observed with samples that were positive for hepatitis viruses, cytomegalovirus, Epstein–Barr virus, varicella zoster virus, influenza, HTLV-1, HTLV-2, or malaria. Conclusions: The ConfiSign HIV Self-Test demonstrated excellent sensitivity, specificity, and robustness across diverse clinical samples, supporting its reliability and practicality as a self-testing option for HIV-1/2 antibody detection. Full article