Search Results (226)

Reticuloendotheliosis virus (REV) is an immunosuppressive virus in poultry that can cause acute reticular neoplasms, chronic lymphoid tumors, stunting syndrome, and secondary infections. In many countries, the lack of effective vaccines has resulted in a high prevalence of REV infections and substantial economic losses. Enzyme-linked immunosorbent assay (ELISA)-based antibody detection is an important tool for monitoring the REV prevalence in poultry farms. ELISA coating antigens generally consist of either whole virus or viral protein; however, most commercially available REV antibody ELISA detection kits use whole virus as the coating antigen, which limits their applicability in certain diagnostic and research settings. In this study, the gp90 protein from a dominant REV strain was expressed and purified using 293F suspension cell eukaryotic expression system. Using recombinant gp90 protein as the coating antigen, an indirect ELISA for detecting gp90 antibodies (gp90-ELISA) was developed. After optimization, the optimal conditions were as follows: coating antigen concentration of 4 µg/mL with overnight incubation at 4 °C; blocking with 5% skim milk at 37 °C for 1.5 h; serum dilution of 1:200 with incubation at 37 °C for 45 min; secondary antibody dilution of 1:1000 with incubation at 37 °C for 30 min; and color development using TMB substrate at room temperature in the dark for 10 min. The cut-off value was defined as an OD₄₅₀ ≥ 0.22 for positive samples and < 0.22 for negative samples. The developed gp90-ELISA specifically detected REV-positive sera at a maximum serum dilution ratio of 1:3200. Intra- and inter-assay variation coefficients were ≤10%, indicating that the gp90-ELISA had good specificity, sensitivity, and reproducibility. Laboratory serum testing showed that the gp90-ELISA successfully detected sera from chickens immunized with the gp90 protein or infected with REV. Furthermore, analysis of clinical serum samples demonstrated 100% concordance between the gp90-ELISA results and a commercial whole-virus-coated ELISA kit. These results indicate that the gp90-ELISA is a reliable supplementary method to whole-virus-coated ELISA and has potential utility in disease surveillance and evaluation of immune responses. Full article

Background: Anti-N-methyl-D-aspartate IgM and IgA antibodies (NMDAR1-abs) are associated with unfavorable stroke outcomes and may be risk factors thereof. However, to utilize NMDAR1-abs serostatus for risk assessment in acute stroke, it is crucial to understand the robustness of serostatus during this phase. Therefore, we investigated the robustness of NMDAR1-abs serostatus and titer levels up to seven days after stroke. Methods: In this exploratory analysis of the multicenter STRAWINSKI trial (identifier: NCT01264549), patients with severe ischemic stroke (NIHSS ≥ 9) in the middle cerebral artery territory were included. The first blood sample was taken within 36 h and then daily from day two to seven after stroke. NMDAR1-abs immunoglobulin (Ig)A and IgM were assessed in serum using cell-based assays. We initially measured NMDAR1-abs in the total cohort on day 1. Subsequently, in samples from seropositive and matched seronegative patients, we measured NMDAR1-abs on each following day. Titer dilutions started from 1:10 up to 1:1000. Seropositivity was defined as any titer > 0. Results: Out of 171 patients (mean age = 76 [SD = 11], median NIHSS = 15 [IQR = 12–18]), 16 (9%) individuals were seropositive. Seropositive patients remained seropositive and matched seronegative participants remained seronegative over sequential measurements. Although titer levels remained largely unchanged, some patients showed fluctuating titers. Conclusions: The status of NMDAR1-abs seropositivity is stable during acute stroke, with little to no variation in titer levels. Full article

Serum tests are valuable sources of information for disease diagnosis. Conventional whole blood cell separation requires many processing steps, including centrifugation, fractionation, lysis, and dilution, and is therefore complex and time consuming. To address the need for the efficient separation of blood cells for on-chip rapid serum assays, we developed a microfluidic chip integrating inertial sorting and deterministic lateral displacement. This chip consists of a helical structure and a deterministic lateral displacement triangular microcolumn array for rapid and efficient separation of blood cells from whole blood samples. After separation, the supernatant is extracted at the exit for subsequent testing or directed to serum test units directly integrated in the chip. Here, the laminar flow and transport modules are coupled using finite element analysis for both multi-component and discrete-phase physical fields to simulate blood flow characteristics in the chip. The influences of flow rate and flux ratio on the sorting efficiency of blood cells were also discussed. Simulation results determined that the microfluidic chip designed in this research can achieve a cell sorting efficiency greater than 98% at suitable flow rates. Experimental results similarly achieved a high sorting effect of above 96%. Therefore, this blood cell sorting microfluidic chip shows strong potential for rapid serum testing applications and can be integrated as a stand-alone blood cell sorting module for various on-chip serum testing systems used to diagnose diseases. Full article

Spring viremia of carp (SVC) is a highly contagious disease that affects cyprinids, resulting in systemic hemorrhage, abdominal distension, exophthalmia, and high mortality in juveniles. This can lead to significant losses in the aquaculture industry. The World Organization for Animal Health (WOAH) recommends a two-step semi-nested reverse transcription polymerase chain reaction (RT-PCR) method for diagnosis. However, this method is labor-intensive, requires large reagent volumes, and is prone to carry-over contamination. Here, we evaluated the detection sensitivity of one-step semi-nested RT-PCR (combining RT and primary amplification in a single tube, followed by a second nested PCR step) against conventional two-step semi-nested RT-PCR. SVC virus (SVCV) subgroup Ia was tested using cell culture, RT-quantitative PCR, and one-step RT-PCR. The two-step semi-nested PCR method detected viral RNA up to a 10⁻² dilution, whereas one-step semi-nested RT-PCR detected it up to a 10⁻⁵ dilution, showing a 1000-fold improvement in sensitivity. Moreover, detection rates increased from 84.2% with two-step semi-nested RT-PCR to 91.7% with one-step semi-nested RT-PCR in fish tissue samples. One-step semi-nested RT-PCR reduces processing time, minimizes handling steps, and contamination risk, and enhances analytical sensitivity. This supports its adoption as a practical, high-throughput diagnostic tool for SVCV and consideration for future WOAH guidelines. Full article

In the present investigation, a novel dispersive liquid–liquid microextraction (DLLME) method was developed for the spectrophotometric determination of Hg²⁺. Fe(II) phthalocyanine (Fe(II)Pc) was employed as the sensor, chloroform (300 µL) as the extraction solvent, and ethanol (700 µL) as the dispersive solvent. Following the formation of the Hg²⁺:Fe(II)Pc complex, the sample was centrifuged at 1000 rpm for 2 min. The aqueous phase was discarded, and the extraction phase was diluted to 250 µL with methanol and transferred into a 250 µL quartz cell for spectrophotometric measurement at 276 nm. The method exhibited a linear range of 1–20 µg/L, with limits of detection (LOD) and quantification (LOQ) calculated as 1.44 µg/L and 4.80 µg/L, respectively. The enrichment factor was determined to be 105, and the optimum pH for the procedure was 2.0. Full article

Background/Objectives: Fungal colonization and biofilm formation on intrauterine device (IUD) strings are known to contribute to recurrent infections and decreased contraceptive efficacy. This study aims to develop a novel approach to prevent Candida reservoir and biofilm formation on IUD strings, thereby lowering the risk of IUD-associated vulvovaginal candidiasis (VVC). Methods: Cervicovaginal samples were collected from human cervix using a sterile cytobrush, avoiding microbial contamination. Cytological examination using the Papanicolaou method was performed to detect the presence of Candida. The antifungal effect of the essential oils (EOs) was determined by broth dilution and disk diffusion methods. Antifungal and biofilm inhibitory effects of Thymus zygis (Tz) EO-coated IUD strings were determined by agar diffusion and crystal violet binding assays, while fungal growth on the coated strings was assessed using Scanning Electron Microscopy (SEM) and Energy-Dispersive X-ray (EDX) analysis. Results: Tz EO exhibited significantly lower minimum inhibitory concentration (MIC ≤ 0.06 µL/mL) and minimum fungicidal concentration (MFC = 0.24 µL/mL) values compared to Melaleuca alternifolia (Ma) EO (MIC > 0.24 µL/mL, MFC = 1.95 µL/mL), along with larger zones of inhibition (ZOI) against both Candida albicans (110.0 ± 6.0 mm vs. 91.3 ± 7.0 mm) and Candida glabrata (84.0 ± 13.1 mm vs. 50.0 ± 9.2 mm), indicating a stronger antifungal potential. On IUD strings coated with 4% (40 μL/g) Tz EO in hypromellose ointment, the biofilm formation of both C. albicans and C. glabrata strains was inhibited by 58.9% and 66.7%, respectively, as confirmed by SEM and EDX. Conclusions: Tz EO-coated IUD strings effectively inhibit Candida growth, suggesting a promising natural strategy to reduce recurrent IUD-associated fungal infections. However, before these results can be translated to clinical practice, additional research is needed. Future investigations may encompass an extended number of Candida isolates, stability and release studies of the EO in relation to the formulation, toxicity to vaginal mucosa, epithelial cells and sperm motility, and the effect on vaginal microbiotia. Full article

Personalized, point-of-care testing of human tears is essential for ocular disease diagnosis, yet it is hampered by picomolar biomarker levels and microliter sample volumes. In this work, we developed an integrated, portable electrochemiluminescence (ECL)-based device for rapid and quantitative detection of tumor necrosis factor alpha (TNF-α), a pivotal inflammatory marker in ocular surface disease, with particular relevance to dry eye syndrome (DES). The device integrates a miniaturized electrochemical cell for ECL reactions and a compact silica photomultiplier for signal measurement. A vertical silica mesochannel (VSM)-coated ITO electrode is also integrated and further functionalized with TNF-α-specific aptamers. The VSM enables the enrichment of ECL luminophores, thus enabling further amplification of ECL signals and enhancing sensitivity. A wide linear range from 0.1 to 200 pg/mL was achieved using 10-fold dilution of 3 μL tear samples. Overall, this study provides a portable, highly sensitive platform for personalized analysis of TNF-α in tear fluid, enabling rapid point-of-care assessment of DES. Full article

Introduction: For pregnant women with a history of fetal and neonatal alloimmune thrombocytopenia (FNAIT) or hemolytic disease of the fetus and newborn (HDFN), prenatal intervention in subsequent pregnancies may be necessary to prevent complications for the fetus. A non-invasive prenatal diagnostic procedure (NIPD) is recommended for fetal blood group genotyping. RT-PCR is used for fetal RHD determination as a reliable screening method with high sensitivity and specificity. For other antigens with variants involving single-base substitutions, droplet digital PCR (ddPCR) and next-generation sequencing (NGS) are recommended to reduce the risk of false-negative results. Only NGS offers the possibility of determining the cell-free fetal DNA (cffDNA) fraction in maternal plasma by sequencing additional gene fragments in parallel, but no standard exists for assay validation. Material and Methods: A custom-made primer panel was designed to target the common platelet and red cell antigens involved in fetal red cell and platelet incompatibilities, as well as additional anonymous single-nucleotide polymorphism (SNP) targets for use as an internal control. Amplicon-based NGS was carried out using semiconductor sequencing. For HPA-1a (HPA*1A, ITGB3) and K (KEL*01.01, KEL) assay validation, the limit of detection (LOD) and limit of quantification (LOQ) were estimated, as were false-positive antithetic alleles, linearity, and inter-assay variation, using cell-free DNA (cfDNA) extracted from the blood samples of healthy blood donors. An additional analysis was performed using 23 diagnostic samples from 21 pregnant women. Results: Regression analysis of dilution series using HPA-1a- and K-positive cell-free plasma samples in antigen-negative donor plasma showed that recovery is definitely feasible up to an HPA*1A and KEL*01.01 allele frequency of 1%. Base calls of false-positive antithetic alleles were detected with a maximum of 0.25% using 21 healthy blood donors. The LOD was estimated to be 0.2057% (mean + 3 SD) for HPA*1A with a LOQ of 0.6298% (mean + 10 SD). For KEL*01.01, the LOD was 0.1706% (mean + 3 SD) and the LOQ was 0.5314% (mean + 10 SD). The analysis of 15 of 21 cases with diagnostic samples from pregnant women with neonatal blood available for confirmatory testing resulted in 100% concordant results. The fetal fraction of these samples was calculated with a median of 11.03% (95% CI: 8.89, 13.20). Conclusions: NGS for non-invasive fetal blood group genotyping is an accurate and reliable method. In-house validation of the used assays can be performed using healthy donors to determine the LOD, LOQ and sensitivity. The threshold for paternally inherited fetal HPA*1A and KEL*01.01 alleles could be set at 1% (i.e., 2% fetal fraction) to obtain reliable test results. Internal controls for assessing the fetal fraction are essential to avoid false-negative test results. Full article

This study presents a green, single-step method for synthesizing nanocomposites based on reduced graphene oxide (RGO) and gold nanoparticles (AuNPs), using sodium citrate as a mild reducing and stabilizing agent. AuNPs were generated from chloroauric acid (HAuCl₄) directly on the surface of graphene oxide (GO), which was simultaneously reduced to RGO. Structural characterization via Transmission Electron Microscopy (TEM), High Resolution TEM (HRTEM) and Selected Area Electron Diffraction (SAED) confirms spherical AuNPs (10–60 nm) distributed on RGO sheets, with indications of nanoparticle aggregation. Dynamic Light Scattering (DLS) and zeta potential analysis support these findings, suggesting colloidal instability with higher RGO content. Biological evaluation demonstrates dose-dependent cytotoxicity in HaCaT keratinocytes, with IC₅₀ values (half maximal inhibitory concentration) decreasing as RGO content is increased. At moderate dilutions (1–25 µL/100 µL), the composites show acceptable cell viability (>70%). Antibacterial assays reveal strong synergistic effects against Escherichia coli, Staphylococcus aureus, and Bacillus subtilis, with sample RGO/Au 0.500/0.175 g/L showing complete E. coli inhibition at low Au content (0.175 g/L). The composite retained activity even in protein-rich media, suggesting potential for antimicrobial applications. These findings highlight the potential of RGO/AuNPs composites as multifunctional materials for biomedical uses, particularly in antimicrobial coatings and targeted therapeutic strategies. Full article

Access to safe water remains a vital public health challenge, especially in low- and middle-income countries like Colombia, where untreated sources lead to severe diarrheal diseases in children under five. Escherichia coli (E. coli), a key indicator of fecal contamination, is often detected using culture-based methods that are time-consuming and rely on specialized infrastructure. To overcome these limitations, we developed an aptamer-based isolation system targeting environmental E. coli. Aptamers were obtained using a Cell-SELEX protocol, and after six enrichment rounds, two candidates—APT-EC-1 and its truncated version APT-EC-MUT—were synthesized and attached to carboxyl-functionalized magnetic nanoparticles (MNP-COOH). Both complexes demonstrated a strong binding affinity and high specificity, successfully isolating E. coli from environmental and ATCC reference strains in the laboratory. Sensitivity tests detected E. coli at dilutions up to 1:10,000, showing reliable performance. In early in-field testing with environmental water samples, APT-EC-1 consistently identified E. coli colonies, while APT-EC-MUT struggled with low bacterial levels, illustrating performance differences. These findings demonstrate the promise of aptamer-functionalized MNPs as the basis for quick, affordable, and portable biosensors for water quality testing, especially in resource-scarce areas. Future efforts will add colorimetric or electrochemical readouts to allow real-time, on-site detection of fecal contamination. Full article

Background: Since liquid-based cytology (LBC) has replaced the conventional Papanicolaou test in cervical cancer screening programs, pre-analytical procedures—particularly the choice of LBC collection media—have become crucial to ensure the accuracy of high-risk (HR) HPV DNA testing. This study aims to evaluate whether the newly developed CytoPath^® LBC medium can serve as a reliable alternative to standard solutions. Methods: This study exploited cell lines to evaluate the stability, integrity, and recovery rate of genomic DNA at different fixation time points (1, 7, 14 and 40 days) and serial dilutions (1:5, 1:10 and 1:20) extracted from cell lines. These samples have also undergone quantitative Real-Time PCR (qPCR) based HR-HPV test to assess the relative performance of the new preservative solution in detecting viral DNA with respect to the standard reference. Results: Cervical cell lines preserved in both media demonstrated consistent DNA stability over time. DNA yields were comparable between the two media. Notably, the DNA Integrity Number (DIN) was higher in samples fixed with the CytoPath^® solution. HR-HPV detection by qPCR showed equivalent performance, regardless of the fixative used. Conclusions: The CytoPath^® fixative solution represents a valid alternative to standard preservation media, offering improved DNA integrity while maintaining equivalent performance in HR-HPV qPCR testing. Full article

Digital PCR, as a nucleic acid absolute quantification method at the single-molecule level, has been widely applied in early cancer screening, single-cell analysis, and other biomedical fields. However, existing digital PCR methods still suffer from high costs, complex operations, and low detection dynamic range, which limit their applications. In the study, we developed a microfluidic chip-based digital PCR with a high-density vertical structure using PDMS (polydimethylsiloxane) flexible material. The chip features a three-layer structure of glass–PDMS–glass, with the PDMS structural layer containing 30,000 reaction chambers, each with a volume of 0.713 nL. This vertical-structured chip can increase the total volume and the total number of chambers by 50% without changing the chip area and chamber volume, thereby significantly enhancing dynamic range and sensitivity of the chip detection. This chip is theoretically capable of achieving a nucleic acid detection dynamic range close to 10⁵. Moreover, the digital PCR quantitative detection results of five different concentrations of serially diluted KRAS plasmid DNA templates using this chip also validated the accuracy and reliability of the nucleic acid quantitative detection results. The vertical-structured digital PCR chip, with its simple manufacturing process, uniform and stable sample partitioning, wide detection dynamic range, and low cost, will promote the widespread application of digital PCR. Full article

Recently, essential rose oils and rose products have gained increasing importance in both the cosmetic and food industries, as well as in the composition of medicinal products. We investigated the in vitro antiviral activity of essential oil and floral water from Rosa damascena Mill against herpes simplex virus type 1 (HSV-1) infection in rabbit retinal cells (RRCs). The composition of the main chemical components in the rose essential oil was determined by means of gas chromatographic analysis. The effect on the viral replication cycle was determined using the cytopathic effect (CPE) inhibition assay. The virucidal activity, the effect on the adsorption stage of the virus to the host cell, and the protective effect on healthy cells were evaluated using the endpoint dilution method. The effects were determined as deviation in the viral titer, Δlg, for the treated cells from the one for the untreated viral control. The identified main active components of rose oil are geraniol (28.73%), citronellol (21.50%), nonadecane (13.13%), nerol (5.51%), heneicosane (4.87%), nonadecene (3.93), heptadecane (2.29), farnesol (2.11%), tricosane (1.29%), eicosane (1.01%), and eugenol (0.85%). The results demonstrated that both rose products do not have a significant effect on the virus replication but directly affect the viral particles and reduce the viral titer by Δlg = 3.25 for floral water and by Δlg = 3.0 for essential oil. Significant inhibition of the viral adsorption stage was also observed, leading to a decrease in the viral titers by Δlg = 2.25 for floral water and by Δlg = 2.0 for essential oil. When pretreating healthy cells with rose products, both samples significantly protected them from subsequent infection with HSV-1. This protective effect was more pronounced for the oil (Δlg = 2.5) compared to the one for the floral water (Δlg = 2.0). We used the in silico molecular docking method to gain insight into the mechanism of hindrance of viral adsorption by the main rose oil compounds (geraniol, citronellol, nerol). These components targeted the HSV-1 gD interaction surface with nectin-1 and HVEM (Herpesvirus Entry Mediator) host cell receptors, at N-, C-ends, and N-end, respectively. These findings could provide a structural framework for further development of anti-HSV-1 therapeutics. Full article

New stable three-dimensional hydrogels were obtained in an inert gas atmosphere in light in an aqueous dispersion of the main components: cod collagen, methyl methacrylate, polyethylene glycol, RbTe_1.5W_0.5O₆ complex oxide, and modifying additives. The analysis of the new hydrogels’ cytotoxicity using the MTT assay showed that the cytotoxicity of the sample extracts was observed in a number of examples, but was decreased with increasing dilution of the extracts. The decrease in cell viability at high concentrations of the extract is likely caused by a decrease in the number of specific components of the complete culture medium used to produce extracts. It is related to the well-known adsorption of medium proteins by the gel component, high-molecular compounds included in the matrix. The stimulating effect of the substances included in its composition was observed with a significant dilution of the extract, i.e., the proliferative activity of the cells increased. The extract of the hydrogel hydrolysate sample and all its dilutions did not show cytotoxicity in the MTT assay examples. It determines the prospect of its use on the wound surface, since hydrogel destruction occurs under the action of body enzymes. The new hydrogel is a promising material for creating wound coverings or scaffolds. Full article

Background: An effective vaccine against Nipah virus (NiV) is crucial due to its high fatality rate and recurrent outbreaks in South and Southeast Asia. Vaccine development is challenged by the lack of validated accessible neutralization assays, as virus culture requires BSL-4 facilities, restricting implementation in resource-limited settings. To address this, we standardized and validated a pseudotyped virus neutralization assay (PNA) for assessing NiV-neutralizing antibodies in BSL-2 laboratories. Methods: The NiV-PNA was validated following international regulatory standards, using a replication-defective recombinant Vesicular stomatitis virus (rVSV) backbone dependent pseudotyped virus. Assessments included sensitivity, specificity, dilutional linearity, relative accuracy, precision, and robustness. The assay was calibrated using the WHO International Standard for anti-NiV antibodies and characterized reference sera to ensure reliable performance. Findings: Preliminary evaluation of the developed NiV-PNA showed 100% sensitivity and specificity across 10 serum samples (5 positive, 5 negative), with a positive correlation to a calibrated reference assay (R² = 0.8461). Dilutional linearity (R² = 0.9940) and accuracy (98.18%) were confirmed across the analytical titer range of 11-1728 IU/mL. The assay also exhibited high precision, with intra-assay and intermediate precision geometric coefficients of variation of 6.66% and 15.63%, respectively. Robustness testing demonstrated minimal variation across different pseudotyped virus lots, incubation times, and cell counts. Conclusions: The validated NiV-PNA is a reproducible and scalable assay platform for quantifying NiV neutralizing antibodies, offering a safer alternative to virus culture. Its validation and integration into the CEPI Centralized Laboratory Network will enhance global capacity for vaccine evaluation and outbreak preparedness. Full article