The prepared Ag-decorated ZFO NPs were characterized via XRD, SEM, EDX, elemental mapping, and FTIR analysis. The antibacterial potential was tested as ZOI and MIC, while antibiofilm activity was estimated by the tube method. The growth curve assay, the effect of UV on the antimicrobial activity, and cell membrane leakage were evaluated, and the antibacterial reaction mechanism was investigated by SEM/EDX analysis. The XRD and FTIR results confirmed the successful preparation of Ag-decorated ZFO NPs. Antibacterial results revealed that the most potent decorated sample was Ag0.75@ZFO NPs, recording the most significant inhibition zone against Staphylococcus vitulinus (24.67 ± 0.577 mm) and low MIC (0.097 μg mL−1) against S. vitulinus. The antibiofilm activity of Ag0.75@ZFO NPs was the highest, recorded as 97.3% for S. aureus and 95.25% for Enterococcus columbae. In the case of UV exposure, bacterial growth reached the lowest grade. Finally, it was seen that the amount of cellular protein released from bacterial cells is directly proportional to the concentration of Ag0.75@ZFO NPs, which clearly explains the formation of pits in the cell membrane. The synthesized nanocomposites may find an application after mixing with operating room paints to reduce the harmful effect of pathogenic microbes and, therefore, eliminate bacterial contamination.This work aimed at the gamma irradiation-assisted synthesis of silver (Ag)-decorated ZnFe 2O4 (ZFO) ferrite nanoparticles (NPs), which were tested for their antibacterial and antibiofilm activities against some pathogenic bacteria from medical operating room surfaces.This work aimed at the gamma irradiation-assisted synthesis of silver (Ag)-decorated ZnFe Bacterial disease affects sensitive patients in the clinical environment in different ways.2 Pathogenic bacteria have been identified to remain on inanimate ‘touch’ surfaces for long periods.3 Touch surfaces usually located in hospital rooms, such as bed tracks, chairs, door handles, and dispensers , are contaminated with some pathogenic bacteria like methicillin-resistant Staphylococcus aureus.4 One of the promising approaches to manage pathogenic bacteria is the application of nanoparticle (NP) therapeutics and nanostructured-based coating materials for controlling bacterial contamination in some medical devices.5A hospital-acquired infection, also identified as a nosocomial infection, is an issue of concern for clinical or other health care equipment.6 A medical laboratory must examine and suggest the antimicrobial tests and agents that are most suitable for bacterial isolation and the site of bacterial infection as a good quality control model.7Owing to the current attraction to find new antimicrobial products that have specific features like the ability to fight multidrug-resistant bacteria, it is essential to improve the successful methods used to screen and quantify the antimicrobial impact of the treatments regarding their applications in human health, agriculture, and the environment.8 The ability of S. aureus to produce biofilms has been wholly related to the production of polysaccharide intracellular adhesion and adhesions termed microbial surface components recognizing adhesive molecule matrix, which become involved as primary agents in biofilm development.9 In current times, the application of inorganic antimicrobial agents has attracted much consideration to control the biofilms produced by pathogenic bacteria.10 Inorganic antimicrobial composites, as compared to their organic equivalents, possess definite benefits such as being more safe and stable at high temperatures.10Biofilms are the usual predominant life form of bacteria in all types of environments, either natural or artificial. Biofilms can grow on a wide variety of surfaces. Biofilms have been associated with food spoilage, water pollution, and infectious diseases.11 Zinc ferrite  is an excellent magnetic material with interesting properties, and is notable for its extraordinary optical, magnetic, and electrical features. Besides, the controllable nature of the ZFO structure can enhance the nanostructure, which leads to superparamagnetic materials.12–14 ZFO NPs have been broadly utilized in numerous areas such as biomedicine,15 energy storage,16 gas sensors,17 drug delivery,18 antimicrobial agents,19 and water remediation.20Nanotechnology is one of the increasingly active topics of research in modern material sciences; consequently, metal NPs are of vital concern regarding their physicochemical properties and particularly optoelectronic features with regard to many areas such as drug distribution, electronic sensing, and photocatalysis.21 water purification,22 medicine,23 and biotechnology.24 Amongst NPs applied so far, silver (Ag) NPs have been notably examined for their unique features such as antibacterial,25 antiviral,26 antifungal,27 anti-inflammatory,28 and anticancer29,30 activities.On the other hand, metal NPs have extraordinary usage in various fields, like electronics,31 Ag NPs are now commonly applied nanomaterials in the healthcare area, with global production yearly estimated to be 500 tons.32Many studies have shown the effectiveness of Ag NPs as a therapeutic agent for treating infectious diseases, and they have also been mixed inside clothes during production for application as an antimicrobial agent.33,34Radiation-induced synthetic methods and especially those that allow the interaction of gamma rays with solutions may have a fantastic potential to fight with approved plans to increase the purposes in terms of synthesis, control in the size, shape, eco-friendly of starting materials, and decreases the production of the toxic materials, overall these benefits are eco-friendly.Overall, this paper reports, for the first time, the gamma irradiation-assisted synthesis of Ag-decorated ZFO NPs (Ag@ZFO). Afterwards, the pure ZFO NPs and Ag-decorated ZFO NPs were tested against pathogenic strains from medical operating room surfaces . The synthesized Ag-decorated ZFO NPs were applied as antibacterial and antibiofilm agents against pathogenic bacteria. So, we assume that the synthesized samples could be used for various purposes in biomedical and industrial applications.2.2.1.3)3·9H2O (98.0%), ZnSO4·7H2O (98%), and AgNO3 (99%) were chosen as precursor materials for Fe, Zn, and Ag, respectively. Also, citric acid  and ethylene glycol  were used in the synthesis of the pure ZFO NPs via a sol–gel method.35–39Fe(NO2.2.3)3·9H2O and ZnSO4·7H2O in their stoichiometric ratio in C6H8O7 and 10 ml of C2H6O2 was obtained via dissolving in deionized water. Further, the resulting solution was heated at 300 °C to initiate gel formation, and then the resulting gel was dried and ground employing agate mortar and pestle to obtain pure ZFO NPs. Furthermore, 5 g of pure ZFO NPs was mixed with different weight ratios of AgNO3 . The resulting suspension solutions were integrated under magnetic stirring for 30 min. The suspension mixture was subjected to a sonication process for 60 min.An aqueous solution of starting precursors Fe, Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD) technique, and scanning electron microscopy (SEM).40,41The pure ZFO NPs and Ag-decorated ZFO NPs were characterized 2.5.2.5.1.36 They were identified as Staphylococcus aureus, Staphylococcus sciuri, Staphylococcus lentus, Staphylococcus vitulinus, and Enterococcus columbae, which were kindly supplied from Drug Microbiology Lab., NCRRT, Cairo, Egypt. For further use, all the isolated and tested bacterial strains were kept on nutrient agar slants at 4 °C. The well diffusion technique was employed to evaluate the effects of pure ZFO NPs and Ag@ZFO NPs with different weight ratios of AgNO3  on the tested pathogens, according to El-Batal et al.42Five pathogenic bacterial strains were isolated from medical operating room surfaces  and identified as mentioned in our published paper.8 CFU ml−1. Bacteria were inoculated on a nutrient agar medium, and 6.0 mm wells were slashed on every nutrient agar plate surface, filled by 100 μl of the tested treatments. Levofloxacin was used as a positive control. All the experiments were performed in triplicate, and the plates were incubated at 37 °C for 24 h. The lack of colonies in the microbial growth was represented as a zone of inhibition (ZOI) measured in mm.To study the antibacterial influence of pure ZFO NPs and Ag@ZFO NPs using the well diffusion method, a bacterial suspension was prepared and adjusted to standard 0.5 McFarland concentration equal to (1–2) × 102.5.2.et al.43 The overnight examined cultures were incubated for 2.0 h at 37 °C. After that, the inoculum of the tested bacteria was adjusted to 0.5 McFarland concentration. A total volume of 100 μl of McFarland's bacterial suspension was inoculated into nutrient agar medium plates. ZFO NPs and Ag@ZFO NPs were serially diluted two-fold with sterile distilled water to different concentrations (μg ml−1) then dipped individually in 6.0 mm wells made on the surface of the inoculated agar plates. The plates had incubated at 37 °C for 24 h, and the zone of inhibition was evaluated in mm. MIC was defined as the minimum concentration of ZFO NPs and Ag@ZFO NPs that will inhibit the apparent growth of bacteria after overnight incubation.The MIC of pure ZFO NPs and Ag@ZFO NPs against the tested bacterial strains was estimated using the well diffusion method according to Roy 2.6.0.75@ZFO NPs  were investigated for their antibiofilm activity against the bacterial test strains by the tubes method as defined by Elbasuney et al.44 Test tubes containing 5.0 ml of nutrient broth were inoculated with 10 μl of 0.5 McFarland (1 × 108 CFU ml−1) of the examined bacteria. Furthermore, 0.5 ml of ZFO NPs and Ag0.75@ZFO NPs was incorporated into the test tubes, while the same volume of water was added to the control tubes. The samples were incubated at 37 °C for 24 h. After the incubation, the media in the sample tubes were eliminated. The tubes were treated with phosphate buffer saline (PBS), pH 7, and finally dehydrated. Sodium acetate (5.0 ml) at a concentration of 3.0% was used for 10 min for stabilizing the adherent bacteria followed by washing with deionized water. The bacterial biofilms were stained with crystal violet (0.1%) for 15 min and then rinsed with deionized water. Finally, 2.0 ml of ethanol was added to dissolve the crystal violet. The biofilms were semi-quantitatively estimated using a UV-visible spectrophotometer at 570 nm. The percentage of inhibition was evaluated using et al.:45c is the absorbance of the control sample (without treatment) and ODt the absorbance of the treated samples.ZFO NPs and Ag2.7.0.75@ZFO NPs on the growth of S. vitulinus was estimated by growth curve method according to Huang et al.46 A bacterial suspension was fixed to 0.5 McFarland (1 × 108 CFU ml−1) in 5 ml of nutrient broth tubes, then 0.5 ml of ZFO NPs or Ag0.75@ZFO NPs was added individually to each of the tested tubes. The absorbance of the bacterial growth after treatment was assessed every two hours up to 24 h at a wavelength of 600 nm. The mean of duplicate readings was plotted against the time intervals to obtain the standard growth curve.The effect of ZFO NPs and Ag2.8.0.75@ZFO NPs without and with UV illumination was assessed against the test pathogenic microbe (S. vitulinus) using the optical density method.47 Bacterial cultures incubated for two hours were adjusted to standard 0.5 McFarland (1 × 108 CFU ml−1). 100 μl of ZFO NPs or Ag0.75@ZFO NPs was added into the tubes. The tubes were grouped into two conditions: tubes with non-illuminated ZFO and Ag0.75@ZnFe2O4 NPs, and tubes with NPs exposed to UV light for 0, 15, 30, 45, 60, and 75 min with an intensity of 6.9 mW cm−2 incident on the samples at 37 °C. The turbidity of the medium was measured at 600 nm. The inhibition percentage of the tested bacterial pathogens was calculated by The antimicrobial activity of ZFO NPs and Ag2.9.8 CFU ml−1) and (100 μl) inoculated in 10 ml of nutrient broth including well-sonicated Ag0.75@ZFO NPs . Ag0.75@ZFO NPs-free broth inoculated with culture was adopted as the control. All the treated solutions were incubated for 5 h at 36 °C and then centrifuged at 5000 rpm for 10 min.48 For each individual sample, 100 μl supernatant was mixed with 1 ml of Bradford reagent. Optical density was estimated at 595 nm following 10 min of incubation (in the dark).48Fresh 18 h bacterial culture was fixed at 0.5 McFarland (1 × 102.10.S. vitulinus) were washed with PBS and fixed with 3.0% glutaraldehyde. Subsequently, the preserved bacterial cells were washed repeatedly with PBS and washed with ethanol for 15.0 min at 25 °C before dehydration. Finally, the bacterial cells were installed and fixed over aluminium stumps for starting the imaging.47 The treated and untreated bacterial cells' morphological and exterior features with the tested Ag0.75@ZFO NPs were examined using SEM imaging. The total elemental analyses of the tested bacterial cells were carried out by EDX.49The examined bacterial cells (2.11.P < 0.05) and determined to be Duncan's multiple ranges investigates and the least significant difference summary.50 The results and data were checked and tested by SPSS software version 15.The statistical analyses of our results were performed using one-way ANOVA (at 3.3.1.Fd3m space group (JCPDS card no. 74-2397 and 89-100).13,51–53 Besides, the XRD patterns confirm the presence of Ag in the Ag-decorated ZFO NPs samples. Four characteristic diffraction peaks were seen at 2θ = 38.32°, 44.58°, 64.51°, and 77.64° and were categorized as the cubic phase of Ag (JCPDS no. 04-0783).54,55 Further, the intensity of Ag peaks increases with an increase in Ag coating ratio, as presented in θ = 35.4° was shifted to higher 2θ for the Ag0.25@ZFO sample and then moved to the lower value with increasing ratio of Ag ions. This behaviour means that the lattice constant (aexp.) was decreased for the Ag0.25@ZFO NPs sample and increased with an increase in the proportion of Ag ions. The lattice constants of pure ZFO and Ag@ZFO NPs samples ranged from 8.2967 Å to 8.4373 Å, as shown in 3+, which has a smaller radius (0.64 Å) than Fe2+ ion (0.74 Å). This process can be ascribed to the interaction of γ-radiation with the outer electrons of the iron contents in the spinel ZFO lattice (γ + Fe2+ ↔ Fe3+ + e−). The reduction in cell volume, on a large scale, can have a noticeable impact on the Ag@ZFO NPs crystallite size.56–58The XRD patterns of pure ZFO NPs, and Ag-decorated ZFO NPs with different Ag NPs ratios are illustrated in via the Scherrer equation  at the expense of disordered grain boundary regions.60Further, the crystallite sizes of pure ZFO and Ag-decorated ZFO NPs samples were estimated tion see , and wer61,62The hopping distance of ions between the A and B sites of pure ZFO NPs and Ag@ZFO NPs samples was determined after applying 0.25@ZFO NPs and then increased with Ag ratio, which was well matched with the behaviour of the lattice constant.61–63 Also, Gingasu et al.64 reported the green synthesis of Ag@CoFe2O4 (Ag@CFO) NPs. They observed that XRD patterns of Ag@CFO showed the formation of CFO and Ag NPs. Further, they studied the effect of annealing temperature on crystallinity. Besides, they found that the (aexp.) values varied between 8.364 Å and 8.378 Å. Also, the crystallite size decreased from 15.8 nm to 14.8 nm. Kooti et al.65 synthesized Ag@CFO NPs via the combustion technique. The XRD patterns confirmed the deposition of Ag NPs on the surface of CFO NPs. Interestingly, the intensity of diffraction peaks for pristine CFO NPs is higher than those for Ag@CFO NPs, which is ascribed to the Ag NPs coating on the CFO surface.It is evident from 3.2.1 and υ2) resulting from the stretching vibration of tetrahedral groups (A-site) and octahedral groups (B-site), respectively.66–68 The peaks around 1105 cm−1 are attributed to the Fe–M ferrite system. Also, the observed bands at ∼2366 cm−1 and 1455 cm−1 were ascribed to H–O–H stretching, interpreted as the presence of free (or absorbed) water.69 Further, the bands detected at 1553 cm−1 correspond to C–H stretching while the peak around 1645 cm−1 corresponds to C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O stretching vibrations.70,71 The intensity of the absorption bands was reduced after coating the ZFO NPs with Ag NPs. Also, the increase in crystallite size after gamma irradiation led to migration of Zn2+ from A-site to B-site and an equal transfer of Fe3+ from B-site to A-site.60 This action leads to the appearance of a shift in the position of the absorption bands, as illustrated in The FTIR spectra of pure ZFO NPs and Ag@ZFO NPs samples are given in 20,72 The elemental composition of pure ZFO NPs was analyzed by EDX  exhibit antibacterial activity against the tested pathogenic bacteria. Also, the antibacterial activity increases with the treatments containing Ag, with the inhibition effect becoming greater on increasing the concentration of Ag. Therefore, Ag0.75@ZFO NPs recorded the most significant zone of inhibition against the bacteria strains, with S. vitulinus being most affected by Ag0.75@ZFO NPs treatment (24.67 ± 0.577 mm).The well diffusion test was carried out to assess the antibacterial action of ZFO NPs and Ag@ZFO NPs, where levofloxacin was used as a positive control, against 3.3.2.−1 using a well diffusion technique, and the results are reported in −1 against S. sciuri. In agreement with the well diffusion assay results, Ag@ZFO NPs were a more efficient antimicrobial agent than ZFO NPs. The MIC values for the three tested Ag concentrations were less than those for ZFO NPs. At the same time, Ag0.75@ZFO NPs recorded the lowest MIC. For Ag0.75@ZFO NPs, the MIC was less against S. vitulinus (0.097 μg ml−1). Ag0.75@ZFO NPs have dual behaviour, acting as bacteriostatic at lower concentration and, on the other hand, behaving as bactericidal at higher concentration. The complete inhibition of bacteria occurred by raising the concentration of Ag0.75@ZFO NPs.The MIC analyses of pure ZFO NPs and Ag@ZFO NPs were conducted. This experiment was conducted on ZFO NPs, and Ag@ZFO NPs at different concentrations of Ag as μg ml3.4.0.75@ZFO NPs were evaluated for their ability to inhibit biofilm formation of the tested bacterial strains. 0.5 ml of pure ZFO NPs or Ag0.75@ZFO NPs was incorporated into test tubes containing 5.0 ml of nutrient broth inoculated with 10 μl of 0.5 McFarland (1 × 108 CFU ml−1) of the examined bacteria. At the same time, the equivalent volume of water was added to the control tubes. It is clearly shown in 0.75@ZFO NPs were more efficient in inhibiting the biofilms of all the tested bacteria compared with pure ZFO NPs. The antibiofilm activity of Ag0.75@ZFO NPs was the highest, registering at 97.3% for S. aureus and 95.25% for E. columbae. Since the antimicrobial strength of NPs dramatically depends on the particle size, the smaller dimensions of Ag0.75@ZFO NPs provide potent antibiofilm activity. It has been recognized that biofilms are a common infection cause, and about 80% of bacterial infections in the world are connected with biofilms.76 Several mechanisms describe how the biofilm is manufactured on a surface, related to genes responsible for the adhesion program and the generation of extracellular polymeric substance. Environmental requirements may also influence biofilm development.77 The environmental requirements and expression of particular genes produced by adhesion may theoretically control biofilms' metabolic action.78 The low cellular metabolism protects the antimicrobial factors that progress through bacterial growth.79ZFO NPs and Ag3.5.0.75@ZFO NPs on the growth of S. vitulinus is shown in S. vitulinus in the control sample occurred rapidly, with the highest optical density values at λ = 600 nm (OD600) reaching about 2.21 nm. In contrast, the OD600 values of the ZFO NPs and Ag0.75@ZFO NPs were lower, indicating the inhibition effect on the growth of S. vitulinus. Ag0.75@ZFO NPs exert an additional suppressing influence compared with pure ZFO NPs that may be explained by the high antibacterial activity of Ag, as reported previously by several researchers.80–82The effect of pure ZFO NPs and Ag80,81 The ZFO NPs-generated ROS cause protein oxidation, DNA damage, and lipid peroxidation that can kill bacteria without affecting the non-bacterial cells.82 Also, the bacterial cell membrane has a negative charge, while the metal ions released from ZFO NPs (Zn2+ and Fe3+) have a positive charge. So, they get in direct connection leading to damage to DNA replication, denaturation of proteins, and death of bacteria.83On the surface of the NPs, the photogeneration of reactive oxygen species (ROS) has been reported in previous research.84 An additional potential reason can be the size, shape, and surface charge of the ZFO NPs, which could be more favorable for contact with Gram-positive bacteria. Previously, NPs like oleoyl-chitosan NPs have been recorded as changing the membrane permeability, damaging the S. aureus cell membrane.85 Fei et al.86 also found that Ag NPs clusters crinkled and pierced the bacterial membrane, producing a significant leakage of cytoplasmic constituents and ultimately inducing bacterial death. The Gram-positive bacteria have essentially too thick peptidoglycans with a cell wall of ∼80 nm, which functions as a barrier protecting molecules such as proteins from quickly leaking out following the disruption of the cell membrane.87 Xu et al.88 showed that ZFO NPs, after irradiation for 80 min, ruptured the cell membrane of Escherichia coli, meaning that disinfection was completed. The ZFO NPs exhibited antibacterial activity against various bacterial strains, S. aureus, Pseudomonas aeruginosa, E. coli and Bacillus subtilis.89The higher sensitivity of the Gram-positive bacteria to the NPs may be described as a result of the lower stiffness of the cell membrane.3.6.S. vitulinus is more sensitive to UV light, increasing its sensitivity when the time of exposure was prolonged. The exposure period of 0 to 75 min with 15 min time intervals has an impact on the growth of S. vitulinus. The growth of S. vitulinus was successfully affected by the treatment of Ag0.75@ZFO NPs compared with the untreated control. With UV exposure, the bacterial growth reached the lowest grade due to the activation by UV illumination. Because of that, 6 min of UV exposure was selected to maximize the potential for photoactivation of Ag0.75@ZFO NPs.The results presented in i.e. NB and MRS). As presented in 600 values for NB and MRS broths. A study of the impact of UV light on the various microorganisms applied in this research was conducted in the second case to distinguish whether the exposure time of UV light on the bacteria affected their growth. Ag0.75@ZFO NPs are a potent disinfectant once stimulated by UV light. In water and air conditions, NPs absorb photons, ending in the development of active hydroxyl (.OH) and additional ROS  and in the appearance of O2 and H2O.90 The ROS generated, especially H2O2, can cross cell membranes and create active oxidative hydroxyl species in a reaction inside bacterial cells that eventually leads to disinfection.80,81Also, it is essential to show that UV light did not impact the turbidity of the culture broths used in the research (3.7.91 From S. vitulinus) is directly proportional to the concentration of Ag0.75@ZFO NPs and reached 186.25 μg ml−1 after treatment with 1.0 mg ml−1 of the tested Ag0.75@ZFO NPs, which confirms the antibacterial nature of the synthesized NPs, which clearly leads to the formation of pits in the cell membrane of bacterial cells resulting in the leakage of the proteins from the cytoplasm of the bacterial cell. These experimental results showed that Ag0.75@ZFO NPs enhanced the permeability of S. vitulinus cell membranes. So it could be inferred that disruption of membranous permeability would be an essential factor for the inhibition of bacterial growth. Related research92,93 reported similar results with synthesized metal NPs showing concentration-dependent destabilization in the cell membrane of bacterial cells and leading to leakage of their intracellular material into the extracellular matrix (cell suspension).The amounts of protein released in a suspension of the treated bacterial cells were estimated using the Bradford assay.et al.94 confirmed that the change in bacterial cell membrane permeability was expressed in terms of percentage change in relative electric conductivity. It was revealed that the relative electric conductivities of all samples increase with an increase in the concentration of the nanocomposite. The integrity of the bacterial cell membrane was determined by measurement of the release of cell constituents of the bacteria like proteins; the leakage increased with time as there was irreversible cell membrane damage that led to the leakage of cell constituents leading to cell death.94Recently, Paul 3.8.S. vitulinus, as seen in S. vitulinus in the absence of Ag-decorated ZFO NPs, exhibited bacterial groups that had continually grown with standard typical bacterial exterior and semi-formed biofilm  with bacterial elements like C and O, along with Cl from the microelement bacterial medium. All detected aspects were found at the abnormal pores and the outer surface of the S. vitulinus cells, confirming the established Ag0.75@ZFO NPs' completion  and distributing all intracellular compositions like plasmid, DNA, and other essential organelles. Ultimately, cellular toxicity happens due to the oxidative stress caused by the generation of ROS. Finally, ZFO NPs withstand the bacterial cells' acidic state, and exchange did not occur,47 but the antibacterial impact is maintained by changing the signal transduction pathways.The schematic representation in 95–100 are presented in Results of related comparative studies4.via XRD and FTIR. Also, the EDX spectra proved that the fundamental elements were uniformly distributed through the Ag@ZFO NPs without any foreign constituents, which established the purity of the synthesized Ag@ZFO NPs. Ag-decorated ZFO NPs showed the well-known decisive antibacterial action towards all the chosen pathogenic bacteria. The activity of Ag0.75@ZFO NPs declines in the following order: S. vitulinus (24.60 mm), > E. columbae (23.5 mm) > S. aureus (21.90 mm) > S. lentus (15.00 mm) > S. sciuri (13.00 nm). For Ag0.75@ZFO NPs, the MIC was less against S. vitulinus (0.097 μg ml−1). The antibiofilm activity of Ag0.75@ZFO NPs was the highest, registering at 97.3% for S. aureus and 95.25% for E. columbae. Since the antimicrobial potency of NPs dramatically depends on the particle size, the smaller dimensions of Ag0.75@ZFO NPs give potent antibiofilm activity against bacteria. Ag0.75@ZFO NPs are a highly effective disinfectant once activated by UV light. The cellular protein released from S. vitulinus is directly proportional to the concentration of Ag0.75@ZFO NPs and reached 186.25 μg ml−1 after treatment with 1.0 mg ml−1, which clearly explains the formation of pits in the cell membrane of bacterial cells resulting in the leakage of the proteins from the cytoplasm of S. vitulinus cells. It is suggested that Ag-decorated ZFO NPs could substitute for some disinfectant solutions applied for surface cleaning in clinics and some paints included in medical operating chambers to save the atmosphere from penetrating pathogenic microbes. Moreover, Ag-decorated ZFO NPs may be acceptable as a constituent in some cosmetics and pharmaceuticals for biomedical treatments. Finally, Ag-decorated ZFO NPs may be used in manufacturing and environmental purposes like rainwater treatment for contaminants and preserve the environment from dangerous pathogens and hazardous pigments.For the first time, this work exhibits that gamma irradiation assisted the synthesis of Ag-decorated ZFO NPs. XRD and FTIR analyses have confirmed the successful preparation of Ag@ZFO NPs. The successful identification of the samples was confirmed The authors declare that they have no conflict of interest.


Escherichia coli (ETEC) is one of the top aetiologic agents of diarrhea in children under the age of 5 in low-middle income countries (LMICs). The lack of point of care diagnostic tools for routine ETEC diagnosis results in limited data regarding the actual burden and epidemiology in the endemic areas. We evaluated performance of the novel Rapid LAMP based Diagnostic Test (RLDT) for detection of ETEC in stool as a point of care diagnostic assay in a resource-limited setting.Enterotoxigenic LT) and heat stable toxins (STh and STp)]. Quantitative PCR was used as the “gold standard” to evaluate the diagnostic sensitivity and specificity of RLDT for detection of ETEC. We additionally described the prevalence and seasonality of ETEC.We conducted a cross-sectional study of 324 randomly selected stool samples from children under 5 presenting with moderate to severe diarrhea (MSD). The samples were collected between November 2012 to September 2013 at selected health facilities in Zambia. The RLDT was evaluated by targeting three ETEC toxin genes [heat labile toxin (LT 28/321(9%), ST 18/321(6%) and LT/ST 16/321(5%). The sensitivity and specificity of the RLDT compared to qPCR using a Ct 35 as the cut-off, were 90.7% and 97.5% for LT, 85.2% and 99.3% for STh and 100% and 99.7% for STp, respectively.The study included 324 participants, 50.6% of which were female. The overall prevalence of ETEC was 19.8% by qPCR and 19.4% by RLDT. The children between 12 to 59 months had the highest prevalence of 22%. The study determined ETEC toxin distribution was The results of this study suggest that RLDT is sufficiently sensitive and specific and easy to implement in the endemic countries. Being rapid and simple, the RLDT also presents as an attractive tool for point-of-care testing at the health facilities and laboratories in the resource-limited settings. ETEC is one of the top causes of diarrheal diseases in low and middle income countries. The advancement of molecular diagnosis has made it possible to accurately detect ETEC in endemic areas. However, the complexity, infrastructure and cost implication of these tests has made it a challenge to routinely incorporate them in health facilities in endemic settings. The ETEC RLDT is a simple and cost-effective molecular tool that can be used to screen for ETEC in resource limited settings. Here, we described the performance of the RLDT against a qPCR as the gold standard. Our findings showed that the ETEC RLDT performs comparable to the qPCR and would be a suitable screening tool in health facilities in recourse limited settings. Escherichia coli (ETEC) is one of the top ten causes of diarrhea [Enterotoxigenic diarrhea  with an diarrhea . Diarrhediarrhea ,4. The Ediarrhea . In low-diarrhea ,7. It isE. coli colonies and then test if the bacterium produces toxins  through the use of phenotypic assays such as dot blotting or through the use of conventional PCR. Quantitative PCR (qPCR) is performed with purified DNA from stool; although more sensitive; however its technology dependent and is difficult to perform without well-equipped laboratories [To accurately diagnose ETEC, one needs to first culture stool, isolate ratories . The comratories ,10 (ii) The complexity of these diagnostic methods results in the underestimation of the burden of ETEC because countries where the infection is endemic cannot afford the infrastructure and expertise required for this . To deveChakraborty et al previously developed a simple diagnostic assay, a Rapid LAMP based Diagnostic Test (RLDT) for detection of ETEC, which is based on Loop mediated Isothermal Amplification (LAMP) [n (LAMP) ,14. The The guardians of the participants in the study gave consent on behalf of the children by filling in consent forms issued in English and Nyanja as these are commonly used languages in Lusaka. An impartial witness was used to interpret for illiterate caregivers and provide a thumbprint signature as evidence of agreement, the impartial witness will attest to the process as being voluntary. For this study, Ethics and regulatory approvals were sought from the University of Zambia Biomedical Research Ethics Committee (UNZABREC) reference number 009–10–18 and the National Health Regulatory Authority (NHRA). Samples were de-identified and given a unique study participant number to maintain confidentiality.This was a retrospective study using 324 randomly selected samples from 1500 stored stool samples collected at various health facilities in the Lusaka district of Zambia. These samples were collected between November 2012 to September 2013 from a previous rotavirus vaccine effectiveness study [Statistics were applied to randomly select 324 retrospectively collected samples. This set of samples represented stool samples with equal distribution of sex and under 5 age groups. The statistician also stratified the participant samples with a 2:1 ratio of symptomatic and asymptomatic diarrhea cases.Samples with collected clinical information of moderate to severe diarrhea and asymptomatic representation were sorted, separated and stored at -80°C before testing.A team from the Johns Hopkins University, United States, Baltimore travelled to Zambia to train staff at the Centre for Infectious Diseases Research in Zambia (CIDRZ) on RLDT and quantitative PCR (qPCR) assays in an effort to build laboratory capacity. The RLDT and qPCR training duration lasted 2 weeks and was followed up by evaluation of performance of the staff.LT, STh and STp genes. One reaction tube was added as the RLDT inhibitor control [RLDT assays were conducted directly from the frozen stool samples using the RLDT kit as described by Chakraborty et al . In shor control . The str control  we used Nucleic Acid extraction: About 100-150mg of bulk stool were added to SK38 bead tubes  containing lysis buffer . The stool suspension was vortexed for 5 minutes, allowed to stand at room temperature for 10 to 15 minutes, then centrifuged at 14,000 rpm for 2 minutes to pellet stool material. About 200μl of the supernatant were transferred into a nuclease-free 1.5ml microcentrifuge tube for extracting nucleic acid using a QIAamp DNA Mini Kit  according to the manufacturer’s instructions.qPCR Amplification: The 25μl reaction mixtures contained 12.5ul Quantitech SYBR Green Master mix , 1uM primer mix 5ul, PCR grade water 5ul  and 2.5ul of samples. PCR was carried out for 40 cycles of 95°C for 15s and 60°C for 1 min [LT+ STh+ STp+) provided by John Hopkins was used for creation of the standard curve for each run similar to what was described in a previous study [or 1 min . qPCR cyor 1 min . Each saus study .LT, STh and STp using stool samples spiked with reference ETEC strain [4 CFU/g of stool which corresponds to qPCR Ct of 28.2, 28.6 and 30.07 for LT, STh and STp). Therefore, we also evaluated the performance of the RLDT using this LOD (Ct 28) as the cut-off.Chakraborty et al previously has established the limit of detection (LOD) of RLDT for ETEC genes C strain , 14. TheDiarrhea (Symptomatic) was defined as the primary caregiver reporting that the child had three or more loose stools within 24 hours.An asymptomatic case was defined as a child presenting to a health facility with other non-diarrhea complications.LT, STh or STp was positive. To compare RLDT with qPCR, a Ct value cut-off of 35 was used. Any sample with Ct value of 35 or less by qPCR was considered as true positive. To avoid incorrectly determining some samples to be false positive by RLDT, samples with Ct values greater than 35 detectable by both qPCR and RLDT were also included as true positive. We also compared RLDT with qPCR with the Ct value cut-off of 28.A minimum sample size of 324 with an assumed ETEC prevalence of 40.7%  producesThe design of the study is described in the flow chart as shown in A total of 324 samples with a mean age of about 30months were included in the analysis, 50.9% were female, 28.4% were asymptomatic, with 3.1% of the symptomatic cases presenting with severe disease according to a modified versikari severity score. Overall, ETEC prevalence was about 19% with both the assays, RLDT and qPCR and the highest prevalence was observed in children between 12–59 months of about 22%  There waLT, STh and STp toxin genes sensitivity, specificity, Positive Predictive value (PPV) and Negative Predictive Value (NPV) of the RLDT using a Ct 28 value cut-off with a 95% confidence interval were, 97.3% (85.5–99.9), 96.4% (93.6–98.3), 78.3% (63.6–89.1) and 99.6% (98–100); 95.8% (78.9–99), 99.3% (97.6–99.9), 92% (74–99) and 99.7% (98.1–100); 100% (59.0–100), 99.7% (98.3–100), 87.5% (47.3–99.7) and 100% (98.8–100). Whereas, for evaluation using Ct cut-off 35 sensitivity, specificity, PPV and NPV for the LT, STh and STp toxin genes were, 90.7% (77.9–97.4), 97.5% (94.8–99), 84.8% (71.1–93.7) and 98.5% (96.3–99.6); 85.2% (66.3–95.8), 99.3% (97.5–99.9), 92% (74–99) and 98.6% (96.5–99.6); 100% (59.0–100),99.7% (98.3–100), 87.5% (47.3–99.7) and 100% (98.8–100) respectively.The performance of the RLDT against qPCR is shown in LT, STh and STp toxin genes sensitivity, specificity, PPV and NPV of the RLDT using the Ct value cut-off of 35 with a 95% confidence interval was 91.4% (76.9–99.7), 96.8% (93.2–98.8),84.2% (68.7–94) and 98.4% (95.4–99.7); 85.7% (63.7–97.0), 99% (96.5–99.9), 90% (68.3–98.8) and 98.5% (95.7–99.7); 100% (54.1–100), 100% (98.3–100), 100% (54.1–100) and 100% (98.3–100) respectively. Similar results observed when asymptomatic cases were evaluated for sensitivity, specificity, PPV and NPV of LT, STh and STp 87.5% (47.4–99.7), 98.8% (93.5–100), 87.5% (47.3–99.7) and 98.8% (93.5–100); 83.3% (35.9–99.6), 100% (95.8–100), 100% (47.8–100) and 98.8% (93.7–100); 100% (2.5–100), 98.9% (94–100), 50% (1.3–98.7) and 100% (96–100) respectively. We compared the Area Under the Curve (AUC) for the RLDT and qPCR in detecting ETEC using clinical representation (asymptomatic and symptomatic) as a proxy reference, we found there was no significant difference between the performance of the RLDT to qPCR  had a frequency of 44/321 (14%) being the dominant gene. Followed by 34/321 (11%) strains presenting with only the Heat stable toxin (ST) genes. The frequency of ETEC with the combination of both LT/ST toxins was 16/321 (5%) as shown in ETEC with the presence of heat Labile toxin  and a minor peak between April and May (dry season) .LT, STh and STp. The performance of the RLDT was similar among ETEC positive diarrhea and asymptomatic cases. These findings are important as they support the use of the RLDT for screening for ETEC among children presenting with diarrhea at health facilities. In addition, its turnaround time and simplicity (not requiring skilled laboratory personnel for testing and results interpretation) makes it ideal for resource-limited settings. The RLDT could also be implemented in these countries for ETEC disease surveillance which is crucial to obtain meaningful disease burden data to inform policymakers and healthcare professionals for developing control and prevention programs.This study is the first field evaluation of ETEC RLDT and establishes that it performed equally as the qPCR, as demonstrated by the specificity, sensitivity, PPV, NPV and AUC curves for each toxin gene STh gene in the TaqMan array was 105CFU/gm of stool [STh was found to be Ct value of 26.2 (corresponding to 2.0x107 CFU/g of stool). The LOD of ETEC in the RLDT assay was approximately 2 logs lower than the diarrhea associated quantity defined in the above study. As demonstrated in this study, RLDT is enough sensitive to detect ETEC diarrhea cases and clinically relevant asymptomatic cases.In this study we evaluated the RLDT with the two qPCR Ct cut-offs, Ct35 and Ct28. In the enteric pathogens TaqMan array used in the reanalysis of the GEMS and MALED studies , Ct35 waof stool  which isof stool . It shouWe determined that across the stratified age groups, the children between 12 to 59 months were at the highest risk of getting ETEC infection with prevalence of ~ 22%. The overall prevalence of ETEC under 5 years old, in this study was ~19%. The isolation rates of ETEC in our study is similar to previous studies that have reported the prevalence of ETEC in developing countries from Bangladesh, Turkey, Peru, Mexico, Egypt, Argentina, India, Nicaragua, and Tunisia which indicated a rate of 18–38% in children –24. HoweThe seasonal prevalence observed in our study is similar to what was reported in Kenya  which reLT-ETEC strains was the highest followed by ST-ETEC and LT+ST-ETEC strains. The least detected ETEC strains were STp-ETEC. A similar distribution of toxin genes among ETEC strains was reported from Bolivia  [LT+STh being the most common toxin combination and LT+STh+STp being the least common in Zambia [We also found that in Lusaka, Zambia, among the circulating ETEC strains, the  STh 7%) . Michelon Zambia . This suThis study has several strengths firstly the results of this study were obtained from samples that were collected from several health facilities across the city of Lusaka, which means that our findings can be generalized across Lusaka Province. In addition, this study included symptomatic and asymptomatic cases that were stratified by age. This sampling method accounted for some biases in the computation of the true prevalence. We conducted qPCR assays with samples run in duplicate using average Ct values increasing the accuracy of the study results. This study’s novelty evaluates the ETEC RLDT platform, demonstrating a successful set-up in a resource-limited setting, which is comparable to qPCR.This study has a couple of limitations which include but are not limited to the cross-sectional design only provides prevalence data but does not provide incidence data, which is critical for vaccine trial design and planning control and prevention efforts.The two assays qPCR and RLDT, compared here, used different sample preparation methods. RLDT was done directly from the stool with minimum sample treatment; qPCR was done from purified DNA; Therefore, the sensitivity of these assays depends not only on the amplification technology but also on the starting material. In addition, while qPCR is quantitative, RLDT is semi-quantitative and was used qualitatively. This study was only carried out in outpatient health facilities but it would be good to evaluate the performance of the RLDT assay in an in-patient facility where there are more severe cases. The RLDT although useful in the diagnostic laboratory is not yet commercially available, however this data will contribute to the commercialization of the assay.LT-ETEC and ST-ETEC strains were highly prevalent and ETEC positivity was highest in the warm rainy season.We found that the RLDT performed comparable to the qPCR assay. Additionally, the observed specificity and sensitivity are high enough to suggest that the RLDT could be used in a field setting to rapidly detect ETEC among patients presenting with diarrhea in the health facilities. This study justifies a broader application of the RLDT as a simple and rapid diagnostic test for ETEC in the endemic countries where such simple assays are critically needed. We also determined that S1 Fig(DOCX)Click here for additional data file.


Background and Objectives: Vertebral artery hypoplasia (VAH) is a controversial risk factor for cerebral infarction. The aim of this study was to analyze the prevalence of vertebral artery hypoplasia and to evaluate its association with vertebrobasilar cerebrovascular accidents. Materials and Methods: The study was conducted in the Neurology Departments of the Republican Vilnius University Hospital from 2015 to 2020. Data of 742 subjects , 80 patients with anterior circulation infarction (ACI) and 529 control subjects with no symptoms of cerebrovascular accident) were analyzed. Ultrasound examination of the extracranial internal carotid and vertebral arteries (VA) was performed, risk factors were recorded. Results: The mean age of the subjects was 64.51 ± 13.02 years. In subjects with PCI/VAS the diameter of VA was smaller, and the prevalence of VAH was higher compared to those in subjects with ACI and in the control group. A higher degree of VAH in subjects younger than 65 years of age increased the risk of PCI/VAS. Subjects with non-dominant VA diameter of 2.7–2.9 mm had 2.21 times higher risk of PCI/VAS, subjects with non-dominant VA diameter of 2.5–2.6 mm had 2.36 times higher risk of PCI/VAS, and subjects with non-dominant VA diameter of 2.2–2.4 mm had 4.12 times higher risk of PCI/VAS compared with subjects with non-dominant VA diameter of ≥3 mm. Among patients with PCI/VAS those with VAH had lower rates of ischemic heart disease compared with patients with normal VA diameter. There was no difference in the rates of other risk factors between PCI/VAS patients with and without VAH. Conclusions: Vertebral artery hypoplasia is not a rare finding in individuals without symptoms of cerebrovascular accident, but more frequent in patients with vertebrobasilar cerebral infarction or vertebrobasilar artery syndrome. Vertebral artery hypoplasia can be considered a risk factor for posterior circulation infarction in subjects under 65 years of age. Cerebral infarction is one of the most acute social, economic and health care problems in Lithuania. The etiology of ~30% of cerebral infarctions remains unknown ,2. AdvanVertebral artery hypoplasia is a controversial risk factor for cerebral infarction: discussions continue regarding what vertebral artery diameter can be considered hypoplastic and affecting cerebral blood flow, and whether vertebral artery hypoplasia is an independent risk factor for cerebral infarction. The pathogenetic mechanism of cerebral infarction in vertebral artery hypoplasia is also not completely understood.The aim of this study was to analyze the prevalence of vertebral artery hypoplasia and to evaluate its association with vertebrobasilar cerebrovascular accident.Subjects hospitalized in the Neurology Departments of the Republican Vilnius University Hospital from 2015 to 2020 and diagnosed with vertebrobasilar (brainstem) artery syndrome (ICD-10-AM G45.0) or cerebral infarction due to cerebral artery thrombosis (ICD-10-AM I63.3) were invited to participate in the study.Adult subjects of both sexes;Diagnosed with vertebrobasilar artery syndrome or cerebral infarction;Agreed to participate in the investigation and signed an informed consent form.Inclusion criteria:Patients with intracerebral hemorrhage (ICD-10-AM I61);Patients with recurrent cerebral infarction (ICD-10-AM I63);Internal carotid artery stenosis > 50% or occlusion.Exclusion criteria:Individuals examined but not diagnosed with cerebrovascular accident were included into the control group.Ultrasound examination of the extracranial internal carotid and vertebral arteries was performed with Aloka Prosound F75 diagnostic ultrasound system using a 7.5 MHz linear array transducer. The extracranial portion of VA was assessed along its entire length. VA diameter was measured in the V2 segment  to avoid larger artifacts, possible in the V3  and V1  segments. Patients with cerebrovascular accident were examined by magnetic resonance imaging (MRI) and magnetic resonance angiography (MRA) using a 1.5 Tesla MRI (GE Optima MR450w 1.5T MRI System).We evaluated different groups of VA diameters: less than 2.2 mm, 2.2–2.4 mm, 2.5–2.6 mm, and 2.7–2.9 mm and less than 3 mm.Risk factors  were recorded.The study sample calculation was based on assumption that the prevalence of VAH among adults without neurological symptoms is 9.5% when VA diameter is measured by ultrasound, and the prevalence of VAH among adults with cerebral infarction is 19.1% ,4. The lData were analyzed using IBM SPSS Statistics  version 20 and Microsoft Excel. In the description of data frequency and relative frequency for categorical variables, mean and standard deviation, 95% of the confidence intervals (CI) for continuous variables are provided. The chi-square test was used to compare distribution of categorical variables between groups. One-way analysis of variance procedure (ANOVA) was used to compare means of three or more independent samples. Least significant difference (LSD) was used for multiple post hoc comparisons. Linear regression analysis was used to evaluate the effect of an independent variables (predictors) on the dependent variable. Logistic regression analysis was used to determine variables predicting the binary outcome. A significance level of 0.05 was chosen.Vilnius Regional Biomedical Research Ethics Committee granted approval No. 158200-15-767-281 to conduct the study on 13 January 2015.A total of 1109 individuals were invited to participate in the study. A total of 367 individuals did not meet the inclusion criteria. Data of 742 subjects were analyzed. A total of 307 (41.4%) subjects were men and 435 (58.6%) women.A total of 213 subjects experienced cerebrovascular accident: A total of 133 subjects had posterior circulation infarction or VAS (PCI/VAS) and 80 subjects had anterior circulation infarction (ACI). A total of 529 control subjects had no evidence of cerebrovascular accident.p < 0.001) and with subjects without symptoms of cerebrovascular accident (p < 0.001)  and with those of the control group  (The mean age of the subjects was 64.51 ± 13.02 years. Subjects with PCI/VAS were younger compared with subjects with ACI (< 0.001) . In subjctively) . The diactively) .The prevalence of non-dominant VA diameter of <3 mm was 56% among subjects without symptoms of cerebrovascular accident .p < 0.001, p < 0.001 and p < 0.001, respectively) and compared to patients with ACI   . We did ctively) . The prep < 0.001) and in the control group (β = 0.008 (95% CI 0.005–0.012), p < 0.001) (The diameter of non-dominant VA increased with age in the group of patients with cerebrovascular accident (β = 0.01 (95% CI 0.005–0.015), < 0.001) .p < 0.001 and age OR = 0.97; 95% CI: 0.96–0.99; p = 0.004). Further analysis demonstrated that a higher degree of VAH in subjects younger than 65 years increased the risk of PCI/VAS. Subjects with non-dominant VA diameter of 2.7–2.9 mm had 2.21 times higher risk of PCI/VAS compared with subjects with non-dominant VA of ≥3 mm. Subjects with non-dominant VA diameter of 2.5–2.6 mm had 2.36 times higher risk of PCI/VAS and subjects with non-dominant VA of 2.2–2.4 mm had 4.12 times higher risk of PCI/VAS compared with subjects with non-dominant VA ≥ 3 mm. In subjects ages 65 years and older no such association was found , diabetes  ischemic heart disease  and atrial fibrillation  compared to those in subjects younger than 65 years.A total of 177 (83.1%) patients with cerebrovascular accident had arterial hypertension, 31 (14.6%) had diabetes, 50 (23.5%) had ischemic heart disease and 27 (12.7%) had atrial fibrillation. Subjects 65+ years of age more frequently had hypertension . There was no statistically significant difference in the rates of other risk factors between PCI/VAS patients having VAH and without VAH.Among patients with PCI/VAS those with VA diameter of <3 mm had lower rates of ischemic heart disease compared with patients with normal VA diameter  is an independent risk factor for having PCI, increasing PCI odds ratio two times . A retroThe results of our study demonstrate that a higher degree of VAH was associated with an increased risk of PCI/VAS development: subjects with non-dominant VA diameter of 2.7–2.9 mm had 2.21 times higher risk of PCI/VAS, subjects with non-dominant VA diameter of 2.5–2.6 mm had 2.36 times higher risk of PCI/VAS and subjects with non-dominant VA diameter of 2.2–2.4 mm had 4.12 times higher risk of PCI/VAS compared to subjects with VA diameter of ≥3 mm, but such association was noticed only in the group of subjects younger than 65 years. This finding is likely to be associated with the fact that in younger patients with low impact of other risk factors the hypoperfusal circulatory disorder mechanism is the main risk factor of PCI/VAS. In older people, atherosclerosis related risk factors were the main contributors leading to circulatory disorders, rather than the decreased blood flow through VA. It is worth to mention that our investigation revealed that the diameter of non-dominant VA increased with age in groups of patients with cerebrovascular accident and in subjects without evidence of cerebrovascular accident. Positive correlation between age and the diameter of VA was reported by Chen et al. in a study of 1000 subjects and by Jeng et al. in 447 subjects .p = 0.012, respectively). Szarazova et al. did not find correlation between conventional vascular risk factors  and presence or absence of VAH in patients with PCI [We did not find any statistically significant differences in the rates of hypertension, diabetes, atrial fibrillation between PCI/VAS patients with and without VAH. Patients with PCI/VAS with VA diameter of <3 mm had lower rates of ischemic heart disease compared to patients with normal VA diameter (11.1% and 29.4%, with PCI . Peren ewith PCI . Hypertewith PCI . Two facThere were several limitations in this study. This was retrospective study conducted at single medical center. No data on VA blood flow velocity were collected. Ultrasound measurement is highly examiner dependent. Vascular risk factors were not collected for subjects in control group.Vertebral artery hypoplasia is not a rare finding in individuals without symptoms of cerebrovascular accident, but more frequent in patients with vertebrobasilar cerebral infarction or vertebrobasilar artery syndrome. Vertebral artery hypoplasia can be considered a risk factor for posterior circulation infarction in subjects under 65 years of age.


Purpose: To determine the safety and outcome profile of five-fraction stereotactic radiotherapy (FSRT) for brain metastases (BM), either as a definitive or adjuvant treatment. Methods: We assessed clinical data of patients receiving five fractions of 7 Gy each  to BM or surgical cavities. The primary endpoints were toxicity and radiation necrosis (RN) rates. Secondary endpoints were 1-year cumulative local control rate (LCR) and estimated overall survival (OS). Results: A total of 36 eligible patients receiving FSRT to a total of 49 targets were identified and included. The median follow up was 9 (1.1–56.2) months. The median age was 64.5 (34–92) years, the median ECOG score was 1, and the median Diagnostic-Specific Graded Prognostic Assessment (DS-GPA) score was 2. Treatment was well tolerated and there were no grade 3 adverse events or higher. The overall RN rate was 14.3% and the median time to RN was 12.9 (1.8–23.8) months. RN occurrence was associated with immunotherapy, young age (≤45 years), and large PTV. The cumulative 1-year local control rate was 83.1% and the estimated median local progression free-survival was 18.8 months. The estimated median overall survival was 11 (1.1–56.2) months and significantly superior in those patients presenting with RN. Conclusions: FSRT with 5 × 7 Gy represents a feasible, safe, and efficient fast track approach of intensified FSRT with acceptable LC and comparable RN rates for both the adjuvant and definitive RT settings. Brain metastases (BM) occur syn- or metachronously in up to 40% of patients with solid tumors ,2. Due tEven though initial prospective data suggest acceptable toxicity and comparable outcomes , this paThe objective of this study was to report the safety and efficiency profile of using 5 × 7 Gy to treat BM in either an adjuvant or definitive setting.For this monocentric retrospective study, all consecutive patients with histologically confirmed solid tumors receiving either adjuvant or definitive FSRT with 5 × 7 Gy  to treat BM at University Hospital Bonn between 2016 and 2018 were assessed for eligibility. The inclusion criteria included age over 18 years, a pathology-confirmed malignant primary tumor, an ECOG score ≤ 2, and a total number of BM ≤ 10. The exclusion criteria were previous SRT of the same volume and simultaneous primary intracranial tumors.Clinical data were extracted from the clinical database and corresponding patient reports using SQL queries. The parameters of the treatments performed were extracted directly from the Eclipse planning system used for irradiation planning . Diagnostic-Specific Graded Prognostic Assessment (DS-GPA)  scores wmin of 95% and a D100% < 99% of the prescription dose. Organ at risk Dmax constraints were defined as 22.5 Gy for optic nerves, 22.5 Gy for the optic chiasm, and 31.0 Gy for the brain stem.Following interdisciplinary evaluation, linear accelerator-based FSRT was administered with intensity-modulated image-guided techniques, employing a 6 to 10 MV energy of 7-Gy single doses to a cumulative 35-Gy dose. The baseline magnetic resonance imaging (MRI) T1-Gd scan with 1 mm slice thickness was co-registered to planning computer tomography (CT) in all cases. The latter was acquired in a neutral supine position with patient fixation ensured by a thermoformed framed mask system. Gross tumor volume (GTV) was defined either as any T1-Gd contrast enhancing lesion or the resection cavity including any possible residual contrast enhancement. A 2 mm margin was added for the planning target volume (PTV) in both scenarios, as per institutional standards. Eclipse software was used for treatment planning and ExacTrac  was used for positioning matching. PTV coverage parameters included a DFollow-up (FU) visits included a physical examination and MRI imaging. Adverse events (AE) were assessed and graded by clinicians according to the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE), Version 5.0 . Acute tThe primary endpoints were RN and neurological adverse event rates according to the CTCAE criteria. Secondary endpoints included estimated OS and cumulative 1-year local control rates (LCRs). OS was defined as the time interval between the first day of SRT and the date of either the last FU (censoring) or death. LC was defined as an absence of MRI-radiographic progression in the previously irradiated metastatic volume. In case of re-resection and a pathologic confirmation of RN, this event was not considered a progression. Patients that were lost to FU or deceased prior to radiographic progression were censored at the last FU time point.p < 0.05 were considered statistically significant. Specifically, statistical tests and analyses were performed as indicated in the respective figure legends. Figures were generated using GraphPad Prism 9 and Adobe Illustrator 2021 .Data analysis was performed with GraphPad Prism 9 . If not stated otherwise, the Mann–Whitney test was employed to determine significance. The Chi-square test was used to assess the significance of contingency tables. For statistical comparison of high and low variable values, the collective was divided into the respective groups by its median. The Log-rank test was used for statistical assessment of survival and control rates and is presented according to the Kaplan–Meier method. Results with A total of 36 patients receiving FSRT to a total of 49 BM or resection cavities were screened and included. The median age was 64.5 (34–92) years, the median ECOG score was 1 (0–2), and the median DS-GPA score was 2 (0–4). The most frequent histology was non-small cell lung cancer , followed by melanoma (22.2%) and breast cancer (11.1%). Additionally, 9 of 36 (25%) patients had been treated with immunotherapy before RT and 24.5% had previously received RT, with five of these cases being WBRT. A total of six patients (16.7%) had received sequential SRS to distant lesions, three patients (8.3%) had received sequential FSRT, and five patients (13.9%) had received sequential WBRT. Further patient characteristics are depicted in median dose was 35.8 (±1.28) Gy. The median PTV was 13.0 cc (0.7–74.4), with 8.2 cc (0.7–28.8) for definitive FSRT and 25.4 cc (5.6–74.4) for adjuvant treatment (p < 0.001). The PTV was significantly larger in the adjuvant RT subgroup than in the definitive RT subgroup (p < 0.001). A median of 2 (1–5) planning fields was used for RT planning. The median conformity index was 1.1 (0.2–3.5) and did not differ significantly between definitive and adjuvant SRT (p = 0.31). The median D0.1cc was 37.4 Gy (36.0–42.1) and median brain V50% was 40.9 cc (8.2–229.1). Further dosimetric features are described in All patients completed treatment. The median treatment time was 5 (5–8) days. The mean PTVA total of 54 events were recorded . Of thesp = 0.05; p = 0.0003). The PTV significantly correlated with RN , while conformity index, V10Gy, and V20Gy were not significantly different (p > 0.05). V100% of the brain was significantly larger in RN developing lesions . Of these, one was pathologically confirmed. No patient had symptomatic RN ≥ grade 3. Four RN cases were located in the parietal lobe. Thus, 57.1% of patients with parietal metastatic localization developed RN during FU. The other RN cases occurred in the frontal, temporal, and occipital lobes. The tumor histology of RN patients was melanoma in three patients (RN incidence 37.5%), lung cancer (16.7%) in two patients, and breast and renal cancer in one patient. Median time to RN was 387 (53–726) days. Additionally, 57.1% of patients with RN had received immunotherapy (IT) prior to RT, and 71.4% of the patients had received SRT as an adjuvant treatment. A total of 75% of the RN patients receiving IT suffered from severe associated immunologic side effects (such as pancreatitis or hypophysitis) that eventually caused treatment interruption. Further characteristics of the RN patients can be found in p = 0.86), as it was restricted to the first 18 months of FU. The 2-year LCR was 43.8% in the adjuvant RT subgroup vs. 59% for the definitive RT subgroup. While local control was superior in the subgroup of FSRT with smaller PTV, this difference was not significant. There was no significant difference for LC in brain V100% and V50%, but patients with lower PTV D0.1cc had a significantly higher LCR (p = 0.037). RN was significantly associated with better local control  was 83.1%, while the 2-year LCR was 50%. After 3 years, the LCR was 41.7%. The median PFS was 18.8 months. The 1-year LCR was 100% for adjuvant RT vs. 70.8% for definitive RT. However, this control benefit for adjuvant RT was not significant (p = 0.023).Only one patient (2.8%) died due to radiology-confirmed local intracerebral progression, whereas 19.4% died of systemic progression. In addition, 53.1% of patients developed distant cerebral metastases and 63.3% progressed systemically, as confirmed by radiologic FU imaging. Median OS was 11 months, and OS was significantly superior in the RN subgroup (39.9 months) compared to the non-RN subgroup (7.0 months)  with subsequent activation of microglia, macrophages, and CD3+ T-cells has been established [Notably, no grade 3 AEs occurred under or after FSRT. Compacted treatment time and dose escalation were not associated with a higher risk of AEs compared to more commonly applied schemes. This holds not only true for acute toxicity and late toxicity, but also for the anticipated RN risk. Even though SRS was associated with a higher risk of RN in previous series , we did ablished ,60. FSRTablished ,62; furtablished . FavorinAlthough this study is the most comprehensive manuscript to date reporting on 5 × 7 Gy FSRT, it carries several limitations. Data were collected retrospectively from a rather heterogeneous patient collective undergoing adjuvant and definitive RT for several different entities. The fractionation regimen was at the physicians’ discretion, and for most patients the decision between FSRT and SRS was based on lesion volumes. Other reasons for FSRT included glucocorticoid intolerance of any kind , which were routinely administered preceding SRS in our clinic during the study period. Low average performance and prognostic scores caused detriments in FU and PFS, and thus rather poor OS outcomes. Taken together, patient selection bias might have led to discordant outcomes when compared to the available literature. However, this collective represents an average patient population at a German university hospital and reflects a real-world situation instead of a carefully selected study population. Prospective randomized trials are needed to bring further insight into the optimal fractionation scheme for BM. The first randomized controlled phase III trial investigating SRS vs. FSRT for BM with a diameter of 2 cm to 4 cm was recently initiated . FurtherFive fractions of 7 Gy each appears to be a safe and effective alternative to more protracted fractionation schemes. Intensified FSRT might yield acceptable local control and toxicity rates in both the adjuvant and definitive RT settings while also maintaining short treatment times. These hypothesis-generating findings should be further studied within a clinical trial.


Additionally, serum and central nervous system samples from 72 horses, including horses with neurological disorders resulting in a fatal outcome or horses which had contact with them, were tested by real-time reverse transcription–polymerase chain reaction (RT-qPCR) for both viruses. Adopting the criterion of four-fold antibody titer difference, 89 (20.4%) horses presented neutralizing antibodies for SLEV and five (1.1%) for WNV. No evidence of SLEV and WNV infection was detected by RT-qPCR and, thus, such infection could not be confirmed in the additional samples. Our findings indicate that horses from Rio de Janeiro were exposed to both SLEV and WNV, contributing to the current knowledge on the distribution of these viruses flaviviruses in Brazil.Infections with arboviruses are reported worldwide. Saint Louis encephalitis (SLEV) and West Nile viruses (WNV) are closely related flaviviruses affecting humans and animals. SLEV has been sporadically detected in humans, and corresponding antibodies have been frequently detected in horses throughout Brazil. WNV was first reported in western Brazil over a decade ago, has been associated with neurological disorders in humans and equines and its prevalence is increasing nationwide. Herein, we investigated by molecular and serological methods the presence or evidence of SLEV and WNV in equines from Rio de Janeiro. A total of 435 serum samples were collected from healthy horses and tested for specific neutralizing antibodies by plaque reduction neutralization test (PRNT Emerging and reemerging viruses are mostly zoonotic, and among these, viruses transmitted by hematophagous arthropods (arboviruses) are of major importance, affecting both animals and humans . ArbovirFlaviviridae family comprises the genera Flavivirus, Pestivirus, Hepacivirus and Pegivirus, but only Flavivirus contains arboviruses. West Nile virus (WNV) and Saint Louis encephalitis virus (SLEV) are species of the Japanese Encephalitis (JE) serocomplex [The ocomplex  and are ocomplex ,5.Culex, as vectors, and some species of birds, as amplifying hosts [SLEV and WNV are maintained in enzootic cycles of transmission mainly involving mosquito species of the genus ng hosts ,7. Humanng hosts ,9. Desping hosts ,11,12,13ng hosts ,15. WNV ng hosts .SLEV is an arbovirus native to the Americas and was first reported in the 1930s during a human outbreak of neurological disorder in Saint Louis, Missouri, United States ,18. CurrThe first evidence of WNV circulation in Brazil emerged in 2011 when specific neutralizing antibodies were detected in healthy horses from the Pantanal, a South American floodplain located in western Brazil . In 2014Aedes-borne arboviruses [Haemagogus spp. and Sabethes spp. mosquitoes [Brazil has environmental and socioeconomic characteristics ideal for the circulation and spread of arboviruses ,45,46. Poviruses ,49,50, asquitoes . RJ is dsquitoes . In Nortsquitoes , which csquitoes .The exposure of horses from RJ to flaviviruses as SLEV and WNV has been rarely assessed and results of mostly based on ELISA serosurveys are ambiguous. In a ELISA-based serosurvey conducted in the South Fluminense mesoregion, no evidence of WNV was found in local horses . In our The study was approved by the Animal Ethics Committee of Fundação Oswaldo Cruz License  in compliance with the requirements of Brazilian Law 11794.2008, which regulates the scientific use of animals, including the principles of the Brazilian society of science in laboratory animals.For the present study, blood samples were collected from horses of stud farms, ranches and horse training centers located in different mesoregions of RJ. Additionally, we also collected and tested samples from horses that presented acute neurological syndrome without a defined cause, and horses that had close contact with sick.The sample size for the serological evaluation was calculated based on the expectance of detection of at least one WNV- or SLEV-seropositive horse through the software OpenEpi . We usedBlood samples from 435 healthy horses were collected between August 2015 and March 2017 from 21 equine properties located in 16 municipalities of six different mesoregions of RJ. At least two municipalities within each mesoregion were selected. These mesoregions included Metropolitan, Northwest Fluminense, North Fluminense, Coast, Centre Fluminense and South Fluminense . During The 435 blood samples were collected from the jugular vein of healthy animals in tubes without anticoagulants and transported to the Laboratório de Flavivírus at Fundação Oswaldo Cruz (Fiocruz), the regional center of reference for arbovirus diagnostics belonging to the national network of laboratories of the Brazilian Ministry of Health. Blood samples were then centrifuged, and serum collected and kept at −70 °C.Mesoregions sampled included Metropolitan (114 samples), Northwest Fluminense (80 samples), North Fluminense (76 samples), Coast (76 samples), Centre Fluminense (49 samples) and South Fluminense (40 samples) .Horses were classified in categories according to their activities, including sport, recreation, reproduction and work. Animals were also divided by age groups including Group 1 (≤6 months old), Group 2 (>6 months and ≤24 months old), Group 3 (>24 months and ≤72 months old), Group 4 (>72 months and ≤120 months old) and Group 5 (>120 months old).A total of 72 samples from 30 horses that presented acute neurological syndrome without a defined cause, including 11 dead horses and 42 samples from equines that had close contact with sick equines, were collected from seven properties in RJ between August 2015 and May 2021. All animals had a history of vaccination for at least one of the following viruses: rabies, Madariaga, Western equine encephalitis, equid influenza and herpesvirus.Sampled municipalities included Teresópolis, Saquarema, Friburgo, Duas Barras, Maricá and Rio de Janeiro . Some prDifferent types of clinical samples were collected . Blood sSerum samples of healthy horses were tested for WNV and SLEV using the plaque reduction neutralization test (PRNT). Shortly thereafter, the presence of specific neutralizing antibodies in serum samples was determined by screening followed by endpoint titer PRNT, as previously described .2 atmosphere. Cell plates inoculated with viral suspensions were assigned as viral control and used to determine the percentage of virus neutralization from each sample. Cell plates inoculated with viral suspension and diluted sera were overlaid by a 0.5% agarose solution. Overlaid plates were incubated for four days and then overlaid again with a neutral red agarose solution. The next day, the number of plaques was counted and compared to the viral control. Serum samples were initially screened at a single dilution (1:10). Samples that neutralized 90% or more plaques (PRNT90) were then tested in duplicate at higher dilutions, in serial two-fold dilutions from 1:10 to 1:2560. The endpoint titers were determined as the reciprocal of the highest dilution that had 90% or more plaques neutralized. Archived serum samples of horses that were seropositive and seronegative for SLEV and WNV in previous studies were used as positive and negative controls, respectively.Samples were heat-inactivated in a water bath at 56 °C for 30 min. After that, samples were two-fold serially diluted in medium 199 containing 5% bovine fetal serum. Viral stock of SLEV and WNV were diluted. Diluted samples were mixed in equal volume of viral suspension containing ~80 plaque forming units (PFU), and then incubated for 1 h at 37 °C. After incubation, 100 µL serum and virus solution was inoculated in six-well plates with VERO cells (ATCC CCL-81) and further incubated for 1 h at 37 °C in 5% CO90 titers equal to or higher than 10 for WNV and SLEV were then sequentially tested for the following other flaviviruses: Zika (ZIKV), Dengue (DENV-1) and Ilheus (ILHV), as previously described [Serum samples that presented PRNTescribed ,32.90 titer equal to or higher than 20 for WNV or SLEV and <10 for all other flaviviruses tested were considered seropositive in monotypic reactions. Additionally, samples that presented heterologous reactions, with PRNT90 titers for SLEV or WNV at least four times higher than that observed for the other flaviviruses tested, were also considered seropositive [90 titer 10 for any flavivirus tested—SLEV, WNV, DENV-1, ILHV or ZIKV—were considered inconclusive. Samples with PRNT90 < 10 for all flaviviruses were considered seronegative  for WNV and SLEV. Equines that had close contact with sick equines were also sampled and tested. In brief, samples of solid tissues (30 mg) were macerated in 600 μL of lysis buffer, centrifuged and the supernatant submitted to RNA extraction using QIAamp Viral RNA Mini Kit  according to the manufacturer’s instructions. Extracted RNA was then tested by specific RT-qPCR for WNV and SLEV based on the amplification of a region of the envelope gene, as previously described ,60.p  <  0.05. All analyses were performed using RStudio Software (version 2021.09.2) [Categorical variables including seropositivity for WNV and SLEV in horses, mesoregion, function, sex and horses’ age groups are displayed as numbers and percentages (%). We chose to examine the relation between seropositivity and the other categorical variables with Pearson’s Chi-squared test and Fisher’s exact row test to compare the prevalence between groups . Statistical significance was defined as values of two-tailed 21.09.2)  and ExceA total of 435 healthy horses were sampled between August 2015 and March 2017 in 16 municipalities of six different mesoregions of RJ . HealthyNo animal had a history of journey outside of the given mesoregion where they were sampled. Furthermore, despite their existence and use since 2002 in some parts of the world, no vaccines for WNV are licensed in Brazil by the Office Veterinary Service. Thus, no animal had history of vaccination for WNV.n = 310) were female and 28.7% (n = 125) male. The horses were classified according to their activities: 218 (50.1%) individuals were for sport, 40 (9.2%) for recreation, 175 (40.2%) for reproduction and 2 (0.5%) for work.Of the 435 healthy horses sampled, 71.3%  belonged to Group 1 (up to 6 months old), 63 (14.5%) to Group 2 (between 7 and 24 months old), 130 (29.9%) to Group 3 (between 25 and 72 months old), 141 (32.4%) to Group 4 (between 73 and 120 months old), and 94 (21.6%) to Group 5 .A total of 72 serum samples were collected from the horses with history of neurological disorder or those in contact with such horses. Other samples from these horses included six samples of CNS tissues, five of CSF and six of spinal cords . Among tn = 165) presented PRNT90 ≥ 10 for WNV and/or SLEV and the corresponding samples were selected for determining endpoint titers, while 62% (n = 270) presented PRNT90 < 10 and were considered seronegative.Among the 435 screened healthy horses, 38% (n = 143) were reactive for SLEV, with 19.3% (n = 84) being monotypic for SLEV. Regarding WNV, 18.4% (n = 80) were reactive in the screening and among these, 4.8% (n = 21) had WNV monotypic results. A total of 13.5% (n = 59) of all serum samples tested presented seropositivity for both SLEV and WNV.Among the samples that were reactive in the screening, 32.9% (n = 94) were seropositive for SLEV  or WNV . A total of 7.4% (n = 32) of samples were seropositive for an undifferentiated flavivirus, and 6.4% (n = 28) of samples were inconclusive. Among the samples that were seropositive for SLEV or WNV, 70.8% (n = 63) and 60.0% (n = 3), respectively, were monotypic results. From the total of 435 samples, 14.5% (n = 63) were seropositive for SLEV and 0.7% (n = 3) were seropositive for WNV.Among individuals reactive in the screening, 21.6% (n = 281) were seronegative. These samples were also seronegative for ZIKV, DENV-1 and ILHV  monotypic and 34.9% (n = 26) heterotypic reactions. Of the 63 samples with monotypic reaction for SLEV, 22 had a neutralizing antibody titer of 20, 16 a titer of 40, 13 a titer of 80, 10 a titer of 160 and two samples presented titer of 320. Among the 26 heterotypic samples, the titers ranged between 40 (n = 9), 80 (n = 11), 160 (n = 5) and 320 (n = 1). Furthermore, 23% (n = 6) of them presented anti-WNV antibody titers ≥ 20, one sample with 80, another with 40 and four samples with 20 titer. The others 20 samples had titers lower than 20 , 320 (n = 2) and 2560 (n = 1), with 60% (n = 3) monotypic and 40% (n = 2) heterotypic reactions. Serum specimens from the two horses with serum antibody titers of 40, as well as one of the two samples that had a titer of 320, had monotypic reactions for WNV. The other serum samples with a titer of 40, one with a titer of 2560 and one with a titer of 10 for SLEV had heterotypic reactions.The five samples seropositive for WNV had serum neutralizing antibody titers of 20 (n = 14), 20 (n = 7) and 40 (n = 11) for SLEV; and 10 (n = 17), 20 (n = 10), 40 (n = 2) and 80 (n = 3) for WNV  had antibody titers ≥ 20 for SLEV, and 15 (46.8%) for WNV. Serum neutralizing antibody titers were: 10 ( for WNV .n = 23), Northwest Fluminense with 27.5% (n = 22), Coast with 23.7% (n = 18), Centre Fluminense with 30.6% (n = 15) and Metropolitan with 9.6% (n = 11). The differences between regions were statistically significant (p < 0.001). In these regions, the corresponding seropositive samples with monotypic reactions were: 65.2% (n = 15), 81.8 % (n = 18), 66.6% (n = 12), 60% (n = 9) and 81.8% (n = 9), respectively. In all municipalities where samples were collected from, except for São Fidélis in the North Fluminense region, there were positive monotypic and heterotypic results for SLEV (Mesoregions that had SLEV-seropositive equines were North Fluminense with 30% (for SLEV .n = 1), Bom Jesus do Itabapoana in Northwest Fluminense with 2.5% (n = 2) and Saquarema and Araruama in Coast mesoregion with 2.5% (n = 2). The differences between regions were not statistically significant (p = 0.39). The monotypic reactions among the seropositive samples in each mesoregion were 50% (n = 1) in Northwest Fluminense, São Fidélis and 100% (n = 2) in the Coast mesoregion in Saquarema and Araruama , Northwest Fluminense with 10.0% (n = 8), Coast with 6.6% (n = 5), Centre Fluminense with 4.1% (n = 2), Metropolitan with 5.3% (n = 6) and South Fluminense with 5.0% (n = 2); the differences were not statistically significant (p = 0.42)  , Table 3n = 44) were classified “for reproduction”, 42% (n = 37) “for sport”, and 9% (n = 8) “for recreation”. Neither of the two horses classified as “for work” were seropositive for SLEV (n = 44) were seropositive for SLEV, with 63.6% (n = 28) presenting monotypic reactions. Meanwhile, 20% (n = 8) and 17% (n = 37) of the horses classified as “for recreation” (n = 40) and “sport” (n = 218), respectively, were seropositive for SLEV. Seropositive samples had 87.5% (n = 7) and 75.6% (n = 28) monotypic reactions, respectively. The seroprevalence for SLEV was substantially higher in reproduction horses when compared to work horses although the difference was not statistically significant (p = 0.21). However, of 175 horses classified as “for reproduction”, only 2.3% (n = 4) were seropositive for WNV, with 50% (n = 2) having monotypic reactions. From the two horses classified as “for work”, one was seropositive (50%) and had a monotypic reaction (n = 100%).Regarding horse function, of 89 SLEV-seropositive horses, 49% (for SLEV . Of 175 n = 2) in Group 1, 12.3% (n = 11) in Group 2, 22.4% (n = 20) in Group 3, 38.2% (n = 34) in Group 4 and 25% (n = 22) in Group 5 (p > 0.05). Within each group, horses with monotypic antibodies against SLEV were: 100% (n = 2) for Group 1, 90.9% (n = 10) for Group 2, 60% (n = 12) for Group 3, 70.5 % (n = 24) for Group 4 and 68.1% (n = 15) for Group 5. Among the horses seropositive for SLEV, 62 (70%) were female and 27 (30%) were male. Of all the 310 females tested, 62 (20%)—and of all the 125 male horses, 27 (21.6%)—were seropositive for SLEV. The difference between the two groups was not statistically significant (p > 0.05) (n = 44) of the females and 70.3% (n = 19) of the males had monotypic reactions.The seroprevalence for SLEV was 2.2% ( > 0.05) . Among tp > 0.05). Among these, two horses classified as “for reproduction” (50%) and one classified as “for work” (100%) had monotypic reactions. Regarding age, among the five WNV-seropositive horses, three (60%) were from Group 4 and two (40%) from Group 5 (p > 0.05), and within each group, 66.6% (n = 2) from Group 4 and 50% (n = 1) from Group 5 had a monotypic reaction for WNV. Of five horses that were seropositive for WNV, one (20%) was male and four (80%) were female. Among 125 males, 1 (0.8%) was seropositive for WNV, while among 310 females, 4 (1.3%) were seropositive for WNV (p > 0.05)  was from the group of two horses classified as “for work” and four (80%) from the group of 175 horses classified as “for reproduction” ( > 0.05) . Among tA total of 130 animals, including 32 horses with neurological disorder and 98 horses which had contact with them, tested negative for SLEV and WNV as assessed by RT-qPCR.The recent outbreaks of flaviviruses highlight their transmission potential and may be a cause for a dynamic state of emergence. Flaviviruses and other arboviruses need to be identified and continuously monitored as an instrumental public health strategy, which will provide us with measures to respond rapidly to emerging and re-emerging viral epidemics.90 in horses from different mesoregions of RJ. We used a conservative criterion of seropositivity to reduce the chances of cross-reactivity and the most specific serological method for the detection of flavivirus-neutralizing antibodies [Human and equine cases of SLEV and WNV infection have been reported in Brazil ,37,62,63tibodies . We repotibodies .We considered monotypic serologic responses to be the most reliable, as these samples reacted with just one of all viruses employed in the tests with no indication of cross-reaction. This stringent criterion is particularly valuable in areas where several flaviviruses co-circulate, because of the high potential for cross-reactivity mainly on secondary infections . SamplesThe prevalence of undifferentiated flavivirus results can be attributed to the cross-reactive commonly observed among flaviviruses . In addin = 9), 80 (n = 11), 160 (n = 5) and 320 (n = 1). Most of these samples (n = 20) also had a low titer for WNV with only six having a titer ≥ 20, more precisely: 80 (n = 1), 40 (n = 1) and 20 (n = 4). The same happened with the heterotypic samples seropositive for WNV—one of them, with a high titer for WNV (2560), also had low titer for SLEV (10). Another one, with a titer of 320 for WNV, also presented a titer of 40 for SLEV, indicating an infection with SLEV. In all cases, some seropositive samples might have been overlooked in the seroprevalence calculation.Of the 32 horses seropositive for an undetermined species of flavivirus, 18 (56.2%) had antibody titers for SLEV ≥ 20, and 15 (46.8%) had antibody titers for WNV ≥ 20. Furthermore, these samples might have had contact with both viruses, SLEV and WNV. Among the 89 seropositive samples to SLEV, 26 (29.2%) had heterotypic reactions and their titers were 40  equines were confirmed seropositive for SLEV, and five (1%) for WNV. Similar rates of infection with SLEV and WNV were reported by other studies applying the same conservative criterion of seropositivity . DespiteRegarding WNV, reports on its circulation throughout the country are emerging. If the increase of reports is related to a more sensitive surveillance or to a recent dissemination of WNV throughout the country remains unknown. Recent cases have been reported in humans and horses mainly from the northeast ,40,41,72Aedes aegypti, a vector that feeds preferentially on humans. Conversely, ILHV, which is an enzootic flavivirus, has presented high seroprevalence in other serosurveys of arboviruses conducted on horses in western Brazil [In 2018, WNV was detected in horses with neurological disorders in Espírito Santo, a state that geographically borders RJ . The sern Brazil ,65. The n Brazil .In this study, 435 samples were collected from horses in all the mesoregions of RJ, regions with different climatic, economic and social characteristics . No horsp < 0.001). In the same mesoregions, including the north of RJ, we observed higher prevalence of monotypic reactions. Besides high humidity and temperature, both of these mesoregions are also characterized by recent increases in development and urbanization [Despite variation in seropositivity for both viruses among the mesoregions, differences were statistically significant only for SLEV. The mesoregions with the highest seroprevalence for SLEV and WNV were Northwest Fluminense and Coast . The analysis of sex as risk factor for WNV infection was not performed because of the low number of samples.Regarding sex as a risk factor, we found no association between sex and seropositivity for SLEV (p = 0.21). The difference in exposure to WNV between animals destined for work (50%) and reproduction (2.3%) was statistically significant (p < 0.001). The difference in seroprevalence between the two groups could be attributed to the management of horses belonging to each group. This variation could ultimately increase or decrease exposure to vectors, which is an important risk factor for arthropod-borne virus infections [The prevalence of SLEV infection was 25.1%, 20% and 17% in animals destined for reproduction, recreation and sports, respectively (fections . FurtherThe seroprevalence for SLEV, WNV and undifferentiated flavivirus was directly proportional to the age group. Seroprevalence increased with age, which can be related to the longer exposure to both arboviruses during the lifetime. Serological surveys conducted in the southern region of Brazil had similar results, suggesting that age is a risk factor for detection of antibodies . ComparaThe negative results obtained when using molecular methods can be attributed to the absence of a current infection or to the brief and low viremia usually observed in dead-end hosts . In the Epizootiological surveillance for arboviruses maintained in bird–mosquito cycles should always comprise not only molecular detection, but also serological investigation in local birds. Species of birds that act as amplifying hosts for WNV have long-lasting high-level viremia and persistent viral infection, and therefore, are easier to identify by molecular methods ,80. In 2Several reports on the circulation of WNV in Brazil emerged in the last decade, which could indicate the establishment of enzootic transmission cycles in the country. The higher prevalence for SLEV and WNV found in horses from the North Fluminense, Northwest Fluminense and Litoral mesoregions from RJ may also be related to local climatic and ecological conditions that favor enzootic cycles of transmission. The findings presented herein demonstrate the need to intensify the epidemiological surveillance of these arboviruses in Brazil and highlight the importance of future epizootiological investigations including both vectors and hosts, with particular interest in regions where cases of neurological syndromes have occurred in horses. The results obtained in this study imply SLEV and WNV previously circulated in horses from RJ, and should serve as an alarm to human and veterinary health professionals.