Enzyme-linked Immunosorbent Assay Test Research Articles

Tetrastigma hemsleyanum Ethanolic Extract Inhibited the Growth of Nonsmall Cell Lung Cancer Cells by Suppressing Hypoxia-Inducible Factor-1α-Dependent Glycolysis and Angiogenesis

Background:The ethanolic extract of Tetrastigma hemsleyanum Diels et Gilg ( T hemsleyanum ethanolic extract [Te-EtOH]) showed positive effects against various tumors. However, there are few studies on the effects of Te-EtOH on nonsmall cell lung cancer (NSCLC). We attempted to examine the inhibiting effect of Te-EtOH on NSCLC cells and to elucidate the relevant mechanisms. Methods: A549 and H1299 cells were pretreated with Te-EtOH at different concentrations. Cell viability was analyzed by Cell Counting Kit-8, flow cytometry, and the 3-dimensional spheroid model; RNA-sequencing was also performed. Moreover, enzyme-linked immunosorbent assay and Western blot tests were performed to determine the metabolic capability, the expressions of energy metabolism-related proteins, and the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/hypoxia-inducible factor-1α (HIF-1α) pathway. Additionally, under hypoxic conditions, the ability of Te-EtOH to inhibit HIF-1α expression and the metabolic capability of NSCLC cells was tested. Results: Te-EtOH considerably repressed cell viability in a dose-dependent manner. RNA-sequencing revealed that Te-EtOH's inhibition of NSCLC cells activity was related to metabolism. In addition, Te-EtOH significantly inhibited glycolysis, and adenosine triphosphate and lactate accumulation in NSCLC cells. Furthermore, we found that Te-EtOH could block PI3K/AKT/HIF-1α pathway activation. Moreover, Te-EtOH significantly inhibited hypoxia-induced expression of HIF-1α, vascular endothelial growth factor, and metabolic capability. Conclusions: Our results suggested that Te-EtOH inhibited the growth of NSCLC cells by suppressing HIF-1α-dependent glycolysis and angiogenesis.

Molecular and serological detection of avian infectious bronchitis virus in vaccinated and non- vaccinated chickens in Wasit province/Iraq

The present study was conducted to evaluate the efficacy of IBV vaccine by detecting presence of the infectious bronchitis virus (IBV) by enzyme linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR) techniques. 180 serum sample were collected from chicken farms including (154) samples were collected from clinically infected vaccinated broiler farm and (26) serum samples were collected from clinically infected non-vaccinated broiler farm. Samples submitted for ELISA assay, The results were as follow 90(98%)positive and 64 sample negative in ELISA test from vaccinated farms, ELISA results showed also 2(7.6%) sample positive and 24(92.3%) negative from non-vaccinated farms. Eighty sample(60 sample were positive and 20 were negative with ELISA test) were submitted for the presence of IBV by RT-PCR technique. Virus was detected in 37(45.25 %), while 43( 66.25%) did not have detectable IBV according to RT-PCR results.

Anti-tissue Transglutaminase Enzyme-Linked Immunosorbent Assay for Screening of Celiac Disease in Moroccan Pediatric Patients

Celiac disease (CD) is an immune-mediated systemic disorder elicited by gluten which induces an enteropathy with atrophy of intestinal villi. No prevalence information was available for CD in Moroccan children because the diagnosis is based on multiple invasive duodenal biopsies. To simplify CD diagnosis, the objective of this work was to develop an Enzyme-linked Immunosorbent Assay (ELISA) for IgA and IgG tissue transglutaminase (tTG) antibodies detection suitable for Moroccan children. The concentration of the coated antigen and serum dilutions were optimized for the development of IgA anti- tTG and IgG anti-tTG ELISA detection in serum. The cut-off was calculated and the developed ELISAs were used for detecting IgA and IgG-tTG antibodies in 97 serum samples from 97 children aged 1 to 13 years, with variable clinical characteristics. After adjusting optimal working conditions of ELISA, the cut-offs were 0.04 and 0.156 AU for detecting anti-tTG and IgA anti-tTG IgG respectively. The use of IgG anti-tTG as CD marker (100 % of sensitivity) was better than the use of IgA anti-tTG in ELISA tests (90.3% sensitivity). In addition, the ELISA test shows a clear correlation between the IgG tTG antibodies titer and the degree of duodenal damage, estimated by the Marsh stages classification in patients with confirmed CD. Our results suggest that the assessment of the adapted IgG anti-tTG ELISA test can be used for CD in initial screening in Moroccan children after validation with a larger number of samples with CD for confirmation.

Detection of Serum Antibody Responses in Nipah Virus-Infected Pigs.

Nipah virus (NiV) is an emerging, zoonotic paramyxovirus that is among the most pathogenic of viruses in humans. During the first reported outbreak of NiV in Malaysia and Singapore in the late 1990s, pigs served as an intermediate host, which enabled the transmission to humans. Although subsequent outbreaks in Asia only reported direct bat-to-human and human-to-human transmission, pigs are still considered a potential source for viral dissemination in the epidemiology of the disease. Thus, serological assays such as Enzyme-linked immunosorbent assay (ELISA) or virus neutralization test (VNT) represent powerful tools to characterize the serum antibody responses in NiV-infected pigs as well as to perform seroepidemiological surveillance studies on the potential circulation of NiV or NiV-related viruses among pig populations worldwide. This chapter describes both methods in detail. Furthermore, we discuss some of the major pitfalls and indicate how to avoid them.

Assessing the Efficacy of Recombinant Newcastle Disease Virus Vaccines (Double-Insert vHVT-IBD-ND and Single-Insert vHVT-ND) Followed by a Vaccination with a Live Newcastle Disease Vaccine Against a Moroccan Velogenic Newcastle Disease Challenge in Commercial Broilers.

The advent of turkey herpesvirus (HVT) vector vaccine technology (vHVT) has made a huge improvement in the prevention and control of several poultry diseases. The objective of this study was to compare, under experimental conditions, the protection conferred by different vaccination programs based on an HVT double-insert (infectious bursal disease {IBD] and Newcastle disease [ND]) vector vaccine (vHVT-IBD-ND) and an HVT single-insert (vHVT-ND) vector vaccine followed by a vaccination with a live ND vaccine at Day 1 only or at Days 1 and 14. Commercial broilers were vaccinated by the recombinant ND virus vaccines subcutaneously at 1 day old, in the hatchery, and challenged at 30 days of age using the Moroccan ND virus velogenic viscerotropic JEL strain. The results showed that the tested vaccine induced 95% to 100% clinical protection against mortality and clinical signs. The humoral immune response to vaccination was detected from 3 wk of age using enzyme-linked immunosorbent assay and hemagglutination inhibition tests. ND challenge virus shedding was significantly reduced in the vaccinated birds as compared to controls. Significant reduction of the cloacal shedding suggests that the vHVT-IBD-ND vaccine stimulates actively the immunity against the tested ND challenge virus. No significant differences were found between the vaccination programs based on vHVT-IBD-ND or on vHVT-ND.

Accuracy of serological tests for COVID-19: A systematic review and meta-analysis.

To determine the diagnostic accuracy of serological tests for coronavirus disease-2019 (COVID-19). PubMed, Embase and the Cochrane Library were searched from January 1 2020 to September 2 2022. We included studies that measured the sensitivity, specificity or both qualities of a COVID-19 serological test and a reference standard of a viral culture or reverse transcriptase polymerase chain reaction (RT-PCR). The risk of bias was assessed by using quality assessment of diagnostic accuracy studies 2 (QUADAS-2). The primary outcomes included overall sensitivity and specificity, as stratified by the methods of serological testing [enzyme-linked immunosorbent assays (ELISAs), lateral flow immunoassays (LFIAs) or chemiluminescent immunoassays (CLIAs)] and immunoglobulin classes (IgG, IgM, or both). Secondary outcomes were stratum-specific sensitivity and specificity within the subgroups, as defined by study or participant characteristics, which included the time from the onset of symptoms, testing via commercial kits or an in-house assay, antigen target, clinical setting, serological kit as the index test and the type of specimen for the RT-PCR reference test. Eight thousand seven hundred and eighty-five references were identified and 169 studies included. Overall, we judged the risk of bias to be high in 47.9 % (81/169) of the studies, and a low risk of applicability concerns was found in 100% (169/169) of the studies. For each method of testing, the pooled sensitivity of the ELISAs ranged from 81 to 82%, with sensitivities ranging from 69 to 70% for the LFIAs and 77% to 79% for the CLIAs. Among the evaluated tests, IgG (80-81%)-based tests exhibited better sensitivities than IgM-based tests (66-68%). IgG/IgM-based CLIA had the highest sensitivity [87% (86-88%)]. All of the tests displayed high specificity (97-98%). Heterogeneity was observed in all of the analyses. The detection of nucleocapsid protein (77-80%) as the antigen target was found to offer higher sensitivity results than surface protein detection (66-68%). Sensitivity was higher in the in-house assays (78-79%) than in the commercial kits (47-48%). Among the evaluated tests, ELISA and CLIA tests performed better in terms of sensitivity than did the LFIA. IgG-based tests had higher sensitivity than IgM-based tests, and combined IgG/IgM test-based CLIA tests had the best overall diagnostic test accuracy. The type of sample, serological kit and timing of use of the specific tests were associated with the diagnostic accuracy. Due to the limitations of the serological tests, other techniques should be quickly approved to provide guidance for the correct diagnosis of COVID-19.

Identification and characterization of natural PR-1 protein as major allergen from Humulus japonicus pollen

BackgroundThe Humulus japonicus pollen is one of the most common allergenic pollens in China. However, little is unveiled regarding the allergenic components in Humulus japonicus pollen. Our study aimed to purify and identify the pathogenesis-related 1 (PR-1) protein from Humulus japonicus pollen, and to characterize the molecular and immunochemical properties of this novel allergen. MethodsThe natural PR-1 protein (named as Hum j PR-1) was purified from Humulus japonicus pollen extracts with a combined strategy of chromatography, and identified by mass spectrometry. The coding sequence of Hum j PR-1 was confirmed by cDNA cloning. The recombinant Hum j PR-1 was expressed and purified from Escherichia coli. The allergenicity was assessed by immunoblot, enzyme-linked immunosorbent assay (ELISA), inhibition ELISA, and basophil activation test using Humulus japonicus allergic patients’ whole blood. The physicochemical properties and 3-dimensional structure of it were comprehensively characterized by in silico methods. ResultsThe allergenicity analysis revealed that 76.6 % (23/30) of the Humulus japonicus pollen allergic patients displayed specific IgE recognition of the natural Hum j PR-1. The cDNA sequence of Hum j PR-1 had a 516-bp open reading frame encoding 171 amino acids. Physicochemical analysis indicated that Hum j PR-1 was a stable and relatively thermostable protein. Hum j PR-1 shared a similar 3-dimensional folding pattern with other homologous allergens, which was a unique αβα sandwich structure containing 4 α-helices and 6 antiparallel β-sheets, encompassing 4 conserved CAP domain. ConclusionThe natural PR-1 was firstly purified and characterized as a major allergenic allergen in Humulus japonicus pollen. These findings would contribute to developing diagnostic and therapeutic strategies for Humulus japonicus pollinosis.

Evaluation of three Staphylococcus aureus proteins involved in the adhesion process as antigens for the detection of bovine intramammary infections.

Introduction. Fast and accurate diagnosis is one of the key strategies in the successful control of intramammary infections caused by Staphylococcus aureus. Immunoassays are one of the diagnostic tools that have been proposed for the detection of S. aureus infection because they offer an advantage in terms of cost and are fast and easy to use compared to other diagnostic tests.Gap statement. The main challenge of the immunoassays is to identify antigens or serological markers that allow accurate discrimination between infected and uninfected cows with S. aureus, since this bacterium can naturally colonize different areas of the animal body.Aim. To evaluate three S. aureus proteins (IsdA, ClfA, SdrD) involved in the adhesion process as antigens to detect indicator antibodies of bovine intramammary infections.Methodology. Ninety-six cows in lactation and not vaccinated against S. aureus were included. Forty-eight of these cows were infected with S. aureus, while the rest (n=48 cows) were uninfected. Blood and milk samples were collected from each animal to recover serum and whey. IgG titres against the three proteins individually and combined (Mix) were measured in each sample using an enzyme-linked immunosorbent assay (ELISA) test.Results. Significant differences in the IgG response against the proteins evaluated were observed, highlighting the antigenic potential of IsdA and demonstrating that some antigens can detect specific antibodies of infection better than others. According to receiver operating characteristic (ROC) curve analysis, the combined proteins showed the most remarkable capacity (sensitivity of 79 % and specificity of 77 %) to differentiate between infected and uninfected cows when blood samples were used. In addition, the combined proteins also showed the highest specificity (94 %) when using milk samples.Conclusion. Our findings provide information on the usefulness of three adhesion-associated S. aureus proteins in detecting serological markers of intramammary infections in bovines.

Study of influence of metal ions in the diagnosis of recombinant hepatitis B surface antigen (HBsAg) using ELISA technique

ObjectivesPreliminary diagnosis of hepatitis B is performed by measuring the levels of serological markers using enzyme-linked immunosorbent assay (ELISA). Errors in diagnosis of the disease using serum markers might occur due to the presence of metallic salts in diagnostic samples. The present study was designed to evaluate the effects of various metal ions on the diagnosis of recombinant hepatitis B surface antigen (HBsAg). MethodsA simple step sandwich ELISA technique was used to evaluate the effects of metal ions on the diagnosis of HBsAg. Further, the zeta potential, electrical conductivity and redox potential of solutions with proteins and metal salts were measured to evaluate the interaction of these metal ions with the protein. ResultsThe results showed that recombinant HBsAg significantly interacted with the tested metal ions, with lithium ion showing the highest interaction, followed by barium, aluminium, and nickel as the percent decrease in the free HBsAg concentration in presence of these metal ions were found to be 95.5, 94.1, 93.5, and 91.6%, respectively. Zeta potential of the protein in reaction mixture also changed considerably and marked decrease in the concentration of free HBsAg was observed. This decrease in the concentration of free HBsAg resulted into error in the ELISA test which was negative in presence of these metal ions due to strong interaction with the HBsAg protein. ConclusionsThe findings of this study clearly demonstrated that the presence of metal ions in pathological samples can result in interaction with the antigen leading to possible diagnostic errors.

Alpha-defensin as a diagnostic tool in revision total knee arthroplasties with unexpected positive intraoperative cultures and unexpected culture negative intraoperative cultures.

Unexpected-positive-intraoperative-cultures (UPIC) are common in presumed aseptic revision-total-knee-arthroplasties (rTKA). However, the clinical significance is not entirely clear. In contrast, in some presumably septic rTKA, identification of an underlying pathogen was not possible, so-called unexpected-negative-intraoperative-cultures (UNIC). The purpose of this study was to evaluate the potential use of synovial alpha-defensin (AD) levels in these patients. Synovial AD levels from 143 rTKAs were evaluated retrospectively from our prospectively maintained institutional periprostetic joint infection (PJI) biobank and database. The 2018-International Consensus Meeting (ICM) criteria was used to define the study groups. Samples from UPICs with a minimum of one positive intraoperative culture (ICM 2- ≥ 6) (n = 20) and UNIC's (ICM ≥ 6) (n = 14) were compared to 34 septic culture-positive samples (ICM ≥ 6) and 75 aseptic culture-negative (ICM 0-1). Moreover, AD-lateral-flow-assay (ADLF) and an enzyme-linked-immunosorbent-assay (ELISA) in detecting the presence of AD in native and centrifuged synovial fluid specimens was performed. Concentration of AD determined by ELISA and ADLF methods, as well as microbiological, and histopathological results, serum and synovial parameters along with demographic factors were analysed. AD was positive in 31/34 (91.2%) samples from the septic culture-positive group and in 14/14 (100%) samples in the UNIC group. All UPIC samples showed a negative AD result. Positive AD samples were highly associated with culture positive and histopathological results (p < 0.001). No high-virulent microorganisms (0/20) were present in the UPIC group, compared to infected-group (19/34; 55.9%). High virulent microorganisms showed a positive AD result in 89.5% (17/19) of the cases. Methicillin resistant Staphylococcus epidermis (MRSE) infections had significantly higher AD levels than with methicillin susceptible S. epidermdis (MSSE) (p = 0.003). ELISA and ADLF tests were positive with centrifuged (8/8) and native (8/8) synovial fluid. AD showed a solid diagnostic performance in infected and non-infected revisions, and it provided an additional value in the diagnosis of UPIC and UNIC associated to rTKAs. Pathogen virulence as well as antibiotic resistance pattern may have an effect on AD levels. Centrifugation of synovial fluid had no influence on ADLF results.

An Array SPRi Biosensor for Simultaneous VEGF-A and FGF-2 Determination in Biological Samples

A new method was developed for the simultaneous determination of vascular endothelial growth factor (VEGF-A) and fibroblast growth factor-2 (FGF-2) in blood serum, using biosensors with array Surface Plasmon Resonance imaging (SPRi) detection. It can be applied as a single method for simultaneous VEGF-A and FGF-2 determination or as two separate methods for testing only one selected protein in each case. Validation was carried out for each method. Limit of detection (LOD) and limit of quantification (LOQ) values were determined and were found not to differ significantly from the parameters obtained in comparisons with commercial enzyme-linked immunosorbent assay (ELISA) tests. Tests were carried out to check the robustness of the method. The results indicate a lack of robustness of the analytical method to elevated temperature and pH values other than those recommended by the manufacturers of the reagents (recommended pH = 7.40). The values of recoveries were determined and confirmed the reliability of the results obtained with the use of the newly developed method. The selectivity studies showed no negative influence of other proteins present in the matrix of the tested samples on the results of the VEGF-A and FGF-2 concentration measurements. The developed method is also characterized by high reproducibility of the results obtained and agreement with the VEGF-A and FGF-2 concentration values obtained with commercial ELISA tests. The proposed method offers fast, reproducible, and accurate simultaneous quantification of VEGF-A and FGF-2 in human body fluids. Only 4 µL of test sample are required for simultaneous analysis. The total time for simultaneous analysis of both biomarkers does not exceed 20 min. The developed analytical method is superior to ELISA in terms of analysis time and sample volume for analysis, and it offers lower LOD and LOQ values and allows for the simultaneous analysis of two biomarkers. There is also no need to collect a large number of samples. Standard ELISAs usually have 96 reaction wells. The proposed biosensor can be used to analyse only one sample, without the need to waste reagents on unused reaction sites. In addition, it is possible to regenerate the biosensor and reuse it.

Sensitivity and specificity for African horse sickness antibodies detection using monovalent and polyvalent vaccine antigen-based dot blotting.

The immune responses of animals infected with African horse sickness (AHS) virus are determined by enzyme-linked immunosorbent assay (ELISA), complement fixation, and virus neutralization test. During the outbreaks of AHS in Thailand, the immune response after vaccination has been monitored using commercial test kits such as blocking ELISA, which are expensive imported products unavailable commercially in Thailand. This study aimed to assess the sensitivity and specificity of anti-AHS virus antibodies using dot blotting based on monovalent and polyvalent strains of live attenuated AHS vaccine. A total of 186 horse sera, namely, 93 AHS-unvaccinated samples and 93 AHS-vaccinated samples, were used in this study. All sera underwent antibodies detection using commercial blocking ELISA and in-house dot blotting based on monovalent and polyvalent strains of live attenuated AHS vaccine. The numbers of true positive, false positive, true negative, and false negative results in the dot blotting were compared with those in blocking ELISA and the sensitivity and specificity of dot blotting were assessed. For the monovalent antigen, there were 78, 19, 74, and 15 true positive, false positive, true negative, and false negative results, respectively, while for the polyvalent antigen, the corresponding numbers were 84, 34, 58, and 9. Meanwhile, the diagnostic sensitivity and specificity for monovalent antigen were 83.87% and 79.57%, respectively, but 90.32% and 62.37% for polyvalent antigen. Dot blotting for AHS antibodies detection using vaccine antigen showed high sensitivity and rather a high specificity compared with the findings with the commercial ELISA test kit. In countries where commercial ELISA test kits are not available and when the size of a serum sample is small, dot blotting could become a good alternative test given its advantages, including its simplicity, rapidity, and convenience. To the best of our knowledge, these findings are the first report on the use of dot blotting for detecting AHS antibodies in horses. In conclusion, monovalent antigen-based dot blotting could be used as a reliable alternative serodiagnostic test for monitoring AHS humoral immune response, especially in vaccinated horses.

PD01 Using ELISA Tests For Monitoring Response To Anti-TNFs In Rheumatoid Arthritis: Findings From A NICE Health Technology Assessment

IntroductionPatients with severe rheumatoid arthritis (RA) disease may be treated with tumor necrosis factor (TNF)-α inhibitors. However, their efficacy is reduced by the presence of anti-drug antibodies. The objective of this HTA was to conduct a systematic review to investigate the effectiveness of using enzyme-linked immunosorbent assay (ELISA) tests (Promonitor, IDKmonitor, LISA-TRACKER, RIDASCREEN, MabTrack, and Sanquin Diagnostic Services) to measure levels of the drug and anti-drug antibodies for monitoring response to TNF-α inhibitors in patients with RA.MethodsA range of bibliographic databases including MEDLINE, EMBASE and CENTRAL were searched from inception to November 2018. Studies were eligible for inclusion if they investigated ELISA tests in RA patients receiving treatment with a TNF-α inhibitor who had achieved the treatment target (remission or low disease activity [LDA]) or experienced a primary or a secondary non-response. The tests must compare with standard care where treatment decisions are based on clinical judgements and monitoring using a composite score such as the disease activity score 28 joints (DAS28). Risk of bias was assessed using the Cochrane (ROBINS-1) tool for non-randomized studies.ResultsTwo studies were included. One non-randomized controlled trial (non-RCT) compared standard care with therapeutic drug monitoring using Promonitor test kits in RA patients in remission/LDA receiving adalimumab, and a historically controlled study investigated Sanquin ELISA kits. The non-RCT study showed that there was a non-significant reduction in the risk of flare in the intervention group compared with the control group. Patients’ health-related quality of life outcomes were higher in the intervention group at all visits compared with the control group. However, this study had serious limitations because analyses were not performed using an intention-to-treat approach. The historically controlled study did not provide valid evidence on whether ELISA-based monitoring is clinically effective.ConclusionsThere is only limited, poor evidence available. There are considerable uncertainties on the effectiveness of using ELISA tests for monitoring response to TNF-α inhibitors in RA. Further controlled trials are required.

BDNF-overexpressing MSCs delivered by hydrogel in acute ischemic stroke treatment.

Ischemic stroke treatment is a challenge worldwide. The efficacy and safety of mesenchymal stem cells (MSCs) for stroke have been confirmed. However, poor survival of MSCs in the ischemic environment limits the therapy efficacy. Changes in MSC status in the ischemic environment after transplantation is difficult to monitor. This study aimed to deliver brain-derived neurotrophic factor (BDNF)-overexpressing MSCs by hydrogel (H-B-MSCs) to promote recovery after ischemic stroke. MSCs were transfected with lentivirus carrying luc2 and BDNF cassette. The properties of hydrogel were tested after synthesis with thiolated gelatin (Gel-SH), thiolated hyaluronic acid (HA-SH), and polyethylene glycol diacrylate (PEGDA). Oxygen-glucose deprivation (OGD) test was carried out to confirm the protective effects of hydrogel in the ischemic environment. Three days after stroke induction, H-B-MSCs, hydrogel carrying MSCs (H-MSCs), or phosphate-buffered saline (PBS) was injected into the brains of mice, respectively. Bioluminescence imaging (BLI) was performed at 3, 7, 14, and 21 days post-cell-transplantation to monitor the dynamic status of MSCs. In the meantime, histology, quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbentassay (ELISA), western blot, and behavior tests were carried out at different time points. Hydrogel with good biocompatibility was synthesized. Lentivirus transfection significantly increased the expression of BDNF. BDNF-MSCs could be tracked by BLI in vitro. In vitro OGD/reperfusion (OGD/R) test results suggested that MSCs carried by hydrogel could survive longer in an environment with low oxygen and glucose. H-B-MSCs significantly improved functional recovery after ischemic stroke. Furthermore, H-B-MSCs treatment promoted neurogenesis, white matter recovery, and angiogenesis after ischemic stroke. MSC dynamics could be monitored in vivo with BLI. We effectively established a robust MSC delivery system with hydrogel. Prolonged survival of transplanted BDNF-MSCs with a hydrogel delivery system could promote the recovery of ischemic stroke via the continuous release of BDNF.

Serum Levels of Eosinophil-Derived Neurotoxin, Platelet-Activating Factor and Vascular Endothelial Growth Factor in Adult Patients with Atopic Dermatitis-A Pilot Study.

Atopic dermatitis (AD) is a chronic, highly pruritic, relapsing-remitting inflammatory skin disease. The etiology of AD has not been fully explained yet and complex interactions of various small molecules are still being taken into account. The aim of this research was to investigate the serum eosinophil-derived neurotoxin (EDN), platelet activating factor (PAF) and vascular endothelial growth factor (VEGF) concentrations in relation to the disease severity and pruritus intensity in adult patients with AD. This pilot study was performed on 30 participants (15 patients with AD and 15 healthy controls). Blood samples were taken to examine the serum levels of EDN, PAF and VEGF using the enzyme-linked immunosorbent assay (ELISA) test. The severity of disease was assessed by the Scoring Atopic Dermatitis (SCORAD) index. The intensity of pruritus, as a subjective symptom, was determined by the Visual Analogue Scale (VAS). Obtained results revealed that the EDN (p = 0.016) and VEGF (p = 0.032), but not PAF (p = 0.841) concentrations were significantly higher in patients with AD compared with those of the control group. There was positive correlation between the EDN level and the SCORAD index in patients with AD (r = -0.9, p = 0.037) which was not found for the PAF and VEGF levels. Circulating EDN, PAF and VEGF levels were not significantly correlated with the severity of pruritus. Our results suggest that the END and VEGF serum levels are significantly increased in patients with AD compared to control group. Moreover, EDN might be useful to reflect the severity of symptoms.

Randomized clinical trial to assess the impact of oral intervention with cetylpyridinium chloride to reduce salivary SARS-CoV-2 viral load.

Aerosols released from the oral cavity help spread the SARS-CoV-2 virus. The use of a mouthwash formulated with an antiviral agent could reduce the viral load in saliva, helping to lower the spread of the virus. The aim of this study was to assess the efficacy of a mouthwash with 0.07% cetylpyridinium chloride (CPC) to reduce the viral load in the saliva of Coronavirus disease 2019 (COVID-19) patients. In this multi-centre, single-blind, randomized, parallel group clinical trial, 80 COVID-19 patients were enrolled and randomized to two groups, namely test (n=40) and placebo (n=40). Saliva samples were collected at baseline and 2h after rinsing. The samples were analysed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and an enzyme-linked immunosorbent assay test specific for the nucleocapsid (N) protein of SARS-CoV-2. With RT-qPCR, no significant differences were observed between the placebo group and the test group. However, 2h after a single rinse, N protein concentration in saliva was significantly higher in the test group, indicating an increase in lysed virus. The use of 0.07% CPC mouthwash induced a significant increase in N protein detection in the saliva of COVID-19 patients. Lysis of the virus in the mouth could help reduce the transmission of SARS-CoV-2. However, more studies are required to prove this.

Preparation and evaluation of a lateral flow immunochromatographic nanogold diagnostic kit for brucellosis in sheep.

Brucellosis is a zoonotic disease with a worldwide distribution. It has a serious impact on the health of humans and animals, along with a negative impact on the economy. This study aimed to prepare and evaluate the diagnostic performance of a lateral flow immunochromatographic test (LFIT) nanogold diagnostic kit for detecting brucellosis in sheep. A rapidly developed LFIT, in which lipopolysaccharide conjugates with nanogold molecules, was placed on the conjugate pad. One hundred ovine serum samples were tested to detect Brucella antibodies (Ab) using the prepared lateral flow immunochromatography assay (LFA) kit and Rose Bengal test. The evaluation of specificity, sensitivity, and accuracy for LFIT and Rose Bengal plate test was conducted using the P04310-10 IDEXX brucellosis ovine/caprine Ab enzyme-linked immunosorbent assay (ELISA) test (gold standard). The lower amount of Brucella Ab in the ovine serum samples was detected and was 1.58 S/P ratio ELISA titer/100 μL using LFIT and with Rose Bengal to detect 1.86 S/P ratio ELISA. The results showed that the developed LFIT had high specificity with no cross-reactivity with other tested bacteria. The calculated sensitivity, specificity, and accuracy of LFIT and Rose Bengal test using the P04310-10 IDEXX brucellosis ovine/caprine Ab ELISA test (gold standard) were 74% and 89%, 81% and 59%, and 76.9% and 66%, respectively. The present results showed interesting results implying that the LFIA strip test could be used as a substantial diagnostic tool for field screening ovine Brucella as an essential step in the control of brucellosis. However, further studies for the validation of the present findings are necessary.

Competitive ELISA test system for the detection of antibodies to the rabies virus in animals (Rhabdoviridae: Lyssavirus)

The main approach to the rabies prevention is the vaccination of domestic and wild carnivores. For the routine evaluation the anti-rabies vaccination effectiveness, World Organization for Animal Health (OIE) recommends various enzyme-linked immunosorbent assays (ELISA).The aim of the study was to design and validate a competitive ELISA (cELISA) test system for the detection of antibodies to the rabies virus (RABV). The development of the cELISA was carried out following the OIE recommendations. The repeatability of the cELISA results within one laboratory was satisfactory (coefficient of variation 7.95-13.61%). The coefficient of determination (CD) between the results of the virus neutralization reaction (FAVN) and cELISA was 0.988, p &lt; 0.001. The lower threshold for antibody detection was less than 0.02 IU/ml. The cELISA did not demonstrate cross-reactivity against antibodies to canine distemper virus, parainfluenza virus, parvovirus, coronavirus, and canine adenovirus (types I and II). During the study of 137 dog blood sera, diagnostic specificity (DSp) and diagnostic sensitivity (DSe) for the cELISA were 83.1% and 94.9%, respectively, and CD between the cELISA and FAVN results was 0.968, p &lt; 0.001. Indirect ELISA test systems for determining the level of antibodies to RABV are not sensitive enough compared to reference tests, unlike cELISA. The developed test system is not inferior for its DSp and DSe to the commercial cELISA BioPro ELISA Rabies Ab (DSp 66.7%, DSe 94.4%). The developed cELISA test system can be used to detect antibodies to RABV in the blood serum of dogs for evaluating the effectiveness of mass vaccination programs.

Starry Sky on MRI Brain: Unusually Observed in a Pediatric Case of Japanese Encephalitis

A 13-year-old girl presented with fever, nausea and projectile vomiting and altered sensorium. On physical examination, the meningeal signs were presented without focal neurological deficits and Grade Ⅱ papilledema was presented on fundus examination. The patient was found to be positive for Japanese encephalitis virus in cerebrospinal fluid. Focal neurological deficit in the form of right hemiparesis was noticed at day 6 of hospitalisation. Multiple lesions with minimal enhancement with perilesional edema were observed in cerebral cortex in contrast with enhanced magnetic resonance imaging of the brain, givingrise to starry sky appearance. No tests for neurotuberculosis were found positive. Serum IgG enzyme-linked immunosorbent assay test was negative for Cysticercosis, but magnetic resonance spectroscopy was suggestive of cysticercal meningitis. During treatment, she had intermittent episodes of nausea and vomiting with waxing and waning sensorium. Subsequently, it was planned to start Albendazole after steroids coverage and anticonvulsants but the patient did not improve and succumbed to her illness.

Seroprevalence and Risk Factors of Brucellosis in Ruminants in Dhofar Province in Southern Oman.

The aim of the present work was to raise awareness of Brucella infection and emphasize the use of serological tests for screening and confirmation of the presence of the infection in different localities in the Dhofar region in the Sultanate of Oman. A seroprevalence of Brucella infection in naturally infected livestock was undertaken in 50 farms (a total of 434 sera, 207 goats, 84 sheep, 54 cattle, and 89 camels) from different wilayat of the Dhofar region in the southern part of Oman. Rose Bengal (RBT), complement fixation (CFT), and indirect enzyme-linked immunosorbent assay (I-ELISA) tests were used to determine the presence of Brucella antibodies. Statistical analysis (Pearson chi-square, binary logistic regression, and univariate logistic regression) was used to investigate the significance between the prevalence and the categorical risk factors individually, with two or more levels (animal species, animal condition, and or location). Our results show that the overall seroprevalence based on CFT, RBT, and I-ELISA was 3% (13/424, CI: 1.8-5.1%), 4.8% (21/434, CI: 3.1-7.3%), and 8% (35/434, CI: 5.8-10.9%), respectively. The highest seroprevalence was reported in goats (13% (27/207)) and animals from East Jabal (13% (21/161)), whereas the lowest was recorded in camels (3.4% (3/89)) and animals from deserts (1.4% (1/69)). Parameters such as the positive predictive value (PPV) and the negative predictive value (NPV) showed that the sensitivity of I-ELISA and CFT based on the RBT test was 61.9% and 57.1%, respectively, whereas the specificity of I-ELISA (94.6%) was less than that of CFT (97.33%). We concluded that three tests are confirmatory for the presence of Brucella infection.