Enzyme-linked Immunosorbent Assay Test Research Articles

Preparation of colloidal gold test strips for the detection of antibodies to peste des petits ruminants based on monoclonal antibodies to N protein

A simple, fast, and visual method for detecting antibodies against peste des petits ruminants virus (PPRV) using colloidal gold strips was developed. In this study, the pET-32a-N was transformed into Escherichia coli Rosetta (DE3) for expression. Hybridoma cell lines were generated by fusing SP2/0 myeloma cells with splenocytes from immunized mice with the expressed and purified N protein of PPRV. The PPRV N protein was labeled with colloidal gold particles as the gold-labeled antigen. The N protein served as the gold standard antigen and as the test (T) line-coated antigen, while the monoclonal antibody served as the quality control (C) line-coated antibody to assemble the colloidal gold immunochromatographic test strips for detecting antibodies against the N protein of PPRV. Hybridoma cell line designated as 1F1 was able to stably secrete the monoclonal antibody against the N protein of PPRV. The titer of 1F1 monoclonal antibody in ascites was 1:128 000 determined by indirect enzyme-linked immunosorbent assays (ELISA), and the immunoglobulin subtype of the monoclonal antibody was IgG1, with kappa chain. The obtained monoclonal antibody was able to specifically recognize the N protein of PPRV, as shown by Western blotting and indirect immunofluorescent assay (IFA). The developed colloidal gold test strip method was able to detect PPRV antibodies specifically, and there was no difference between different batches of the test strips. Testing of a total of 122 clinical sera showed that the compliance rate of the test strip with ELISA test was 97.6%.The test strip assay developed in this study has good specificity, reproducibility, and sensitivity, and it can be used for the rapid detection of PPRV antibodies.

Evaluation of humoral immune responses of experimental animals to the recombinant SARS-CoV-2 spike ectodomain with the ISCOM adjuvant

Scientific relevance. The use of recombinant antigens in vaccine production is limited because vaccines based on such antigens tend to have low immunogenicity. However, a COVID-19 vaccine that combines recombinant SARS-CoV-2 spike glycoprotein as its antigen and virus-like immune-stimulating complexes (ISCOMs) as its adjuvant (Nuvaxovid) induces a protective virus-neutralising response. The State Research Center of Virology and Biotechnology “Vector” (hereinafter, Vector) has developed the ISCOM adjuvant Matrix-V, which plays a key role in inducing virus-neutralising antibodies. Studying Matrix-V will provide for the wide use of recombinant antigens combined with this adjuvant in the development and production of novel Russian vaccines.Aim. This study aimed to evaluate the humoral immune responses of experimental animals to intramuscular injections of a complex combining the recombinant Wuhan-type SARS-CoV-2 spike RBD antigen and the virus-like ISCOM adjuvant containing Quillaja saponaria saponins.Materials and methods. The Matrix-V ISCOM adjuvant was produced using Vector’s proprietary technology, which involves cross-flow filtration through Sartorius VivaFlow cassettes. To determine the saponin and residual detergent concentrations in Matrix-V, the authors conducted high-performance liquid chromatography. Having produced the recombinant SARS-CoV-2 RBD antigen, the authors used electron microscopy to analyse the ultrastructure of the ISCOM–antigen complex. In the study of the ISCOM–antigen complex, 25 female Balb/c mice (5 groups) and 15 male and female outbred guinea pigs (3 groups) received two intramuscular injections with a 14-day interval. Serum tests relied on virus neutralisation (VN) and enzyme-linked immunosorbent assay (ELISA) methods and used antigens of 8 SARS-CoV-2 variants (State Collection of Viruses and Rickettsia, Vector). The authors used Statistica 10 to analyse the results.Results. Two injections of the SARS-CoV-2 RBD antigen (mice: 7 μg, guinea pigs: 1 μg) alone did not induce statistically significant virus-neutralising antibody responses, as shown by the VN results. Two injections of the SARS-CoV-2 RBD antigen (mice: 7 μg, guinea pigs: 1 μg) adjuvanted with Matrix-V (25 μg) resulted in geometric mean antibody titres of 1:83–1:178 (mice) and 1:174–1:587 (guinea pigs) in the VN tests with the Wuhan variant. One injection of the antigen (1 μg or 7 μg) with Matrix-V (25 μg) induced antibodies only in individual cases, as demonstrated by the VN and/or ELISA results. The most intensive immune response was observed in ELISA tests with the Delta variant after two injections of the Ecto-S-Wuhan (1 μg) and Matrix-V (25 μg) complex. Immune responses did not differ between the group that received two injections of the Ecto-S-Wuhan antigen (1 μg) without the ISCOM adjuvant and the negative control group (titres below 1:100; p=0.95). Two injections of the SARS-CoV-2 RBD antigen (7 μg) without the ISCOM adjuvant induced antibodies in mice (titres between 1:248 and 1:1477).Conclusions. Two intramuscular injections of the complex containing the recombinant SARS-CoV-2 RBD antigen and the Matrix-V ISCOM adjuvant induce virus-neutralising antibodies. The approach proposed by the authors has the potential for use in the development of immunobiological medicinal products to prevent and treat a wide range of infectious diseases.

SEROPREVALENCE OF IgG ANTIBODIES OF HUMAN TOXOCARIASIS AND ASOCIATED RISK FACTORS IN KHANH HOA PROVINCE

Background: Human toxocariasis is a zoonotic parasitic disease caused by Toxocara canis and T. cati roundworm larvae. While many studies have shown that dog and cat owners are at a higher risk of acquiring Toxocara spp. infection, there is no available evidence regarding the seroprevalence of Toxocara spp. infection among dog and cat owners in Khanh Hoa. Therefore, this study aims to investigate the prevalence of anti-IgG of Toxocara spp. infection and associated risk factors for visceral larval migrans among dog owners in three different areas of Khanh Hoa province..Subjects and methods: A descriptive cross-sectional study was conducted from 2022 to 2024 in three localities representing different socio-geographic areas (urban, rural, and mountainous) of Khanh Hoa province. A total of 1502 blood samples were collected from local individuals aged 5 to 75 for this study. Serum anti-T.canis IgG antibodies were detected using a commercial enzyme-linked immunosorbent assay (ELISA) kit: NovaLisaTM, Toxocara canis IgG ELISA. Simultaneously, a survey using structured questionnaire about information on risk factors associated with Toxocariasis was conducted among 720 people (aged 18 and over) from 1,502 individuals whose bloods were taken for testing.Results and conclusions: A total of 1,502 blood samples were examined for the presence of antibodies to dog and cat roundworm larval infection. Results revealed an overall positive rate of 57.66% across the three localities, with variations depending on geography. In Khanh Vinh, a mountainous district, the infection rate was 75.44%, and in its commune Khanh Trung, it was 81.20%. For the two urban wards, the rates were significantly lower (Phuoc Long Ward: 44.40% and Van Thanh Ward: 54.18%). A structured questionnaire was used to investigate risk factors among 720 individuals (aged 18 years old and over) out of the 1,502 individuals whose bloods were tested. Results indicated that 389 of them tested positive for the ELISA test. Infection rates varied among age groups: under 25 years old (6.94%), 25-50 years old (66.78%), and over 50 years old (37.28%). Notably, 49.87% of infected individuals owned dogs or cats, 93.32% regularly consumed raw vegetables, and 25.71% frequently handled dogs or cats. The high infection rate of dog and cat roundworm larvae in Khanh Hoa underscores the presence of several identified high-risk factors.

Analysis of subgingival bacterial and fungal diversity in patients with peri-implantitis based on 16sRNA and internal transcribed spacer sequencing.

Aim: To analyze subgingival fungal diversity in peri-implant inflammation patients and their relationship with bacteria. Methods: We collected saliva samples from four groups. 16sRNA and internal transcribed spacer sequencing was performed preceded by quantitative PCR and enzyme-linked immunosorbent assay tests. Analyses were done usingR and Cytoscape software. Results: Significant differences were observed in the Abundance-based Coverage Estimator(ACE) index between control and peri-implantitis samples. Basidiomycota was the dominant fungal species, while Firmicutes dominated thebacteria. The most abundant fungal and bacterial species were 's_unclassified g Apiotrichum' and 's_unclassified g Streptococcus', respectively. Dothiorella was strongly associated with immunoglobulin G levels, with positive correlations between specific microorganisms and peri-implantitis in Q-PCR. Conclusion: Our findings have significant clinical implications, suggesting specific fungal and bacterial taxaroles in peri-implant inflammation.

A single synthetic lipid antigen for improved serological diagnosis of Buruli ulcer

SETTING: The diagnosis of Buruli ulcer (BU) is frequently made by experienced health workers in rural regions. This leads to long turnaround times to confirm the diagnosis as it requires specialised laboratory infrastructure to perform confirmatory testing.BACKGROUND: Given the lack of success with protein antigens to detect BU in human sera, the aim of this study was to evaluate a range of single synthetic lipid antigens using an enzyme-linked immunosorbent assay (ELISA). The ELISA system used was initially developed to detect TB using single synthetic lipid antigens.METHODS: Thirty polymerase chain reaction (PCR) positive BU samples and 30 PCR-negative healthy contact samples collected from Asante Akim North and Ahafo Ano North Districts, Ghana, that are endemic for BU between 2013 and 2016 were used to evaluate the synthetic lipid antigen ELISA. A Quantikine ELISA was also conducted on a randomly blinded sub-set of 30 samples.RESULTS: The synthetic lipid ELISA evaluated here outperforms all other ELISA tests using protein antigens to detect BU to date and has shown potential as a fast (2 h) test for BU which may be adapted for use at the point of care. A sensitivity of 63% and specificity of 80% was observed for 30 BU-positive and 30 BU-negative samples, with significantly reduced interleukin-8 (IL-8) levels in a subset of patients with BU.CONCLUSION: A single lipid was shown for the first time to have the ability to distinguish between PCR-positive BU and negative sera using ELISA. The low lipid antibody load detected may be a result of immune suppression caused by the presence of mycolactone in patients with BU, given that levels of IL-8 were significantly reduced in patients with BU compared to the control serum samples.

Rapid immunochromatographic test: An evolving tool for diagnosis of scrub typhus

Background: Scrub typhus is prevalent in many districts of South Bengal throughout the year where an average temperature of 20–35°C, which contributes to the spread of Leptotrombidium deliense. However, its diagnosis remains complicated by the lack of readily available and validated assays, the non-specificity of clinical symptoms on admission, and even non-availability of the pathognomonic eschar in most of the cases. Aims and Objectives: This study was carried out to evaluate the rapid immunochromatographic test (RICT) for early detection of scrub typhus for using it as an early diagnostic tool at the field level. Materials and Methods: This cross-sectional study in which 181 serum samples from clinically suspected cases (after excluding dengue, malaria, Japanese encephalitis, and typhoid fever) collected over 13 months were processed for the detection of immunoglobulin M (IgM) antibodies for scrub typhus by enzyme-linked immunosorbent assay (ELISA) and rapid test. Results: Considering IgM ELISA for scrub typhus as the gold standard, the sensitivity, specificity, positive predictive value, and negative predictive value for RICT were found to be 100%, 86.87%, 50%, and 100%, respectively. Conclusion: RICT is a simple, rapid, and reliable assay for diagnosis of scrub typhus, capable of providing accurate results quickly and is highly suitable for field deployment in remote areas with limited medical support.

Prevalence and Genotype Distribution of Hepatitis C Virus Infection at Zonal Hospital in Northern India

Abstract Background: Hepatitis C virus (HCV) infection is a major public health concern. Persons living with HCV infection are at risk of developing liver cirrhosis and progressing to end-stage liver disease and liver cancer (hepatocellular carcinoma). The present study was undertaken to assess the prevalence of HCV infection in patients admitted as well as outpatient department services of a Zonal Hospital. Methods: The present study was cross-sectional, observational study, conducted in patients >18 years of age, clinically suspected acute infectious hepatitis or admitted to the intensive care unit, and willing to participate in the study. Results: In the present study, 472 patients were studied for hepatitis C infection. Majority of cases were from 51 to 60 years age group (34.5%), followed by 61–70 years age group (26.7%). Female cases were (51.3%) slightly more than male (48.7%). Rapid method and enzyme-linked immunosorbent assay (ELISA) methods detected HCV prevalence as 5.7% and 12.5%, respectively. After HCV genotype testing, the prevalence was 12.1%. Majority had 3A genotype (69.49%), followed by 1A genotype (16.95%), 1B genotype (8.47%), 3B genotype (3.39%), and 4 genotypes (1.69%). ELISA testing was significantly correlated with HCV genotype testing (P = 0.017) while the rapid method was not correlated with HCV genotype testing (P = 0.937). Conclusion: There is a high prevalence of HCV among the suspected, genotype 3 being the most common.

Biological Evaluation of the Osteoinductive Potential of Dry Teeth after Chemical Demineralization Treatment Using the Tooth Transformer Device.

Several studies have already demonstrated the biocompatibility of a tooth as a grafting material in the regeneration of bone tissue, showing its osteoconductive potential, while no studies have verified whether the osteoinductive potential of a tooth remains constant or is altered after its treatment with the Tooth Transformer (TT) device. The aim of the study was to demonstrate that the treatment with the TT device did not alter the osteoinductivity of an extracted tooth that was stored dry. Twelve extracted human teeth were collected from real patients. Caries, tartar and filling materials were removed from each tooth; each tooth was coarsely cut and stored at room temperature (RT) until use. Each sample was shredded, demineralized and disinfected, using the TT device. Protein extraction was carried out for each sample, and Western Blot analysis was performed to test the presence of mineralization protein LIM-1 and transforming growth factor-β. The presence of the human Bone Morphogenetic Protein 2 (BMP-2) and human collagen Type I (COL-I) was found in dry tooth samples processed with the TT device and subjected to Enzyme-Linked Immunosorbent Assay (ELISA) testing. The treatment of chemical demineralization using the TT device does not alter the osteoinductive potential of a dry tooth.

FEEDING PATTERN EFFECT ON ORAL HEALTH PARAMETERS AND SALIVARY IL-6 LEVEL AMONG PRESCHOOL CHILDREN

Background: Interleukin-6 plays a role in both regenerative and anti-inflammatory processes as well as inflammatory reactions. Various cytokines can be produced as a result of caries, which can then trigger oral immune reactions. Different inflammatory states, such as gingivitis, periodontitis, and oral lichen planus, were discovered to have high cytokine levels. This study aimed to evaluate the effect of feeding patterns on oral health parameters (dental caries, gingival health status) and age by measuring the level of salivary IL-6. Material and methods: The study sample included 61 children aged 4-6 years old divided into two groups (breastfeeding group and formula feeding group). The examination of the oral cavity was done on the basic method of oral health surveys of the World Health Organization by using dmfs, plaque, and gingival index according to recording protocols to measure the severity of dental caries and gingivitis. Saliva samples were collected and subjected to an enzyme-linked immunosorbent assay (ELISA) test to measure the level of IL-6. Results: Data of the present study showed a highly significant difference in salivary IL-6 levels between the breastfeeding group and formula-feeding group, and the median level of IL-6 was 5.5 in the breastfeeding group while in the formula-feeding group, it was 32.2. The results were statistically significant in the breastfeeding group between IL-6 and caries rate dmfs (p0.048), IL-6 and gingival index (p0.035), and dmfs with age (p0.049), while for formula feeding group these results were statistically non-significant. Conclusion: In preschool-aged caries rate (dmfs) is strongly correlated to the salivary IL-6 level. IL-6 level has a potent relation to the gingival index with a positive direction so it can be used as an early indicator for the presence of gingivitis and carious lesion. Breastfeeding has a direct effect to reduce the level of salivary IL-6 so it will affect the correlation between IL-6 levels and oral health parameters.

Developing a Novel Non-invasive Serum-based Diagnostic Test for Early Detection of Colorectal Cancer

Abstract Introduction/Objective Colorectal cancer (CRC) is the third-most common cancer worldwide, and early detection is vital for successful treatment and improved outcomes. Current screening methods such as colonoscopy, fecal occult blood tests, and stool DNA tests have limitations such as patient discomfort, cost, and low sensitivity and specificity most commonly diagnosed cancer worldwide with a high mortality rate. Early detection remains a crucial factor in reducing the burden of CRC. Currently, the gold standard for CRC diagnosis is colonoscopy, but it may not be feasible for widespread screening due to its invasiveness, cost, and patient compliance issues. Therefore, there is a great need to identify reliable biomarkers that can detect CRC at an early stage. This study aimed to evaluate the effectiveness of updated novel biomarkers for early detection of CRC. Methods/Case Report This study was conducted on 158 patients with suspected or confirmed CRC. The study population included patients of both genders, aged above 18 years, and with a history of CRC or other relevant risk factors. Blood samples were collected from all included patients, and the biomarkers (CTCS, miRNAs, ctDNA, and Methylated DNA markers ) were measured using enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) tests. Novel biomarkers compared their diagnostic performance with existing biomarkers such as fecal occult blood tests (FOBTs), fecal immunochemical tests (FITs), and CEA. Results (if a Case Study enter NA) The studied biomarkers (CTCs, microRNAs, ctDNA, and Methylated DNA markers) have high sensitivity from 78.8 % to 96.72% and accuracy from 83.76% to 94.83% as well as their cut-off which approved the objective of this study. Overall, these novel biomarkers show promise for early detection of colorectal cancer and may improve screening and early detection efforts. Conclusion The evaluation of the studied biomarkers (Circulating Tumor Cells, microRNAs, Circulating tumor DNA, and Methylated DNA markers) for early detection of CRC has substantial clinical and public health implications. The identification of reliable biomarkers for CRC screening can avoid unnecessary colonoscopies, reduce the burden on healthcare costs, and improve the detection of early-stage CRC.

Limited value of current and new in silico predicted oocyst-specific proteins of Toxoplasma gondii for source-attributing serology

Toxoplasma gondii is a zoonotic parasite infecting all warm-blooded animals, including humans. The contribution of environmental contamination by T. gondii oocysts to infections is understudied. The aim of the current work was to explore T. gondii serology as a means of attributing the source of infection using a robust stepwise approach. We identified in silico thirty-two promising oocyst-specific antigens from T. gondii ´omics data, recombinantly expressed and purified them and validated whether serology based on these proteins could discriminate oocyst- from tissue cyst-driven experimental infections. For this, three well-characterized serum panels, sampled from 0 to 6 weeks post-infection, from pigs and sheep experimentally infected with T. gondii oocysts or tissue cysts, were used. Candidate proteins were initially screened by Western blot with sera from pigs or sheep, infected for different times, either with oocysts or tissue cysts, as well as non-infected animals. Only the recombinant proteins TgCCp5A and TgSR1 provoked seroconversion upon infection and appeared to discriminate between oocyst- and tissue cyst-driven infections with pig sera. They were subsequently used to develop an enzyme-linked immunosorbent assay test for pigs. Based on this assay and Western blot analyses, a lack of stage specificity and low antigenicity was observed with all pig sera. The same was true for proteins TgERP, TgSporoSAG, TgOWP1 and TgOWP8, previously described as source-attributing antigens, when analyzed using the whole panels of sera. We conclude that there is currently no antigen that allows the discrimination of T. gondii infections acquired from either oocysts or tissue cysts by serological tests. This work provides robust new knowledge that can inform further research and development toward source-attributing T. gondii serology.

Efficacy of curcumin gel as an adjunct to scaling and root planing on salivary procalcitonin level in the treatment of patients with chronic periodontitis: a randomized controlled clinical trial

The aim of the studyTo evaluate the effect of curcumin gel combined with scaling and root planing (SRP) on salivary procalcitonin in periodontitis treatment.Materials and methodsseventy patients were selected from the Department of Oral Medicine and Periodontology, Faculty of Dentistry, Mansoura University, and sixteen patients were excluded. Patients in groups II and III included stage II grade A periodontitis. The participants were classified into three groups: group I as a negative control group (individuals with healthy gingiva), group II (SRP) were treated with SRP, and group III (curcumin gel) which was applied weekly for four weeks after SRP. Clinical indices (plaque index (PI), gingival index (GI), clinical attachment level (CAL), and probing depth (PD)) and saliva samples for procalcitonin (PCT) assessment using an enzyme-linked immunosorbent assay (ELISA) test were collected and measured at both baselines and after six weeks.ResultsThis randomized controlled clinical trial registered on ClinicalTrials.gov (NCT05667376) and first posted at 28/12/2022 included Fifty-four patients (20 male; 34 female). Regarding the age and sex distribution, there was no statistically significant difference between the three studied groups (p > 0.05). There was no significant statistical difference regarding PI, GI, PPD, and CAL between group II and group III at baseline p (> 0.05). However, there was a significant statistical difference regarding the clinical parameters at baseline of both group II and group III as compared to group I (p ≤ 0.05). At six weeks after treatment, group III showed greater improvement in the PI, PD, and CAL as opposed to group II (p ≤ 0.05). Regarding PCT values, at baseline, there wasn’t a statistically significant difference between group II and group III (p > 0.05). However, there was a significant statistical difference between group II, group III, and group I (p ≤ 0.05). At six weeks after treatment, there was a statistically significant decrease in PCT levels of both group II and III (p ≤ 0.05).ConclusionThe application of curcumin gel was found to have a significant effect on all clinical indices as opposed to SRP.

Local nasal immunotherapy with birch pollen-galactomannan conjugate-containing ointment in mice and humans

BackgroundAllergen immunotherapy (AIT) is the only disease-modifying treatment for immunoglobulin (Ig) E-mediated allergy. Owing to the high prevalence and early onset of hay fever and pollen-food allergy syndrome (PFAS), a safer and simpler treatment method than conventional AIT is needed. To develop a local nasal immunotherapy using an ointment containing hypoallergenic pollen and assess its efficacy in mice and healthy humans. MethodsHypoallergenicity was achieved by combining pollen and galactomannan through the Maillard reaction to create birch pollen-galactomannan conjugate (BP-GMC). The binding of galactomannan to Bet v 1 was confirmed using electrophoresis and Western blotting (WB). Binding of specific IgE antibodies to BP-GMC was verified using enzyme-linked immunosorbent assay (ELISA) and basophil activation test (BAT). The localization of BP-GMC absorption was confirmed using a BALB/c mouse model. BP-GMC mixed with white petrolatum was intranasally administered to 10 healthy individuals (active drugs, 8; placebo, 2) for 14 days. ResultsIn electrophoresis and WB, no 17-kDa band was observed. In ELISA and BAT, BP-GMC did not react to specific IgE but was bound to IgA and IgG. In the mouse model, BP-GMC was detected in nasopharyngeal-associated lymphoid tissues. In the active drug group, the salivary-specific IgA level significantly increased on day 15 (p = 0.0299), while the serum-specific IgG level significantly increased on day 85 (p = 0.0006). ConclusionsThe BP-GMC ointment rapidly produced antagonistic antibodies against IgE; it is safe and easy to use and might serve as a therapeutic antigen for hay fever and PFAS.

Performance of a New Rapid Point-of-Care Test for Infliximab Levels in Patients with Inflammatory Bowel Disease: A Comparison to ELISA

BackgroundTherapeutic drug monitoring of infliximab levels in patients with inflammatory bowel disease (IBD) optimizes patients’ treatment. The reference technique is based on enzyme-linked immunosorbent assay (ELISA) although point of care (POC) assays are being developed.AimsTo assess the performance of a new rapid immunochromatographic POC assay (Promonitor Quick IFX) compared with ELISA technique to measure infliximab levels in patients with IBD.MethodsA prospective, observational, unicentric study was performed on capillary blood samples from patients with IBD before infliximab infusion (trough levels). Infliximab levels and anti-infliximab antibodies were measured using the ELISA technique (Promonitor IFX) and the POC assay. Correlation between both techniques was assessed by Pearson’s coefficient. Quantitative differences were evaluated by Bland–Altman analysis. Samples were stratified according to infliximab therapeutic ranges (< 3 μg/mL, 3–8 μg/mL, and > 8 μg/mL).ResultsA total of 135 experimental samples were assessed. Infliximab levels showed a high correlation between POC and ELISA tests (r = 0.84, P < 0.001). The mean difference between tests was 1.46 μg/mL (P < 0.001), being minimal for concentrations < 8 μg/mL. POC and ELISA assays showed an overall concordance of 87.4%. Most samples were in the same therapeutic range, which lead to equivalent therapeutic decisions. POC and ELISA assays detected the presence of anti-infliximab antibodies in 2.2% and 3.7% of the samples, respectively.ConclusionsPOC assay results in blood samples from patients with IBD were comparable to those obtained with the reference ELISA technique. The POC assay could be considered for routine testing based on its ease of use and rapidity.

Evaluation of Biodegradable Microparticles for Mucosal Vaccination Against Diphtheria Toxoid: Nasal Efficacy Studies in Guinea Pigs.

In this study, poly-(ɛ-caprolactone) (PCL) and poly-(lactic-co-glycolic acid) (PLGA) microparticles encapsulating diphtheria toxoid (DT) were investigated for their potential as a mucosal vaccine delivery system. Antigen-containing microparticles were prepared using the double emulsion (w/o/w) solvent evaporation method. The average geometric diameter of the particles was found to be between 7 and 24 µm, which is suitable for uptake by the antigen-presenting cells in the nasal mucosa. Although the differences were insignificant, the PLGA polymer-containing formulations exhibited the highest encapsulation efficiency. Microparticle formulations, prepared with both PLGA and PCL polymers, were successfully produced at high production yields. The in vitro release profile was presented as a biexponential process with an initial burst effect due to the release of the protein adsorbed on the microsphere surface, and the subsequent sustained release profile is the result of protein diffusion through the channels or pores formed in the polymer matrix. DT-loaded microparticles, DT solution in phosphate-buffered saline (PBS), and empty microparticles (control) were administered via nasal route and subcutaneously to guinea pigs. The antibody content of each serum sample was determined using an enzyme-linked immunosorbent assay (ELISA). Absorbance values of the ELISA test showed that PLGA- and PCL-bearing microparticles could stimulate an adequate systemic immune response with intranasal vaccination. In addition, PLGA and PCL microparticles resulted in significantly increased IgG titers with intranasal administration as a booster dose following subcutaneous administration. PCL polymer elicited a high immune response compared with PLGA polymer (p <0.05).

PSXII-9 Identifying Selection Signatures for Immune Response and Resilience to Aleutian Disease in Mink Using Genotypes Data

Abstract Aleutian disease (AD), a severe immune-complex disease, brings tremendous financial losses to the mink industry. Resilience is the ability of an animal to minimize the influences caused by disturbances and maintain its performance under pathogen exposure. Many AD-positive farms have selected AD-resilient mink based on AD tests and/or AD-resilience indicator traits, such as growth, pelt quality, and reproduction. Selection based on these indicator traits may have changed the patterns of genetic variation and potentially represent a genomic signature that can be used to identify genes subjected to selection. Therefore, the main objective of this study was to identify the selection signatures related to immune response (IRE) and resilience to AD. . A total of 1,411 mink in five color types (black, demi, mahogany, pastel, and stardust) from the Canadian Centre for Fur Animal Research (Truro, Nova Scotia, Canada), which is an AD-positive facility, were genotyped using the Axiom Affymetrix Mink 70K single nucleotide polymorphism (SNP) panel. For the IRE trait, 264 mink were grouped into pairwise groups based on the combined results of counter-immunoelectrophoresis (CIEP) and enzyme-linked immunosorbent assay (ELISA) tests. Individuals in black (n = 29), demi (n = 131), mahogany (n = 76), and pastel (36) color types were also grouped based on combined results of CIEP and ELISA tests, respectively. For resilience traits, two methods were used to group CIEP-positive individuals into pairwise groups: 1) based on their general resilience performance (GRP, n = 30), which was measured by considering individual performance for feed conversion ratio, Kleiber ratio, and pelt quality, and 2) based on female reproductive performance (FRP, CIEP-positive dams only, n = 36), which was measured by the number of kits alive 24h after birth. The pairwise fixation index, nucleotide diversity, and cross-population extended haplotype homozygosity were used to detect selection signatures. Only SNPs detected by at least two methods were considered selection signatures candidates, including 619, 569, and 526 SNPs for IRE, GRP, and FRP traits, respectively. The genes annotated from the candidate SNPs were associated with previously reported traits influenced by AD, including immune system process, growth, reproduction, and pigmentation. Notably, two olfactory-related gene ontology (GO) terms were significant (q &amp;lt; 0.05) for all studied traits, suggesting AD might cause loss of smell in infected mink. Additionally, the differences in genes and GO terms detected across different color types for IRE indicated that mink of different color types may differ in immune response to AD. The mitogen-activated protein kinase (MAPK) signalling pathway was significant (q &amp;lt; 0.05) in Kyoto Encyclopedia of Genes and Genomes pathway analyses for FRP, indicating AD infection may disorder the MAPK signalling pathway, in turn influencing female reproductive performance. Our study has enhanced the understanding of genomic architecture underlying resilience of mink to AD and sheds light on the underlying biological mechanisms involved.

3Ts Score, an Attempt for Less Testing in Thrombocytopenia Patients with Suspected Heparin Induced Thrombocytopenia (HIT): Retrospective Analysis in a University Hospital Setting

Background: Heparin-induced thrombocytopenia (HIT) is a prothrombotic and potentially fatal adverse drug reaction mediated by platelet-activating antibodies against multimolecular complexes of platelet factor 4 (PF4) and heparin. The 4Ts is a pretest scoring system for HIT that was developed to improve and standardize clinical diagnosis. The strength of the 4Ts score lies in the negative predictive value of a low pretest probability score. Those with a 4T score ≥4 is considered to have moderate to high probability of HIT and should undergo confirmatory laboratory test. Delays in treatment are associated with risk of thrombosis. Immediate cessation of heparin and initiation of a non-heparin anticoagulant is therefore standard of care. Method: This is a single institutional retrospective study. We reviewed the charts of 219 patients with thrombocytopenia with suspected HIT to evaluate the appropriateness of ordering the HIT testing based on the pretest probability of the 4Ts scoring system. All patients had enzyme linked immunosorbent assay (ELISA) test for HIT antibodies (the screening test). We studied tests that were sent from 03\16\2021 through 07\03\2023. The serotonin release assay (SRA) which is the confirmatory test for HIT was only sent for patients who had a positive ELISA test. 4T scores were calculated retrospectively using chart's information available to clinicians at the time of the HIT tests were sent. Results: Out of the 219 patients, 80(36.5%), 120(54.7%) and 19(8.6%) patients had a low, intermediate and high pretest probability respectively. The HIT ELISA test was positive in 33 (15.7%) patients and 196 (84.9%) patients had negative results. HIT ELISA had 100% and 93.5% sensitivity and specificity respectively while the positive and negative predicative values were 60% and 100% respectively. A positive SRA test was reported in 23 patients (10.5% of the total study population). Seventy-nine patients (36% of our total sample) scored 4 points which represents intermediate pretest probability. Out of these 79 patients only 6 (11.3%) patients had a positive ELISA test, and 5 (6.3%) patients had a positive SRA test. Aiming to minimize the HIT over testing we examined the increase in the cutoff point of the intermediate pretest probability from 4 points to 5 points which resulted in significant increase in the sensitivity and positive predictive value of the intermediate pretest probability (75.4% and 21% versus 39% and 12.9% respectively). However, the sensitivity dropped from 90% to 65%. The 4th part of 4Ts score gives points based on the probability of HIT versus other pathology as a cause of the thrombocytopenia. Most of the patients with suspected HIT will score one point out of the 4th part of 4Ts score which makes this part of the score is less helpful in distinguishing between patients with suspected HIT and will result in higher overall pretest probability and more testing. We studied the 3Ts score (the 4Ts scoring system after elimination of the 4th part) effect on the intermediate pretest probability. The cutoff points for the 3Ts score are 1-3, 4-5 and 6 for low, intermediate and high pretest probability respectively. The 3Ts score model resulted in significant increase in the specificity and the positive predictive value (76% and 21.7% versus 39.2% and 12.9% respectively) however the sensitivity dropped from 90% to 65%. The average turnaround time for HIT ELISA test and the SRA test was 2.3 days and 4.4 days respectively. Fifteen patients (21% out the patient had low pretest probability on the 4Ts score), 41 patients (59% of the patient who had an intermediate pretest probability on the 4Ts score) and 13 patients (18.8% of the patients who had high pretest probability on the 4Ts score) started on non-heparinoid anticoagulation meanwhile awaiting on the HIT testing result. Conclusion: Over testing of HIT is common secondary to underuse of 4Ts scoring system. Multicomponent educational interventions to increase the awareness and utility of the 4Ts score in addition to implantation of clinical decision support based on HIT computerized score will help to reduce unnecessary diagnostic testing of HIT in patients with low pretest probability. Increasing the cutoff point of the intermediate pretest probability or applying the 3Ts score model resulted in significant increase in the specificity and the positive predictive value of the 4Ts scoring system on expense of the scoring system sensitivity.

Evaluation of the seroprevalence and the demographic and clinical findings of fascioliasis patients in the Dicle River Basin in Turkey: a nine-year experience at a university hospital.

Fasciola hepatica and Fasciola gigantica are liver trematodes that cause fascioliasis in humans and animals. In Turkey, the medical importance of fascioliasis has been increasing in humans, and it continues to cause great economic loss in the field of animal husbandry. Therefore, it is important to diagnose fascioliasis quickly and reliably. The aim of this study is to show that the ELISA test is a reliable and specific method for diagnosing fascioliasis both in the early stage and in the acute stage. In this study, 640 individuals aged 7-75 years who showed one or more symptoms of fascioliasis, such as abdominal pain, fever, weight loss, weakness, fatigue, headache, sweating, nausea, vomiting, allergic urticaria, liver mass, hypereosinophilia, or liver enzyme elevation, were recruited from the Dicle University Research and Application Hospital in southeastern Turkey. Serum and fecal samples were taken from them to investigate if the Fasciola hepatica IgG antibody was present in the serum and if eggs were present in the feces. To detect the IgG antibodies, an enzyme-linked immunosorbent assay (ELISA) kit was used. The stool samples were analyzed for three consecutive days in mini Parasep fecal parasite concentrator tubes using the native-lugol and sedimentation methods. Abdominal ultrasonography and computed tomography were performed in all the patients. Among the subjects of this study, 90 (14%) were positive for fascioliasis, of whom 85 (94.4%) were adults and 5 (5.5%), children; 73 (81.1%) were women and 17 (18.8%), men; 57 (63.3%) lived in the rural areas and 33 (36.6%), in the city center; 90 (14%) were positive for Fasciola hepatica IgG antibodies; (20%) had helminth eggs in their stools; and 85 (94.4%) had a history of eating watercress. According to the epidemiological classification for fascioliasis by Mas-Coma, the Dicle Basin, which is the setting of this study, is indeed a hyperendemic region. Thus, ELISA is a reliable and specific method of diagnosing fascioliasis, both in the early phase and in the acute phase, when the eggs are no longer seen in the stool.

Antibodies to Aedes spp. salivary proteins: a systematic review and pooled analysis

Aedes spp. mosquitos are responsible for transmitting several viruses that pose significant public health risks, including dengue, Zika, yellow fever, chikungunya, and West Nile viruses. However, quantifying the number of individuals at risk and their exposure to Aedes spp. mosquitos over time is challenging due to various factors. Even accurate estimation of mosquito numbers at the population level may not fully capture the fluctuations in human exposure based on factors that affect biting rates of mosquitoes. Measuring the antibody response of humans to mosquito salivary proteins (MSP) has been proposed as a method to assess human exposure to mosquito bites and predict disease risk. The presence of antibodies to MSP can be quantified using the enzyme-linked immunosorbent assay (ELISA). While there is known variability in laboratory methods, the consistency of MSP measurements across different research groups has not been quantitatively examined. Variation in laboratory protocols, antigens used, and the human populations sampled all may contribute to differences observed in measured anti-MSP responses. In this study, we conducted a systematic review of the published literature focusing on antibody responses to MSP in humans and other vertebrate hosts. Whenever possible, we extracted individual-level anti-MSP IgG data from these studies and performed a pooled analysis of quantitative outcomes obtained from ELISAs, specifically optical densities (OD). We analyzed the pooled data to quantify variation between studies and identify sample and study characteristics associated with OD scores. Our candidate list of characteristics included the type of antigen used, age of human subjects, mosquito species, population-level mosquito exposure, collection season, Köppen-Geiger climate classification, and OD reporting method. Our findings revealed that the type of antigen, population-level mosquito exposure, and Köppen-Geiger climate classification were significantly associated with ELISA values. Furthermore, we developed a classification algorithm based on OD scores, which successfully distinguished samples from individuals living in areas where a specific mosquito species was present from those where it was not, with a high degree of accuracy. The pooled analysis we conducted provides a harmonized assessment of ELISA testing, which can be utilized to refine the use of antibody responses as markers for mosquito exposure. In conclusion, our study contributes to the understanding of antibody responses to MSP and their utility as indicators of mosquito exposure. By identifying the factors associated with variations in ELISA values, we have provided valuable insights for future research and the refinement of antibody-based assessments of mosquito exposure.

Leptospirosis seroprevalence and risk factors among slaughterhouse workers in Burkina Faso

ObjectivesThe objectives of this study were to determine the seroprevalence and risk factors of leptospirosis among slaughterhouse workers in Burkina Faso. MethodsWe performed a cross-sectional survey of slaughterhouse workers from Ouagadougou and Bobo Dioulasso between March and April 2021. Blood was collected by venipuncture and serum samples were tested using enzyme-linked immunosorbent assay and microscopic agglutination test. Questionnaires were used to collect information from these workers on sociodemographic characteristics, work activities, knowledge of zoonosis, and risky behaviors. ResultsOf the 172 subjects investigated, 28 (16.28%) were found seropositive for leptospirosis using enzyme-linked immunosorbent assay or microscopic agglutination test. The main Leptospira infecting serogroup were Mini, Autumnalis, Canicola, Copenhageni, L. mayottensis (ND), Icterohaemorrhagiae, Pyrogenes/Tarassovi (cross reaction), Panama, and Ballum. Risk factors according to multivariate analysis, included residence (P = 0.02), working at the bleeding station (P = 0.03), contact with feces and urine (P = 0.04), and the practice of agriculture outside the slaughterhouse (P = 0.05). ConclusionThese findings indicate that a significant proportion of slaughterhouse workers are being exposed to pathogenic Leptospira. Public-health interventions against leptospirosis will need to target this occupational group. Proper personal protective equipment and information about the disease should be disseminated among slaughterhouses.