Enzyme-linked Immunosorbent Assay Test Research Articles

Enhancing Mycotoxin Detection in Table Olives: The Role of Enzyme-Linked Immunosorbent Assay and Method Optimization

The issue of mycotoxin contamination in food is particularly important for consumer safety. The consumption of these fungi can cause genetic mutations or lead to the formation of cancer cells. For years, foods have been studied for this problem, but for table olives, there is currently a significant lack of scientific literature. This study aimed to point out the possible application of the Enzyme-Linked Immunosorbent Assay (ELISA) method for the detection of mycotoxins in table olives, assessing the presence of aflatoxins and ochratoxin A. Different tests were carried out on commercial and inoculated olives to assess the efficacy of the method by comparison with the conventional one, based on Liquid Chromatography–Tandem Mass Spectrometry (LC–MS/MS). Initial unsatisfactory results led us to refine the extraction method via the substitution of the chemical extractant 70% methanol with dichloromethane; this procedure allowed for improving the recovery of inoculated mycotoxins. The results show the effectiveness of the ELISA test, especially in mycotoxin recovery, after the modification of the extraction method. The recoveries increased for aflatoxins from 55% to over 70% and for ochratoxin A from 40% to over 55%. When samples were analyzed with commercial kits, the results showed some false positives, especially for the first sample of ochratoxin A, but the reliability of the ELISA method test was largely verified in the rest of the samples. The validity, the reduced cost, and the speed of application of the ELISA test make this method suitable as a control program for mycotoxin detection in table olives, which can be supported by LC-MS/MS analysis in case of positive results.

Comparison of RT.PCR and ELISA Methods in the Diagnosis of Human Cytomegalovirus in Kidney Transplant Patients

Background: In kidney transplant recipients prone to infections like cytomegalovirus (CMV), a vital need for an accurate diagnostic method is evident. This study at Khorshid Laboratory in Tehran rigorously compares real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) for CMV detection in transplant patients. Methods: In January to March 1400, 70 kidney transplant recipients were assessed for CMV DNA using RT-PCR and concurrent ELISA tests, with statistical analysis aided by SPSS. Results: In kidney transplant patients (average age: 49.40 ± 13.64 years), 4.3% tested positive for CMV via PCR. Strong correlation between serological and molecular methods. IgG and IgM antibody detection both showed high sensitivity and specificity, advocating for efficient CMV diagnosis at Khorshid Laboratory, Tehran. Conclusion: This study emphasizes the need for a quick and efficient CMV diagnostic approach in kidney transplant patients. The strong correlation between molecular and serological methods favors using the faster RT-PCR method, crucial for timely management, especially with the increasing age- related CMV incidence. The findings strongly recommend RT-PCR integration for enhanced sensitivity in kidney transplant patients at Khorshid Laboratory, Tehran.

Assessment of Serum Adiponectin and Leptin in Chronic Preiodontitis Over Healthy Individuals

ABSTRACT Objective: The objective of the research was to ascertain whether serum levels of the biochemical markers adiponectin and leptin are related to clinical periodontal characteristics. Materials and Method: For the study, 20 people with chronic periodontitis (Group A) and 20 healthy people (Group B) were enrolled. Assessments were conducted on periodontal markers, including bleeding on probing (POP), gingival index (GI), plaque index (PI), probing pocket depth (PPD), and clinical attachment level (CAL). An enzyme-linked immunosorbent assay (ELISA) test was used to assess the levels of adiponectin and leptin in serum samples. Result: There was significant elevation in average level of leptin in chronic periodontitis in association to that in healthy group. On the other hand, there was considerable decrease in serum adiponectin in group A in association to healthy group. The ratio of leptin\adiponectin was appreciably greater amongst periodontitis group compared to healthy group. Conclusion: The current investigation leads us to the conclusion that the pathophysiology of periodontitis is significantly influenced by serum levels of adiponectin and leptin.

Critical role of ROCK1 in AD pathogenesis via controlling lysosomal biogenesis and acidification

BackgroundLysosomal homeostasis and functions are essential for the survival of neural cells. Impaired lysosomal biogenesis and acidification in Alzheimer’s disease (AD) pathogenesis leads to proteolytic dysfunction and neurodegeneration. However, the key regulatory factors and mechanisms of lysosomal homeostasis in AD remain poorly understood.MethodsROCK1 expression and its co-localization with LAMP1 and SQSTM1/p62 were detected in post-mortem brains of healthy controls and AD patients. Lysosome-related fluorescence probe staining, transmission electron microscopy and immunoblotting were performed to evaluate the role of ROCK1 in lysosomal biogenesis and acidification in various neural cell types. The interaction between ROCK1 and TFEB was confirmed by surface plasmon resonance and in situ proximity ligation assay (PLA). Moreover, we performed AAV-mediated ROCK1 downregulation followed by immunofluorescence, enzyme-linked immunosorbent assay (ELISA) and behavioral tests to unveil the effects of the ROCK1–TFEB axis on lysosomes in APP/PS1 transgenic mice.ResultsROCK1 level was significantly increased in the brains of AD individuals, and was positively correlated with lysosomal markers and Aβ. Lysosomal proteolysis was largely impaired by the high abundance of ROCK1, while ROCK1 knockdown mitigated the lysosomal dysfunction in neurons and microglia. Moreover, we verified ROCK1 as a previously unknown upstream kinase of TFEB independent of m-TOR or GSK-3β. ROCK1 elevation resulted in abundant extracellular Aβ deposition which in turn bound to Aβ receptors and activated RhoA/ROCK1, thus forming a vicious circle of AD pathogenesis. Genetically downregulating ROCK1 lowered its interference with TFEB, promoted TFEB nuclear distribution, lysosomal biogenesis and lysosome-mediated Aβ clearance, and eventually prevented pathological traits and cognitive deficits in APP/PS1 mice.ConclusionIn summary, our results provide a mechanistic insight into the critical role of ROCK1 in lysosomal regulation and Aβ clearance in AD by acting as a novel upstream serine kinase of TFEB.

Neutrophil Elastase, Neuron-Specific Enolase, and S100B Protein as Potential Markers of Long-Term Complications Caused by COVID-19 in Patients with Type 2 Diabetes Mellitus (T2DM) and Advanced Stage of Diabetic Nephropathy (NfT2DM)-Observational Studies.

Despite numerous studies conducted by various research teams, predicting long-term outcomes (known as Post-COVID-19 Syndrome, PCS) that may result from Coronavirus Disease 2019 (COVID-19) remains challenging. PCS affects over a million people, primarily those with comorbid conditions. Therefore, it is crucial to undertake research aimed at developing a predictive model for early diagnosis of PCS, which in turn would enable faster preventive actions. The aim of this study was to assess the value of measuring and attempt a quantitative evaluation using Enzyme-Linked Immunosorbent Assay (ELISA) tests of three non-serum proteins, whose presence in the blood during COVID-19 was associated with severe disease progression: neutrophil elastase (NE), calcium-binding protein S100B, and neuron-specific enolase (NSE). The concentrations of these proteins were measured in blood serum samples collected before the COVID-19 pandemic from (1) patients with type 2 diabetes (T2DM); (2) advanced stage diabetic nephropathy (NfT2DM); (3) a healthy group; and in blood serum samples collected two years after recovering from COVID-19 from patients with (4) T2DM and (5) NfT2DM. It was found that elevated levels of NE and NSE were significantly more common (p < 0.05) in patients with NfT2DM after recovering from COVID-19 compared to the other groups, while elevated levels of S100B were significantly more frequently observed in patients with T2DM after recovering from COVID-19 (p < 0.05). Demonstrating differences in the prevalence of NE, NSE, and S100B in individuals who recovered from COVID-19 with T2DM and NfT2DM makes these proteins important components of the developing predictive model for early detection of PCS. To our knowledge, this is the first study showing the significance of NE, NSE, and S100B in PCS in the context of T2DM and NfT2DM.

Application of random quality control based on time -series model in ELISA detection of blood donors in Nanjing

This study introduces a completely Random Quality Control (R-QC) method for Enzyme-Linked Immunosorbent Assay (ELISA) tests and compares it with traditional Fixed Quality Control (F-QC) methods across different testing items. A random quality control system was constructed using a Time-series model, enabling fully random positioning of quality control in ELISA tests. The Coefficient of Variation (CV) for R-QC were found to be lower as compared to F-QC in Hepatitis B surface antigen (HBsAg), anti-Human Immunodeficiency Virus (anti-HIV), and Treponema Pallidum (TP) items, and there were significant differences among them. Moreover, the alarm rates and out of control rates in the R-QC groups were notably lower than those in the F-QC groups. The experimentally designed R-QC method demonstrates superior stability, reducing alarm and out of control rates, with has significant implications for improving ELISA testing at the Nanjing Blood Center.

The level of IL-12, IL-10, IL-4, and MMP-13 in cutaneous leishmaniasis patients

Abstract Background: Cutaneous leishmaniasis (CL) is a dermatological condition that arises due to the presence of protozoan parasites. The disease disproportionately impacts those residing in economically disadvantaged regions and is closely linked to factors such as inadequate nutrition, population displacement, substandard living conditions, and compromised immune systems. Objectives: Investigating the most important immunological changes associated with infection in patients with CL by measuring the levels of some cellular kinetics interleukins-12 (IL-12), IL-10, IL-4, and human matrix metalloproteinase-13 (MMP-13). Materials and Methods: This study involved a sample that included 90 individuals, comprising 20 control subjects and 70 patients diagnosed with CL. The age distribution of the participants in the study spanned from 3 months to 65 years, encompassing individuals of both genders. The sample consisted of 50 females and 40 males. The amounts of IL-12, IL-10, IL-4, and MMP-13 were quantified via an enzyme-linked immunosorbent assay (ELISA) test. Results: The current study showed a significant increase in the level of IL-12 in patients with CL (461.5 ± 14.8) compared to the control group (321.6 ± 57.9) at probability 0.05, whereas non-significant differences in levels of IL-10 and MMP-13 in patients with CL (143.5 ± 6.8) (5.7 ± 0.2) compared with the control group (198.0 ± 35.2) (6.9 ± 0.6) at P ≤ 0.05, respectively. As well as a highly significant decrease in the level of IL-4 in patients with CL (15.5 ± 1.5) compared to the control group (46.3 ± 5.7) at P ≤ 0.05. Conclusion: There was an increasing serum of IL-12 and decreased IL-4 in patients with CL compared with control, while non-significant decreases in the level of MMP-13 and IL-10 between patients with CL and control.

Anhydrous microwave synthesis as efficient method for obtaining model advanced glycation end-products.

Advanced glycation end-products (AGEs) are capable of stimulating oxidative stress and inflammation. This study investigates the synthesis of medium crosslinked AGEs (the most optimal form of AGEs because of soluble in water, used in many assays as markers) and their biochemical properties. One of model protein-myoglobin from horse heart muscle (MB) and a chosen respective glycation factor - D-melibiose (mel), acrolein (ACR), D-glucose (glc), 4-hydroksynonenal (4HNE), trans-2-nonenal (T2N), methylglyoxal (MGO) - were subjected to high temperature water synthesis (HTWS) and high temperature microwave synthesis in anhydrous conditions (HTMS). The syntheses were deliberately carried out in two different conditions to check whether adding an additional energy source (microwaves) while lowering the temperature and shortening the reaction time would allow for more effective obtaining of medium-cross-linked AGEs, monitored with SDS-PAGE. Products were analyzed using fluorescence measurements, Enzyme-Linked Immunosorbent Assay (ELISA) and immunoblotting tests and electrophoretic mobility shift assay to evaluate their ability to activate nuclear factor kappa-light-chain-enhancer (NF-κB). Medium cross-linked AGEs were more efficiently obtained in HTMS. Fluorescence was high for MB-ACR, MB-T2N and MB-glc products. Anti-MAGE antibodies showed reactivity towards MB-mels of HTMS and HTWS, and the MB-4HNEs from HTMS. HTWS products, apart from MB-ACR, did not activate NF-κB, whereas MB-ACR, MB-4HNE, MB-mel, and MB-T2N products of HTMS strongly activated this factor that indicates their strong pro-inflammatory properties. HTMS is a fast and efficient method of synthesizing medium cross-linked AGEs.

Improvement of RBD-FC Immunogenicity by Using Alum-Sodium Alginate Adjuvant Against SARS-COV-2.

Adjuvants use several mechanisms to boost immunogenicity and to modulate immune response. The strength of adsorption of antigen by adjuvants can be a determinant factor for significant improvement of immunopotentiation. We expressed recombinant RBD-FC in PichiaPink Strain 4 and examined the vaccination of mice by vaccine formulation with different adjuvants (sodium alginate and aluminum hydroxide, alone and together). Sodium alginate significantly increased the immunogenicity and stability of RBD-FC antigen, so RBD-FC formulated with combined alginate and alum (AlSa) and sodium alginate alone showed higher antibody titer and stability. Immunogenicity of RBD-FC:AlSa was determined by serological assays including direct enzyme-linked immunosorbent assay (ELISA) and surrogate virus neutralization test (sVNT). High levels of IgGs and neutralizing antibodies were measured in serum of mice immunized with the RBD-FC:AlSa formulation. On the other hand, cytokines IL-10 and INF-γ were severely accumulated in response to RBD-FC:AlSa, and after 10 days, their accumulation was significantly declined, whereas IL-4 showed the highest and the lowest accumulation in response to alum and alginate, respectively. Our data may suggest that combination of alum and sodium alginate has a better compatibility with RBD-FC in vaccine formulation.

Molecular characterization and immunopathological investigation of Avian reticuloendotheliosis virus in breeder flocks in Egypt

BackgroundReticuloendotheliosis virus (REV) is an oncogenic immunosuppressive retrovirus that infects different kinds of avian species; posing significant economic losses to the poultry industry worldwide.MethodsIn Egypt, there is an unidentified disease associated with the runting-stunting syndrome with neoplasia, suspected to be REV, that has been continuously monitored in several breeder flocks. To diagnose and analyze REV by cell cultures, enzyme-linked immunosorbent assay (ELISA), histopathological investigation, the polymerase chain reaction (PCR) test, and sequencing analysis, 200 blood samples, and 50 tissue specimens were collected. The current study targets the occurrence and genetic characteristics of a viral neoplastic disease, resembling REV infection, circulating in breeder flocks from 2022 to 2023 in the Ismailia, El-Sharqia, and El-Dakahliya governorates.ResultHere, REV was isolated on chicken embryo fibroblast cell culture; exhibiting cell aggregation, rounding, and cell detachments. Collectively, only 70 serum samples were positive for anti‐REV antibodies with seroprevalence rates of 35% based on the ELISA test. The histopathological observation demonstrated lymphoreticular tumors in the liver, spleen, and other examined organs. The immunohistochemical staining method confirmed the REV-positive signals in all examined organs (liver, kidney, spleen, bursa, ovaries) except for the heart. The PCR assay of the LTR gene assessed 370 base pairs with only 5 positive samples with a percentage of 16.6%. Three positive samples were further sequenced and submitted to the Genbank under accession numbers (PP763709, PP763710, PP763711). Phylogenetic analysis of the REV-LTR gene showed that our three isolates (Sharquia-1-REV, Ismilia-2-REV, Mansoura-3-REV) are REV subtype III which predominantly circulated in breeders in Egypt. These three isolates are highest similar to American, Chinese, and Taiwanese REV reference strains, and other Egyptian strains with nucleotide identity percentages of 100%, 99%, and 99%; respectively, and on the amino acid identity level were with (99–100%), (98%, 99%), (99%, 100%); respectively.ConclusionsThis study established that REV infection was extensively distributed in the breeders and became one of the causes of the clinical outbreaks of tumors, raising awareness of REV as the causative agent of avian oncogenic disease in Egypt.

Camelpox Virus in Western Kazakhstan: Assessment of the Role of Local Fauna as Reservoirs of Infection.

This article investigates the role of local fauna in Western Kazakhstan as potential reservoirs of the camelpox virus (CMLV). The study emphasizes analyzing possible sources and transmission pathways of the virus using polymerase chain reaction (PCR) and serological methods, including virus neutralization tests and enzyme-linked immunosorbent assays (ELISA). Samples were collected from both young and adult camels, as well as rodents, ticks and blood-sucking insects in the Mangystau and Atyrau regions. The PCR results revealed the absence of viral DNA in rodents, ticks and blood-sucking insects; also, the ELISA test did not detect specific antibodies in rodents. These findings suggest that these groups of fauna likely do not play a significant role in the maintenance and spread of CMLV. Consequently, the primary sources of transmission are likely other factors, potentially including the camels themselves. The study's results indicate the need to reassess current hypotheses regarding infection reservoirs and to explore alternative sources to enhance strategies for the control and prevention of the camelpox virus.

Preliminary field evaluation of indirect ELISA test using the recombinant antigen rLicNTPDase-2 for serodiagnosis of canine leishmaniasis in Colombia

Leishmaniasis is a significant public health concern, with dogs as the primary reservoir in urban scenarios and facilitating transmission. Diagnosing infected dogs is a crucial step for public health interventions, and the development of new diagnostic platforms can significantly enhance efforts in various regions worldwide. Given the limited availability of diagnostic methods in Colombia, this study evaluates the effectiveness of an Indirect Enzyme-Linked Immunosorbent Assay (ELISA) based on the recombinant protein rLicNTPDase-2 to detect Leishmania in infected dogs. Serum samples were collected from dogs in both endemic and non-endemic areas and classified as natural standards based on prior parasitological diagnoses. The results revealed 24 true positives (TP) and 9 true negatives (TN). Subsequently, the test was then validated with samples from symptomatic and asymptomatic animals, alongside the standards, yielding a specificity of 96 %, a sensitivity of 81 %, efficiency of 90.6 %, a positive predictive value of 92.8 %, and a negative predictive value of 89.6 %. The positive likelihood ratio (RV+) was 20, while the negative likelihood ratio (RV-) was 0.19, indicating high relevance and a robust clinical utility. The area under the curve (AUC) was 1.00, suggesting that the test has excellent discriminatory ability, significantly deviating from the reference diagonal. This is further supported by the significant difference(p < 0.0001) between TN and TP results determined by Fisher's exact test. Involving 163 animals showed 47 % positive and 46 % negative results with a significant difference (p < 0.05) in the mean optical density (OD) values between positive and negative samples. These findings indicate that the ELISA test effectively differentiates between positive and negative samples based on OD values. This study suggests that ELISA based on the recombinant antigen rLicNTPDase-2 could serve as a viable alternative for the serodiagnosis of leishmaniasis in canines in Colombia.

Otimização do conjugado Anti-IGG canino no kit diagnóstico da Leishmaniose Visceral Canina (LVC)

Introduction: The diagnosis of canine visceral leishmaniasis (CVL) is of great importance in the control of visceral leishmaniasis, a disease that is neglected worldwide and represents a significant public health problem. Enzyme-linked immunosorbent assay (ELISA) test is widely used because of the possibility of large-scale diagnosis and low cost. Objective: The objective of this study was to optimize the stoichiometry of the conjugate used in the EIE/CVL kit; for this purpose, some conjugation and monitoring parameters were modified through molecular exclusion chromatography to improve the final product. Method: The conjugate produced by Bio-Manguinhos was optimized by purification using SEC gel filtration chromatography and evaluated using the EIE-leishmaniasis-visceral-canine-Bio-Manguinhos kit (EIE-LVC). The conjugates were produced with different stoichiometries (IgG X peroxidase): current conjugate, optimized conjugate and optimized and purified conjugate. Results: The results obtained were validated using the manual Canine Visceral Leishmaniasis test. The homogeneity of the conjugate means in the ELISA test was evaluated using the chi-square test, thereby demonstrating the normality and reliability of the data. Based on statistical data, positive samples obtained by the ELISA test showed homogeneity at a significance level of 0.05, i.e. a data reliability level of 95%. The optimization of the production of the canine anti-IgG conjugate, an extremely important component used in the composition of the Bio-Manguinhos EIE LVC KIT, contributed to ensuring the quality of the kit and improving the diagnosis of canine visceral leishmaniasis disease, making it more sensitive and inexpensive for public health. Conclusions: This work demonstrates that it is possible to optimize the anti-IgG canine peroxidase (HRP) conjugate used in the EIE CVL kit to make it more specific and ultimately more economical.

Effects of cowpea aphid-borne mosaic virus infection on the growth and yield parameters of Sesamum indicum plants

The potyvirus cowpea aphid-borne mosaic virus (CABMV) is the main viral agent present in crops of Sesamum indicum in several regions of Paraguay. However, information about the effects of CABMV infection in S. indicum plants is scarce and the critical period for virus infection in S. indicum plants is unknown. This study aimed to evaluate the damage caused by CABMV on the development and yield of S. indicum plants experimentally infected at three phenological stages. Mechanical inoculations of CABMV were performed on S. indicum plants at the early vegetative stage, at the beginning of flowering, and during capsule and seed formation. An enzyme-linked immunosorbent Assay (ELISA) test was used to confirm virus infection on the inoculated plants using specific CABMV antisera. Plants inoculated 30 days after emergence (DAE) had severe systemic symptoms, but they were mild in mid and late-infected plants (60 and 90 DAE). Plant aerial dry weight was significantly affected by CABMV infection, reducing up to 71.6% compared to the control. Mid and late-infected plants experienced a lower level of damage, with an average aerial dry weight reduction of 17 and 14.2%, respectively. Furthermore, plants with early, mid, and late CABMV infections showed an average capsule per plant reduction of 97, 33.3, and 13.3%, compared to healthy plants. Seeds per capsule produced by CABMV-infected plants decreased by 93.5%, 21%, and zero, respectively. Viral infections also affected the 1000-seed weight in early infected plants, with a reduction of up to 93.3%. To prevent economic losses, it is crucial to have effective strategies for managing CABMV, particularly during the early stages of the crop.

&lt;i&gt;Oroxylum indicum&lt;/i&gt; Kurz (L) leaf extract exerted antioxidant and anti-inflammatory effects on LPS-stimulated BV2 microglial cells

Purpose: To investigate the antioxidant and anti-inflammatory effects of Oroxylum indicum (L.) Kurz leaf extract on lipopolysaccharide (LPS)-activated BV2 microglia. Methods: BV2 cells were treated with LPS for 24 h in the presence or absence of Oroxylum indicum (O. indicum) leaf extract. The levels of reactive oxygen species (ROS), nitric oxide (NO), and interleukin 6 (IL-6) were quantified with CM-H2DCFDA, Griess reagent assay, and enzyme-linked immunosorbent assay (ELISA) test, respectively. The antioxidant activity of O. indicum leaf extract was assessed in vitro using DPPH, ABTS and FRAP assays. Results: Treatment with LPS resulted in significant increases in the levels of ROS, NO, and IL-6, when compared to untreated cells. However, co-treatment with O. indicum leaf extract significantly suppressed the production of these inflammatory markers (p &lt; 0.05). In addition, the leaf extract of O. indicum exhibited antioxidant activity in the DPPH, ABTS, and FRAP assays. The contents of flavonoids and phenolics in O. indicum leaf extract were 13.25 ± 0.163 mg QE/g of dried extract and 82.58 ± 1.772 mg QE/g of dried extract, respectively (p &lt; 0.05). Conclusion: The leaf extract of O. indicum exhibits anti-inflammatory and antioxidant properties in LPS-activated BV2 cells. This finding indicates that the leaf extract of O. indicum has promising potentials as a new source of herbal medication with anti- inflammatory properties. However, there is a need for more studies to unravel the fundamental mechanisms of their actions.

Evaluation of urinary podocin and nephrin as markers of podocyturia in dogs with leishmaniosis

BackgroundRenal disease is the main cause of death in canine leishmaniosis. Detection of an active glomerular injury is important to identify early renal damage and to prevent the development of chronic kidney disease. Podocyturia can indicate renal injury, and podocyte-associated molecules such as podocin and nephrin can be used to identify podocyturia. The purpose of the study was to evaluate urinary podocin and nephrin concentrations in dogs with leishmaniosis as markers of podocyturia.MethodsA total of 35 healthy dogs and 37 dogs with leishmaniosis were enrolled in the study. Dogs with leishmaniosis were classified according to the staging of the International Renal Interest Society (IRIS). Urinary podocin and nephrin concentrations were measured in all dogs with a validated enzyme-linked immunosorbent assay test and normalized to creatinine (uPoC and uNeC, respectively). The demographic, clinical, and laboratory data from both groups were analyzed and compared. Subsequently, the laboratory results were analyzed and compared according to IRIS staging in dogs in IRIS stage I and dogs in IRIS stage II + III + IV. The Pearson’s correlation test evaluated the relationship between urinary markers of podocyturia.ResultsCompared with healthy dogs, lower urinary podocin [median values (IQR): 15.10 (11.75–17.87) ng/ml versus 8.63 (7.08–13.56) ng/ml; P < 0.01] and nephrin [median values (IQR): 3.2 (3.62–5.43) ng/ml versus 2.67 (2.06–3.44) ng/ml; P < 0.01] were found in infected sick dogs. No significant differences were observed in the uPoC and uNeC between the two groups. Urinary nephrin and podocin concentrations were higher in healthy dogs and in dogs in IRIS stage I (both P < 0.05) compared with dogs in IRIS stages II + III + IV. No significant differences were found for uPoC and uNeC between healthy dogs and dogs with leishmaniosis in different IRIS clinical stages.ConclusionsDogs with leishmaniosis had a low concentration of podocin and nephrin in more advanced IRIS clinical stages, when kidney disease was more severe compared with healthy dogs and dogs in IRIS stage I with mild disease. Urinary nephrin was detectable for the first time in healthy non-infected dogs.Graphical

First Report of Bean Leafroll Virus in Pea and Chickpea, in Canada.

Bean leafroll virus (BLRV; Bean leafroll virus), a single-stranded RNA virus in the genus Luteovirus, is phloem-limited and primarily transmitted by aphids in a non-propagative, persistent manner (Rashed et al., 2018; Kidanemariam and Abraham, 2023). BLRV infects various legumes and has been reported from major pulse-growing regions worldwide (Agindotan et al., 2019) but not in the Canadian Prairies. Its impact on crop yield varies with plant and virus genotypes and the timing of infection. Some pea fields have experienced disease rates of up to 80% (Clement et al., 2020; Hampton, 1983). Throughout the 2022 growing season (June and July), pulse fields from across Saskatchewan were randomly selected and surveyed, and symptomatic plants demonstrating leaf yellowing and chlorosis were collected and stored at -80°C before processing. Observed symptoms included necrotic spots, chlorosis, leaf mottling, leaf rolling in peas, severe bright yellowing, and leaf marginal necrosis in chickpeas. BLRV detection was performed on 35 leaves of the collected samples using both Enzyme-Linked Immunosorbent Assay (ELISA) and Reverse transcription polymerase chain reaction (RT-PCR). ELISA testing followed the manufacturer's protocol using a commercial kit (Nano Diagnostics, San Jose, CA, USA). Total RNAs were extracted from the frozen samples using TRIzol (Invitrogen, Carlsbad, CA, USA). For the detection of the diverse BLRV isolates, sequences of various isolates were aligned and primers were specifically designed in-house, targeting the virus's highly conserved regions on the GP3 and 3' UTR (see Supplementary material). Additional primers were also designed targeting coat protein (CP) coding regions which were previously used for BLRV detection (Agindotan et al. 2019; Larsen & Webster 1999). PCR testing of 35 symptomatic samples including 12 pea plants and 23 chickpea plants, identified the presence of BLRV in two symptomatic samples, one each from a field pea (Pisum sativum L. var. CDC Inca) and a desi-type chickpea (Cicer arietinum L. var. CDC Leader). The infected pea and chickpea samples were found in Saskatoon, SK (Coordinates: 52°9'27''N,106°34'14"W), and the Leader area, southwest of Saskatchewan, SK (Coordinates: 50°52'14"N,109°23'11"W), respectively. PCR amplicons were purified and sent for Sanger sequencing. The reads were assembled to generate 1666 and 323 nucleotides from pea and chickpea, respectively, with a minimum of 2X coverage. Partial nucleotide sequences of the BLRV isolates obtained from pea (PsSK1) and chickpea (CaSK1) (GenBank accession numbers: PP240429, PP266588) showed (1521/1574 bp) 96.63% and (316/323 bp) 97.83% similarity with a BLRV reference isolate sequence (NC_003369) and to an isolate from Argentina (KR261610) which was reported on Medicago sativa L. with (1555/1574 bp) 98.79% and (319/323 bp) 98.76% similarity, correspondingly. Both infected samples were confirmed to be BLRV-infected through the ELISA and exhibited a high interaction ratio (PsSK1: 0.319 and CsSK1: 0.245) compared to a positive control (0.292) after 30 minutes as measured at 450 nm. This is the first report of BLRV in the pulse-growing region of the Canadian Prairies. In Saskatchewan, there is no history of BLRV despite the large amount of area growing susceptible crops. Therefore, the survey project that this study was part of was not intended to evaluate the severity of BLRV but rather to determine if there is any virus present that might have been overlooked. The samples were therefore taken randomly, with a focus on the number of fields and geographic coverage rather than focusing on multiple plants per field. Moreover, fields were not chosen based on symptoms but rather at random. Although, plants within fields were chosen because they displayed symptoms. Typically, a disease note includes estimates of severity and potential risk; however, that is not possible for this study. Rather, the fact that it was detected indicates a greater risk than previously perceived, since it was assumed that BLRV was not present. These findings highlight the need for further research on the virus's current status, its impact on crop production, and the resistance of pulse varieties grown in Saskatchewan.

Prediabetes Associates with Matrix Metalloproteinase-8 Activation and Contributes to the Rapid Destruction of Periodontal Tissues.

The aim of this cross-sectional study was to investigate the relationship between periodontitis, potential periodontitis oral fluid biomarkers, and prediabetes. This study included 150 Greek adults aged 25 to 78 years who were tested with an Hemoglobin A1C (HBA1c) diagnostic system, an active-matrix metalloproteinase-8 (aMMP-8) point-of-care (PoC) test, and several salivary biomarkers enzyme-linked immunosorbent assay tests and gelatin zymography. A full-mouth clinical examination was performed to assess their periodontal and oral health status. The Kruskal-Wallis test was used to determine the statistically significant difference in the levels of periodontal oral fluid biomarkers between the different periodontitis stages, periodontitis grades, and the stages and grades of periodontitis combined. Spearman's rank correlation was performed to assess the strength and direction of the association between aMMP-8 and HbA1c levels (<5.7 and ≥5.7%) and with the other oral fluid biomarkers among patients with severe periodontitis. A two-sided p-value below 0.05 was considered statistically significant in this study. aMMP-8, but not total MMP-8 or other biomarkers, associated significantly with the stage and grade of periodontitis combined (p < 0.001, Kruskal-Wallis test). Among stage III grade C periodontitis patients, aMMP-8 levels were significantly positively correlated with prediabetes (Spearman's rho = 0.646, p = 0.044), total MMP-8 (rho = 0.636, p = 0.048), PMN Elastase (rho = 0.729, p = 0.017), total MMP-9 (rho = 0.721, p = 0.019), and total MMP-8/TIMP-1 molar ratio (rho = 0.879, p < 0.001). Prediabetic disease development can upregulate MMP-8 expression (total MMP-8) in rapidly progressing, severe periodontitis, where MMP-8 latent species are further activated into their active forms (aMMP-8). Simultaneously, several proinflammatory biomarker levels are elevated in this tissue-destructive biomarker cascade. This development is easily detectable online/in real-time within 5 minutes by aMMP-8 PoC testing at the dentist's office.

Immunoglobulin E and Serum Interleukin-13 and Bacteria in Patients with Alopecia Areata

Alopecia areata (AA) is a form of hair loss that does not result in permanent scars. The etiopathology of it is not completely understood. To evaluate the serum concentration of immunoglobulin E (IgE) and interleukin-13 (IL-13) in patients diagnosed with AA and determine the bacterial strain present in individuals with infections. The study comprised a sample of 60 women afflicted with alopecia areata and 30 women who were in good health, serving as controls.The levels of IgE and IL-13 in the serum were evaluated using an enzyme-linked immunosorbent assay test(ELISA). The microorganisms were identified by conventional techniques and verified using Vitek technology. for IgE showed highly significant differences (p value &lt; 0.1) Between patients and the control group, Regarding IL-13, the results showed significant differences (p value &lt; 0.05), Between patients and the control group, Regarding age As for IgE, there is no significant difference between patients in the three groups (p value &gt; 0.05), but it is different from the control (p value &lt; 0.05).IL-13 There are significant differences (p value &lt; 0.05). Among adults, the level of interleukin is higher than that of middle and young people. The weight-related results demonstrated significant variability across patients (p value &lt; 0.05), with obese individuals exhibiting higher levels of IGE and IL-13 compared to those of medium and underweight. Regarding, In relation to bacteria, the percentages were as stated S.epedermidis (50.98%) , (S.aureus33.33%) mixed , S.aureus + S.epedermidis , (9.8%) E.coli (1.96%) Pseudomonas aeruginosa(1.96 %) , Granulicatella elegans (1.96%) .Total is IgE and IL-13 increased in patients with alopecia areata, Regarding bacteria, we need other studies to prove their role in alopecia areata.

Comparing Point-of-Care Technology to ELISA Testing for Infliximab and Adalimumab Levels in Adult Inflammatory Bowel Disease Patients: A Prospective Pilot Study.

Introduction: Therapeutic drug monitoring (TDM) has proven to be a valuable strategy for optimizing biologic therapies, among which are anti-tumor necrosis factor (anti-TNF) treatments in inflammatory bowel disease (IBD). In particular, reactive TDM has been shown to manage treatment failures more cost-effectively than empirical dose adjustments for anti-TNF drugs. However, several challenges currently impede the widespread adoption of TDM in clinical practice, particularly addressing the delay between sample collection and result availability. To overcome this limitation, the use of point-of-care technology tests (POCTs) is a potential solution. Point-of-care technology tests are medical diagnostic tests performed at the site of patient care to provide immediate results, allowing for quicker decision-making and treatment. The current standard of care (SOC) for drug level measurement relies on the enzyme-linked immunosorbent assay (ELISA), a method that is time-consuming and requires specialized personnel. This study aims to evaluate a novel, user-friendly, and efficient POCT method (ProciseDx Inc.) and compare its performance with the SOC ELISA in assessing infliximab and adalimumab levels in blood samples from IBD patients. Methods: In this prospective, single-center study, we collected blood samples from IBD patients, both CD and UC, receiving infliximab (87 IBD patients; 50% UC and 50% CD) or adalimumab (60 patients; 14% UC and 48% CD) and we analyzed the blood's drugs levels using both the ProciseDx Analyzer POC and the SOC ELISA. We examined the correlation between the two methods using statistical analyses, including the Deming regression test. Additionally, we assessed the ease of use, turnaround time, and overall practicality of the POCT in a clinical setting. Results: The ProciseDx test demonstrated a strong correlation with the SOC ELISA for measuring both infliximab and adalimumab levels. In particular, the overall correlation between the ProciseDx POCT and the ELISA assessments showed an r coefficient of 0.83 with an R squared value of 0.691 (95% CI 0.717-0.902) for IFX measurements, and an r coefficient of 0.85 with an R squared value of 0.739 (95% CI 0.720-0.930). Conclusions: the ProciseDx POC test offers significantly faster turnaround times and is more straightforward to use, making it a viable alternative for routine clinical monitoring. Despite its promising potential, further refinement and validation of the ProciseDx test are necessary to ensure its effectiveness across diverse patient populations and clinical settings. Future research should focus on optimizing the POC tests' performance and evaluating its long-term impact on IBD management.