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Background: Adiponectin is an adipocyte-derived hormone that acts as a marker of insulin sensitivity. Bloodspot sampling by fingerstick onto filter paper may increase the feasibility of large-scale studies of the determinants of insulin sensitivity. We first describe the validation of an enzyme-linked immunoassay (ELISA) for quantifying adiponectin from dried blood spots and then demonstrate its application in a large trial (PROBIT)., Methods: We quantified adiponectin from 3-mm diameter discs (≈3 µL of blood) punched from dried blood spots obtained from: i) whole blood standards (validation); and ii) PROBIT trial samples (application) in which paediatricians collected blood spots from 13,879 children aged 11.5 years from 31 sites across Belarus. We examined the distribution of bloodspot adiponectin by demographic and anthropometric factors, fasting insulin and glucose., Results: In the validation study, mean intra-assay coefficients of variation (n=162) were 15%, 13% and 10% for 'low' (6.78 µg/ml), 'medium' (18.18 µg/ml) and 'high' (33.13 µg/ml) internal quality control (IQC) samples, respectively; the respective inter-assay values (n=40) were 23%, 21% and 14%. The correlation coefficient between 50 paired whole bloodspot versus plasma samples, collected simultaneously, was 0.87 (95% CI: 0.78 to 0.93). Recovery of known quantities of adiponectin (between 4.5 to 36 µg/ml) was 100.3-133%. Bloodspot adiponectin was stable for at least 30 months at -80°C. In PROBIT, we successfully quantified fasting adiponectin from dried blood spots in 13,329 of 13,879 (96%) children. Mean adiponectin (standard deviation) concentrations were 17.34 µg/ml (7.54) in boys and 18.41 µg/ml (7.92) in girls and were inversely associated with body mass index, fat mass, triceps and subscapular skin-fold thickness, waist circumference, height and fasting glucose., Conclusions: Bloodspot ELISA is suitable for measuring adiponectin in very small volumes of blood collected on filter paper and can be applied to large-scale studies.

The World Health Organization (WHO) calls for the development of a rapid, biomarker-based, non-sputum test capable of detecting all forms of tuberculosis (TB) at the point-of-care to enable immediate treatment initiation. Lipoarabinomannan (LAM) is the only WHO-endorsed TB biomarker that can be detected in urine, an easily collected sample matrix. For obtaining optimal sensitivity, we and others have shown that some form of sample pretreatment is necessary to remove background from patient urine samples. A number of systems are paper-based often destined for resource limited settings. Our current work presents incorporation of one such sample pretreatment, proteinase K (ProK) immobilized on paper (IPK) and test its performance in comparison to standard proteinase K (SPK) treatment that involves addition and deactivation at high temperature prior to performing a capture ELISA. Herein, a simple and economical method was developed for using ProK immobilized strips to pretreat urine samples. Simplification and cost reduction of the proposed pretreatment strip were achieved by using Whatman no.1 paper and by minimizing the concentration of ProK (an expensive but necessary reagent) used to pretreat the clinical samples prior to ELISA. To test the applicability of IPK, capture ELISA was carried out on either LAM-spiked urine or the clinical samples after pretreatment with ProK at 400 μg/mL for 30 minutes at room temperature. The optimal conditions and stability of the IPK were tested and validation was performed on a set of 25 previously analyzed archived clinical urine samples with known TB and HIV status. The results of IPK and SPK treated samples were in agreement showing that the urine LAM test currently under development has the potential to reach adult and pediatric patients regardless of HIV status or site of infection, and to facilitate global TB control to improve assay performance and ultimately treatment outcomes., Competing Interests: The authors have declared that no competing interests exist.

Amphimeriasis, a fish-borne zoonotic disease caused by the liver fluke Amphimerus spp., has recently been reported as an emerging disease affecting an indigenous Ameridian group, the Chachi, living in Ecuador. The only method for diagnosing amphimeriasis was the microscopic detection of eggs from the parasite in patients' stool samples with very low sensitivity. Our group developed an ELISA technique for detection of anti-Amphimerus IgG in human sera and a molecular method based on LAMP technology (named LAMPhimerus) for specific and sensitive parasite DNA detection. The LAMPhimerus method showed to be much more sensitive than classical parasitological methods for amphimeriasis diagnosis using human stool samples for analysis. The objective of this work is to demonstrate the feasibility of using dried stool samples on filter paper as source of DNA in combination with the effectiveness of our previously designed LAMPhimerus assay for successfully Amphimerus sp. detection in clinical stool samples. A total of 102 untreated and undiluted stool samples collected from Chachi population were spread as thin layer onto common filter paper for easily transportation to our laboratory and stored at room temperature for one year until DNA extraction. When LAMPhimerus method was applied for Amphimerus sp. DNA detection, a higher number of positive results was detected (61/102; 59.80%) in comparison to parasitological methods (38/102; 37.25%), including 28/61 (45.90%) microscopy-confirmed Amphimerus sp. infections. The diagnostic parameters for the sensitivity and specificity werecalculated for our LAMPhimerus assay, which were 79.17% and 65.98%, respectively. We demonstrate, for the first time, that common filter paper is useful for easy collection and long-term storage of human stool samples for later DNA extraction and molecular analysis of human-parasitic trematode eggs. This simple, economic and easily handling method combined with the specific and sensible LAMPhimerus assay has the potential to beused as an effective molecular large-scale screening test for amphimeriasis-endemic areas. [ABSTRACT FROM AUTHOR]

Background: In large-scale epidemiology, bloodspot sampling by fingerstick onto filter paper has many advantages, including ease and low costs of collection, processing and transport. We describe the development of an enzyme-linked immunoassay (ELISA) for quantifying insulin from dried blood spots and demonstrate its application in a large trial. Methods: We adapted an existing commercial kit (Mercodia Human Insulin ELISA, 10-1113-01) to quantify insulin from two 3-mm diameter discs (≈6 µL of blood) punched from whole blood standards and from trial samples. Paediatricians collected dried blood spots in a follow-up of 13,879 fasted children aged 11.5 years (interquartile range 11.3-11.8 years) from 31 trial sites across Belarus. We quantified bloodspot insulin levels and examined their distribution by demography and anthropometry. Results: Mean intra-assay (n = 157) coefficients of variation were 15% and 6% for 'low' (6.7 mU/L) and 'high' (23.1 mU/L) values, respectively; the respective inter-assay values (n = 33) were 23% and 11%. The intraclass correlation coefficient between 50 paired whole bloodspot versus serum samples, collected simultaneously, was 0.90 (95% confidence interval 0.85 to 0.95). Bloodspot insulin was stable for at least 31 months at -80°C, for one week at +30°C and following four freeze-thaw cycles. Paediatricians collected a median of 8 blood spots from 13,487 (97%) children. The geometric mean insulin (log standard deviation) concentrations amongst 12,812 children were 3.0 mU/L (1.1) in boys and 4.0 mU/L (1.0) in girls and were positively associated with pubertal stage, measures of central and peripheral adiposity, height and fasting glucose. Conclusions: Our simple and convenient bloodspot assay is suitable for the measurement of insulin in very small volumes of blood collected on filter paper cards and can be applied to large-scale epidemiology studies of the early-life determinants of circulating insulin. [ABSTRACT FROM AUTHOR]

Objective: Detection of vascular endothelial growth factor (VEGF) levels in ocular tissue may perhaps provide insight into the role of VEGF in the pathogenesis and progression of diabetic retinopathy (DR). The aim of this study was to evaluate the levels of VEGF in tears and serum amongst type 2 diabetes mellitus (DM) patients. Methods: A comparative cross-sectional study was conducted between August 2016 and May 2018 involving type 2 DM patients with no DR, non-proliferative DR (NPDR), and proliferative DR (PDR). Tear samples were collected using no.41 Whatman filter paper (Schirmer strips) and 5 mL blood samples were drawn by venous puncture. VEGF levels in tears and serum were measured by enzyme-linked immunosorbent assay. Results: A total of 88 type 2 DM patients (no DR: 30 patients, NPDR: 28 patients, PDR: 30 patients) were included in the study. Mean tear VEGF levels were significantly higher in the NPDR and PDR groups (114.4 SD 52.5 pg/mL and 150.8 SD 49.7 pg/mL, respectively) compared to the no DR group (40.4 SD 26.5 pg/mL, p < 0.001). There was no significant difference in the mean serum VEGF levels between the three groups. There was a fair correlation between serum and tear VEGF levels (p = 0.015, r = 0.263). Conclusion: VEGF levels in tears were significantly higher amongst diabetic patients with DR compared to those without DR and were significantly associated with the severity of DR. There was a fair correlation between serum and tear VEGF levels. Detection of VEGF in tears is a good non-invasive predictor test for the severity of DR. A large cohort study is needed for further evaluation. [ABSTRACT FROM AUTHOR]

Background: Antigen specific release of IP-10 is an established marker for infection with M.tuberculosis. Compared to IFN-γ, IP-10 is released in 100-fold higher concentrations enabling the development of novel assays for detection. Dried blood spots are a convenient sample for high throughput newborn screening. Aim: To develop a robust and sensitive ELISA-based assay for IP-10 detection in plasma, dried blood spots (DBS) and dried plasma spots (DPS); to validate the ELISA in clinically relevant samples; and to assess the performance of the assay for detection of Cytomegalovirus (CMV) and M.tuberculosis specific immune responses. Method: We raised mice and rat monoclonal antibodies against human IP-10 and developed an ELISA. The assay was validated and applied to the detection of CMV and M.tuberculosis specific responses in 18 patients with immune reactivity towards M.tuberculosis and 32 healthy controls of which 22 had immune reactivity towards CMV and none towards M.tuberculosis. We compared the performance of this new assay to IFN-γ. Results: The ELISA was reliable for IP-10 detection in both plasma and filter paper samples. The linear range of the ELISA was 2.5-600 pg/ml. IFN-γ was not readily detectable in DPS samples. IP-10 was stabile in filter paper samples for at least 4 weeks at 37°C. The correlation between IP-10 detected in plasma, DPS and DBS samples was excellent (r2>0.97). Conclusions: This newly developed assay is reliable for IP-10 quantification in plasma, DBS and DPS samples from antigen stimulated and non-stimulated whole blood. The filter paper assays enable easy sample acquisition and transport at ambient temperature e.g. via the postal system. The system can potentially simplify diagnostic assays for M.tuberculosis and CMV infection [ABSTRACT FROM AUTHOR]

With current imaging, discriminating tumor progression from treatment effect following immunotherapy or oncolytic virotherapy of glioblastoma (GBM) is challenging. A blood based diagnostic biomarker would therefore be helpful. Axl is a receptor tyrosine kinase that is highly expressed by many cancers including GBM. Axl expression is regulated through enzymatic cleavage of its extracellular domain. The resulting fragment can be detected in serum as soluble Axl (sAxl). sAxl levels can distinguish patients with melanoma, hepatocellular carcinoma, and pancreatic ductal adenocarcinoma from healthy controls. This is a pilot study to determine if sAxl is a candidate biomarker for GBM. The sAxl levels in the serum of 40 healthy volunteers and 20 GBM patients were determined using an enzyme-linked immunosorbent assay (ELISA). Pre- and post- operative sAxl levels were obtained. Volumetric MRI evaluation provided GBM tumor volume metrics. There was no significant difference in the sAxl levels of the volunteers (30.16±1.88 ng/ml) and GBM patients (30.74±1.96 ng/ml) p = 0.27. The postoperative sAxl levels were significantly higher than preoperative levels (32.32±2.26 ng/ml vs 30.74±1.96 ng/ml, p = 0.03). We found no correlation between tumor volume and sAxl levels. Axl expression was low or absent in 6 of 11 (55%) patient derived GBM cell lines. Given the wide range of Axl expression by GBM tumors, sAxl may not be a reliable indicator of GBM. However, given the small sample size in this study, a larger study may be considered. [ABSTRACT FROM AUTHOR]

Public health emergency of SARS-CoV-2 has facilitated diagnostic testing as a related medical countermeasure against COVID-19 outbreak. Numerous serologic antibody tests have become available through an expedited federal emergency use only process. This paper highlights the analytical characteristic of an ELISA based assay by AnshLabs and three random access immunoassay (RAIA) by DiaSorin, Roche, and Abbott that have been approved for emergency use authorization (EUA), at a tertiary academic center in a low disease-prevalence area. The AnshLabs gave higher estimates of sero-prevalence, over the three RAIA methods. For positive results, AnshLabs had 93.3% and 100% agreement with DiaSorin or Abbott and Roche respectively. For negative results, AnshLabs had 74.3% and 78.3% agreement with DiaSorin and Roche or Abbott respectively. All discrepant samples that were positive by AnshLabs and negative by RAIA tested positive by all-in-one step SARS-CoV-2 Total (COV2T) assay performed on the automated Siemens Advia Centaur XPT analyzer. None of these methods, however, are useful in early diagnosis of SARS-CoV-2., Competing Interests: Baylor Scott and White Health provided support for the study in the form of salaries for all authors. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare. All authors have no other potential competing financial, non-financial, professional, or personal interests.

While complex sample pooling strategies have been developed for large-scale experiments with robotic liquid handling, many medium-scale experiments like mycotoxin screening by Enzyme-Linked Immunosorbent Assay (ELISA) are still conducted manually in 48- and 96-well plates. At this scale, the opportunity to save on reagent costs is offset by the increased costs of labor, materials, and risk-of-error caused by increasingly complex pooling strategies. This paper compares one-dimensional (1D), two-dimensional (2D), and Shifted Transversal Design (STD) pooling to study whether pooling affects assay accuracy and experimental cost and to provide guidance for when a human experimentalist might benefit from pooling. We approximated mycotoxin contamination in single corn kernels by fitting statistical distributions to experimental data (432 kernels for aflatoxin and 528 kernels for fumonisin) and used experimentally-validated Monte-Carlo simulation (10,000 iterations) to evaluate assay sensitivity, specificity, reagent cost, and pipetting cost. Based on the validated simulation results, assay sensitivity remains 100% for all four pooling strategies while specificity decreases as prevalence level rises. Reagent cost could be reduced by 70% and 80% in 48- and 96-well plates, with 1D and STD pooling being most reagent-saving respectively. Such a reagent-saving effect is only valid when prevalence level is < 21% for 48-well plates and < 13%-21% for 96-well plates. Pipetting cost will rise by 1.3-3.3 fold for 48-well plates and 1.2-4.3 fold for 96-well plates, with 1D pooling by row requiring the least pipetting. Thus, it is advisable to employ pooling when the expected prevalence level is below 21% and when the likely savings of up to 80% on reagent cost outweighs the increased materials and labor costs of up to 4 fold increases in pipetting., Competing Interests: The authors have declared that no competing interests exist

In a previous paper we showed that the yeast Pichia pastoris X-33 grown in parboiled rice effluent supplemented with glycerol byproduct from the biodiesel industry improved the quality of the effluent. In this paper we show the validation of this yeast (PPE) as probiotic for broilers. Its effect on feed efficiency and immunomodulation was compared with the same yeast grown in yeast peptone dextrose medium (PPY), with Saccharomyces boulardii (SBY) and with the controls fed unsupplemented feed (CON). One-day-old female chicks were vaccinated against infectious bursal disease (IBD) and the titers of anti-IBD antibodies were measured by ELISA. PPE group had the highest mean titres on days 14 and 28 (p<0,05), and at 28 days, 64% of the animals showed seroconvertion. The PPE group also showed the best weight gains at 42 days of age, that, on days 7, 14 and 21 were 19%, 15%, and 8.7% higher, respectively, than the control group. The best feed conversion, 8.2% higher than the control group, was obtained by PPY at 42 days. Histopathological studies did not detect any undesirable effects in the supplemented animals. We concluded that Pichia pastoris X-33 when grown in effluents of the rice parboiling industry supplemented with glycerol byproduct from the biodiesel has probiotic properties for poultry. [ABSTRACT FROM AUTHOR]

The high-affinity interaction between biotin and streptavidin has opened avenues for using recombinant proteins with site-specific biotinylation to achieve efficient and directional immobilization. The site-specific biotinylation of proteins carrying a 15 amino acid long Biotin Acceptor Peptide tag (BAP; also known as AviTag) is effected on a specific lysine either by co-expressing the E. coli BirA enzyme in vivo or by using purified recombinant E. coli BirA enzyme in the presence of ATP and biotin in vitro. In this paper, we have designed a T7 promoter-lac operator-based expression vector for rapid and efficient cloning, and high-level cytosolic expression of proteins carrying a C-terminal BAP tag in E. coli with TEV protease cleavable N-terminal deca-histidine tag, useful for initial purification. Furthermore, a robust three-step purification pipeline integrated with well-optimized protocols for TEV protease-based H10 tag removal, and recombinant BirA enzyme-based site-specific in vitro biotinylation is described to obtain highly pure biotinylated proteins. Most importantly, the paper demonstrates superior sensitivities in indirect ELISA with directional and efficient immobilization of biotin-tagged proteins on streptavidin-coated surfaces in comparison to passive immobilization. The use of biotin-tagged proteins through specific immobilization also allows more efficient selection of binders from a phage-displayed naïve antibody library. In addition, for both these applications, specific immobilization requires much less amount of protein as compared to passive immobilization and can be easily multiplexed. The simplified strategy described here for the production of highly pure biotin-tagged proteins will find use in numerous applications, including those, which may require immobilization of multiple proteins simultaneously on a solid surface. [ABSTRACT FROM AUTHOR]

The techniques used for diagnosis of canine visceral leishmaniasis (CVL) in Brazil ELISA and IFAT have been extensively questioned because of the accuracy of these tests. A recent change in the diagnosis protocol excluded IFAT and included the Dual-Path Platform (DPP). We evaluated the prevalence and incidence rates of Leishmania spp. before and after the change in the protocol. In addition, based on our results, we propose a new alternative that is less expensive for the screening and confirmation of CVL. Plasma samples were obtained from a serobank from dogs evaluated in a cross-sectional study (1,226 dogs) and in a cohort study of susceptible animals (n = 447), followed for 26 months. Serology testing was performed using ELISA, IFAT, and DPP. The incidence and prevalence of CVL were determined by using the protocol of the Visceral Leishmaniasis Control and Surveillance Program until 2012 (ELISA and IFAT using filter paper) and the protocol used after 2012 (DPP and ELISA using plasma). The prevalence was 6.2% and the incidence was 2.8 per 1,000 dog-months for the protocol used until 2012. For the new diagnosis protocol for CVL resulted in an incidence of 5.4 per 1,000 dog-months and a prevalence of 8.1%. Our results showed that the prevalence and incidence of infection were far greater than suggested by the previously used protocol and that the magnitude of infection in endemic areas has been underestimated. As tests are performed sequentially and euthanasia of dogs is carried out when the serological results are positive in both tests, the sequence does not affect the number of animals to be eliminated by the Control Program. Then we suggest to municipalities with a large demand of exams to use ELISA for screening and DPP for confirmation, since this allows easier performance and reduced cost. [ABSTRACT FROM AUTHOR]

In this study, a key issue to be addressed is the safe disposal of hybridoma instability. Hybridoma technology was used to produce anti–O. viverrini monoclonal antibody. Previous studies have shown that antibody production via antibody phage display can sustain the hybridoma technique. This paper presents the utility of antibody phage display technology for producing the phage displayed KKU505 Fab fragment and using experiments in concomitant with molecular simulation for characterization. The phage displayed KKU505 Fab fragment and characterization were successfully carried out. The KKU505 hybridoma cell line producing anti–O. viverrini antibody predicted to bind to myosin was used to synthesize cDNA so as to amplify the heavy chain and the light chain sequences. The KKU505 displayed phage was constructed and characterized by a molecular modeling in which the KKU505 Fab fragment and -O. viverrini myosin head were docked computationally and it is assumed that the Fab fragment was specific to -O. viverrini on the basis of mass spectrometry and Western blot. This complex interaction was confirmed by molecular simulation. Furthermore, the KKU505 displayed phage was validated using indirect enzyme-linked immunosorbent assays (ELISA) and immunohistochemistry. It is worthy to note that ELISA and immunohistochemistry results confirmed that the Fab fragment was specific to the -O. viverrini antigen. Results indicated that the approach presented herein can generate anti–O. viverrini antibody via the phage display technology. This study integrates the use of phage display technology together with molecular simulation for further development of monoclonal antibody production. Furthermore, the presented work has profound implications for antibody production, particularly by solving the problem of hybridoma stability issues. [ABSTRACT FROM AUTHOR]

Background: The imbalance between T helper 17 (Th17) and T regulatory (Treg) cells plays a key role in the progression of multiple myeloma (MM). Methods: The gene expression profiles of MM were acquired and examined from the Gene Expression Omnibus (GEO) database (GSE72213). Our research involved experimental investigations conducted using the MOPC-MM mouse model. Dysregulation of Treg and Th17 cells was evaluated through flow cytometry, while the levels of inflammatory factors were measured using the enzyme-linked immunosorbent assay. Cell proliferation was gauged using the Cell Counting Kit-8 assay, and cell apoptosis was quantified via flow cytometry. Cell metastasis capabilities were determined by conducting transwell assays. To confirm the relationship between miR-27a and PI3K, a dual-luciferase reporter assay was employed. Finally, proteins associated with the PI3K/AKT/mTOR signaling pathway were assessed using western blotting. Results: MiR-27a exhibited reduced expression levels in MM. Moreover, it exerted control over the equilibrium of Th17 and Treg cells while reducing the expression of inflammatory mediators such as TGF-β1 and IL-10 in an in vivo setting. Elevated miR-27a levels led to the inhibition of cell viability, colony formation capacity, migratory and invasive traits in an in vitro context. The PI3K/AKT/mTOR signaling pathway was identified as a direct target of miR-27a and could reverse the effects induced by miR-27a in MM cells. Notably, PI3K was directly targeted by miR-27a. Conclusions: Our study revealed that miR-27a inhibited MM evolution by regulating the Th17/Treg balance. Inhibition of the PI3K/AKT/mTOR signaling pathway by miR-27a may play a potential mechanistic role. [ABSTRACT FROM AUTHOR]

Celiac disease (CD) is an inflammatory disorder of the upper small intestine caused by the ingestion of storage proteins (prolamins and glutelins) from wheat, barley, rye, and, in rare cases, oats. CD patients need to follow a gluten-free diet by consuming gluten-free products with gluten contents of less than 20 mg/kg. Currently, the recommended method for the quantitative determination of gluten is an enzyme-linked immunosorbent assay (ELISA) based on the R5 monoclonal antibody. Because the R5 ELISA mostly detects the prolamin fraction of gluten, a new independent method is required to detect prolamins as well as glutelins. This paper presents the development of a method to quantitate 16 wheat marker peptides derived from all wheat gluten protein types by liquid chromatography tandem mass spectrometry (LC-MS/MS) in the multiple reaction monitoring mode. The quantitation of each marker peptide in the chymotryptic digest of a defined amount of the respective reference wheat protein type resulted in peptide-specific yields. This enabled the conversion of peptide into protein type concentrations. Gluten contents were expressed as sum of all determined protein type concentrations. This new method was applied to quantitate gluten in wheat starches and compared to R5 ELISA and gel-permeation high-performance liquid chromatography with fluorescence detection (GP-HPLC-FLD), which resulted in a strong correlation between LC-MS/MS and the other two methods. [ABSTRACT FROM AUTHOR]

Objective: The main pathological change of myocarditis is an inflammatory injury of cardiomyocytes. Long noncoding RNAs (lncRNAs) are closely related to inflammation, and our previous study showed that differential expression of lncRNAs is associated with myocarditis. This study aimed to investigate the impact of lncRNAs on the onset of myocarditis. Methods: RNA expression was measured by quantitative reverse-transcription polymerase chain reaction (RT-qPCR). Lipopolysaccharide (LPS) was used to induce inflammation in human cardiomyocytes (HCMs). The expression of inflammatory cytokines and myocardial injury markers was detected by enzyme-linked immunosorbent assay (ELISA) and RT-qPCR. Cell viability and apoptosis were measured by the cell counting kit-8 assay and flow cytometry. The binding force between lncRNA NONHSAT122636.2 and microRNA miRNA-2110 was detected using the dual-luciferase assay. Results: NONHSAT122636.2 was dynamically expressed in patients with myocarditis and negatively correlated with inflammation severity. The overexpression of NONHSAT122636.2 improved inflammatory injury in LPS-stimulated HCMs. The study observed that there was a weak binding force between NONHSAT122636.2 and miR-2110. Conclusion: NONHSAT122636.2 attenuates myocardial inflammation and apoptosis in myocarditis. Additionally, its expression decreases in the peripheral blood of children suffering from myocarditis and in patients who are diagnosed for the first time showing higher diagnostic sensitivity and specificity. This decrease is negatively correlated with the degree of inflammation. Overall, the study suggests that NONHSAT122636.2 can be exploited as a potential diagnostic biomarker for pediatric myocarditis. [ABSTRACT FROM AUTHOR]

Initial transmission of severe acute respiratory syndrome virus-2 (SARS-CoV-2) was highest in densely populated regions of Kenya. Transmission gradually trickled down to the less densely populated, remote and underserved regions such as the pastoral regions of Kajiado County which are characterized by poor healthcare systems. Molecular assays that were pivotal for COVID-19 diagnosis were not available in these regions. Serology is an alternative method for retrospectively tracking the transmission of SARS-CoV-2 in such populations. Dry blood spots (DBS) were prepared from consenting patients attending six health facilities in Kajiado County from March 2020 to March 2022. Upon elution, we conducted an enzyme-linked immunosorbent assay (ELISA) for the detection of SARS-Cov-2 IgG antibodies. Of the 908 DBSs we analyzed, 706 (78%) were from female participants. The overall seropositivity to SARS-Cov-2 antibodies was 7.3% (95% CI 5.7–9.1). The elderly (over 60 years) and male participants had a high likelihood of testing positive for SAR-CoV-2 infections. Mashuru (15.6%, 14/90) and Meto (15%, 19/127) health facilities registered the highest proportion of seropositive participants. Evidence of SARS-CoV-2 transmission among pastoralists in the remote and underserved regions of Kajiado County was established by DBS sampling and serologic testing. [ABSTRACT FROM AUTHOR]

Background: Caspase-1 is a crucial component in the inflammasome activation cascade. This study evaluated the potential of serum caspase-1 level as an inflammatory biomarker in patients with adult-onset Still's disease (AOSD). Methods: The study included 51 consecutive patients diagnosed with AOSD based on the Yamaguchi criteria, 66 patients with rheumatoid arthritis (RA) as disease control, and 36 healthy controls (HCs). Serum caspase-1 concentrations were measured using enzyme-linked immunosorbent assay. The serum 69 cytokine levels were analyzed using a multisuspension cytokine array in patients with AOSD, and a cluster analysis of each cytokine was performed to determine specific molecular networks. Results: Patients with AOSD had significantly increased serum caspase-1 levels versus patients with RA (p < 0.001) and HCs (p < 0.001). Additionally, serum caspase-1 demonstrated significant positive correlations with AOSD disease activity score (Pouchot score, r = 0.59, p < 0.001) and serum ferritin (r = 0.54, p < 0.001). Furthermore, among patients with AOSD, significant correlations existed between serum caspase-1 and inflammatory cytokines, including interleukin-18. Immunoblot analysis detected the cleaved form of caspase-1 (p20) in the serum of untreated patients with AOSD, not in those from patients with inactive AOSD receiving immunosuppressive treatments. Conclusions: Caspase-1 is a useful biomarker for AOSD diagnosis and monitoring. Caspase-1 activation could be correlated with the inflammatory component of AOSD, specifically through proinflammatory cytokine induction via inflammasome activation cascades. [ABSTRACT FROM AUTHOR]

At present, the mechanism of fluorosis-induced damage to the hepatic system is unclear. Studies have shown that excess fluoride causes some degree of damage to the liver, including inflammation. The SDF-1/CXCR4 signaling axis has been reported to have an impact on the regulation of inflammation in human cells. In this study, we investigated the role of the SDF-1/CXCR4 signaling axis and related inflammatory factors in fluorosis through in vitro experiments on human hepatic astrocytes (LX-2) cultured with sodium fluoride. CCK-8 assays showed that the median lethal dose at 24 h was 2 mmol/l NaF, and these conditions were used for subsequent enzyme-linked immunosorbent assays (ELISAs) and quantitative real-time polymerase chain reaction (qPCR) analysis. The protein expression levels of SDF-1/CXCR4 and the related inflammatory factors nuclear factor-κB (NF-κB), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and interleukin 1β (IL-1β) were detected by ELISAs from the experimental and control groups. The mRNA expression levels of these inflammatory indicators were also determined by qPCR in both groups. Moreover, the expression levels of these factors were significantly higher in the experimental group than in the control group at both the protein and mRNA levels (P < 0.05). Excess fluorine may stimulate the SDF-1/CXCR4 signaling axis, activating the inflammatory NF-κB signaling pathway and increasing the expression levels of the related inflammatory factors IL-6, TNF-α and IL-1β. Identification of this mechanism is important for elucidating the pathogenesis of fluorosis-induced liver injury. [ABSTRACT FROM AUTHOR]

In this study, we aimed to investigate the protective effects of Panax notoginseng and leech (PL) on renal fibrosis and explore the mechanisms underlying their actions. For this study, we created an adenine-induced renal fibrosis model in SD rats to investigate the protective effect of PL on renal fibrosis and explore its underlying mechanism. Initially, we assessed the renal function in RF rats and found that Scr, BUN, and urine protein content decreased after PL treatment, indicating the protective effect of PL on renal function. Histological analysis using HE and Masson staining revealed that PL reduced inflammatory cell infiltration and decreased collagen fiber deposition in renal tissue. Subsequently, we analyzed the levels of α-SMA, Col-IV, and FN, which are the main components of the extracellular matrix (ECM), using IHC, RT-qPCR, and WB. The results demonstrated that PL was effective in reducing the accumulation of ECM, with PL1-2 showing the highest effectiveness. To further understand the underlying mechanisms, we conducted UPLC-MS/MS analysis on the incoming components of the PL1-2 group. The results revealed several associations between the differential components and antioxidant and mitochondrial functions. This was further confirmed by enzyme-linked immunosorbent assay and biochemical indexes, which showed that PL1-2 ameliorated oxidative stress by reducing ROS and MDA production and increasing GSH and SOD levels. Additionally, transmission electron microscopy results indicated that PL1-2 promoted partial recovery of mitochondrial morphology and cristae. Finally, using RT-qPCR and WB, an increase in the expression of mitochondrial fusion proteins Mfn1, Mfn2, and Opa1 after PL1-2 treatment was observed, coupled with a decline in the expression and phosphorylation of mitochondrial cleavage proteins Fis and Drp1. These findings collectively demonstrate that PL1-2 ameliorates renal fibrosis by reducing oxidative stress and restoring mitochondrial balance. [ABSTRACT FROM AUTHOR]

Purpose: The resurgence of pertussis has occurred around the world. However, the epidemiological profiles of pertussis cannot be well understood by current diseases surveillance. This study was designed to understand the seroepidemiological characteristics of pertussis infection in the general population of Huzhou City, evaluate the prevalence infection of pertussis in the population, and offer insights to inform adjustments in pertussis prevention and control strategies. Methods: From September to October 2023, a cross-sectional serosurvey was conducted in Huzhou City, involving 1015 permanent residents. Serum samples were collected from the study subjects, and pertussis toxin IgG antibodies (Anti-PT-IgG) were quantitatively measured using enzyme-linked immunosorbent assay (ELISA). The analysis included the geometric mean concentration (GMC) of Anti-PT-IgG, rates of GMC≥40IU/mL, ≥100IU/mL, and <5IU/mL. Stratified comparisons were made based on age, vaccination history, and human categories. Results: Among the 1015 surveyed individuals, the geometric mean concentration (GMC) of Anti-PT-IgG was 10.52 (95% CI: 9.96–11.11) IU/mL, with a recent infection rate of 1.58%, a serum positivity rate of 11.43%, and a proportion with <5IU/mL of 40.49%. Among 357 children with clear vaccination history, susceptibility decreased with an increasing number of vaccine doses (Z = -6.793, P < 0.001). The concentration of Anti-PT-IgG exhibited a significant post-vaccination decline over time (Z = -5.143, P < 0.001). In women of childbearing age, the GMC of Anti-PT-IgG was 7.71 (95% CI: 6.90–8.62) IU/mL, with no significant difference in susceptibility among different age groups (χ2 = 0.545, P = 0.909). The annual pertussis infection rate in individuals aged ≥3 years was 9321 (95%CI: 3336–16039) per 100,000, with peak infection rates in the 20–29, 40–49, and 5–9 age groups at 34363 (95%CI: 6327–66918) per 100,000, 22307.72 (95%CI: 1380–47442) per 100,000, and 18020(95%CI: 1093–37266) per 100,000, respectively. Conclusions: In 2023, the actual pertussis infection rate in the population of Huzhou City was relatively high. Vaccine-induced antibodies exhibit a rapid decay, and the estimated serum infection rate increases rapidly from post-school age, peaking in the 20–29 age group. It is recommended to enhance pertussis monitoring in adolescents and adults and refine vaccine immunization strategies. [ABSTRACT FROM AUTHOR]

Background: Hepatitis B (HBV) and C virus (HCV) coinfection are the major causes of liver-related morbidity and mortality among people living with Human Immunodeficiency Virus (HIV). The burden of hepatitis among HIV-positive individuals has not been studied in the Afar region. Therefore, this study aimed to determine the prevalence of HBV and HCV coinfection and associated factors among HIV-positive patients in Afar Regional State, northeast Ethiopia. Methods: A cross-sectional study was conducted on 477 HIV-positive patients between February 2019 and May 2019. A structured and pretested questionnaire was used to collect socio-demographic data and associated factors. Five milliliters of blood was collected, and Hepatitis B surface antigen (HBsAg) and HCV antibodies were detected using rapid test kits. Positive samples were confirmed using enzyme-linked immunosorbent assay (ELISA). Binary and multivariable logistic regression analyses were performed to identify associated factors. Statistical significance was set at P <0.05. Results: Among the 477 study participants, 320/477(67.1%) of them were females and 157(32.9%) males. The overall prevalence of HIV-HBV and HIV-HCV coinfection was 25(5.2%) and 7(1.5%), respectively. Multi-sexual practice was significantly associated with HIV-HBV coinfection (AOR = 5.3; 95% CI: 1.2–24.4, P = 0.032). Conclusion: The prevalence of both HIV-HBV and HIV-HCV coinfection was intermediate. Multi-sexual practice was significantly associated with HIV-HBV coinfection. Screening of all HIV-positive patients for HBV and HCV and health education regarding the transmission modes should be considered. [ABSTRACT FROM AUTHOR]

Analbuminemia is characterized by the near absence of albumin in the plasma. Different methods are available for measuring albumin levels, but they do not necessarily agree with one another. It is a concern that analbuminemic samples could be falsely characterized due to the incorrect estimation of albumin. The objective of the work was to evaluate the performance of different assays in detecting analbuminemia. Albumin knockout (Alb-/-) mouse plasma was used to test the suitability of different albumin assays for their ability to properly characterize extreme albumin deficiency. Bromocresol green (BCG), bromocresol purple (BCP), enzyme-linked immunosorbent assay (ELISA), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and gel electrophoresis were tested. The LC-MS/MS assay exhibited broad coverage of the amino acid sequence of albumin and indicated 8,400-fold lower (P<0.0001) albumin expression in Alb-/- than wildtype (WT), demonstrating its suitability for identifying extreme albumin deficiency. ELISA estimated albumin at 1.5±0.1 g/dL in WT and was below the detection limit in all Alb-/- samples. Gel electrophoresis yielded consistent results with LC-MS/MS and ELISA. The BCG assay overestimated albumin with apparently appreciable albumin concentrations in Alb-/- mice, yet the assay still indicated a significant difference between genotypes (Alb-/-, 1.2±0.05 g/dL, WT, 3.7±0.1 g/dL, P<0.0001). BCP drastically overestimated albumin and could not successfully identify the known analbuminemic phenotype of Alb-/- mice. By using Alb-/- plasma as a reference material and LC-MS/MS as a reference method, ELISA and gel electrophoresis appear appropriate for identifying analbuminemia, while BCG and BCP are not suitable. It is concluded that dye-binding assays should be avoided when extreme hypoalbuminemia or analbuminemia is suspected. [ABSTRACT FROM AUTHOR]

Noroviruses are recognized worldwide as the principal cause of acute, non-bacterial gastroenteritis, resulting in 19-21 million cases of disease every year in the United States. Noroviruses have a very low infectious dose, a short incubation period, high resistance to traditional disinfection techniques and multiple modes of transmission, making early, point-of-care detection essential for controlling the spread of the disease. The traditional diagnostic tools, electron microscopy, RT-PCR and ELISA require sophisticated and expensive instrumentation, and are considered too laborious and slow to be useful during severe outbreaks. In this paper we describe the development of a new, rapid and sensitive lateral-flow assay using labeled phage particles for the detection of the prototypical norovirus GI.1 (Norwalk), with a limit of detection of 107 virus-like particles per mL, one hundred-fold lower than a conventional gold nanoparticle lateral-flow assay using the same antibody pair. [ABSTRACT FROM AUTHOR]

Subclinical infection of laboratory animals with one or more of several pathogens affects the results of experiments on animals. Monitoring the health of laboratory animals encompasses routine surveillance for pathogens, including several viruses. This study aimed to explore the development of an alternative assay to the existing ones for detecting infection of mice and rats with the parvoviruses minute virus of mice (MVM) and Kilham rat virus (KRV), respectively. Full-length VP2 and NS1 proteins of these parvoviruses, besides fragments containing multiple predicted epitopes stitched together, were studied for serological detection. The optimal dilution of full-length proteins and antigenic regions containing predicted epitopes for coating, test sera, and conjugate was determined using a checkerboard titration at each step. The assays were evaluated vis-à-vis commercially available ELISA kits. The results showed that an engineered fusion of fragments containing multiple predicted MVM VP2 and NS1 epitopes was better than either of the full-length proteins for detecting antibodies in 90% of the tested sera samples. For KRV ELISA, full-length VP2 was better compared to other individual recombinant protein fragments or combinations thereof for the detection of antibodies in sera. This report is the first description of an ELISA for KRV and an improved assay for MVM. Importantly, our assays could be exploited with small volumes of sera. The results also demonstrate the utility of immunoinformatics-driven polypeptide engineering in the development of diagnostic assays and the potential to develop better tests for monitoring the health status of laboratory animals. [ABSTRACT FROM AUTHOR]

Objective: Neurofilament light chain (Nf-L) has been used to detect neuroaxonal damage in the brain caused by physical injury or disease. The purpose of this study was to determine if serum Nf-L could be used as a biomarker for pre-symptomatic detection of scrapie in sheep. Methods: Four sheep with prion protein genotype AVQQ were intranasally inoculated with the classical scrapie strain x124. Blood was collected every 4 weeks until 44 weeks post-inoculation, at which point weekly collection commenced. Serum was analyzed using single molecule array (Quanterix SR-X) to evaluate Nf-L concentrations. Results: Scrapie was confirmed in each sheep by testing homogenized brainstem at the level of the obex with a commercially available enzyme immunoassay. Increased serum Nf-L concentrations were identified above the determined cutoff during the last tenth of the respective incubation period for each sheep. Throughout the time course study, PrPSc accumulation was not detected antemortem by immunohistochemistry in rectal tissue at any timepoint for any sheep. RT-QuIC results were inconsistently positive throughout the timepoints tested for each sheep; however, each sheep had at least one timepoint detected positive. When assessing serum Nf-L utility using receiver operator characteristic curves against different clinical parameters, such as asymptomatic and symptomatic (pruritus or neurologic signs), results showed that Nf-L was most useful at being an indicator of disease only late in disease progression when neurologic signs were present. Conclusion: Serum Nf-L concentrations in the cohort of sheep increased as disease progressed; however, serum Nf-L did not increase during the presymptomatic window. The levels increased substantially throughout the final 10% of the animals' scrapie incubation period when other clinical signs were present. Serum Nf-L is not a reliable biomarker for pre-clinical detection of scrapie. [ABSTRACT FROM AUTHOR]

Background: Pancreatic ductal adenocarcinomas (PDAC) have heterogeneous tumor microenvironments relatively devoid of infiltrating immune cells. We aimed to quantitatively assess infiltrating CD3+ and CD8+ lymphocytes in a treatment-naïve patient cohort and assess associations with overall survival and microenvironment inflammatory proteins. Methods: Tissue microarrays were immunohistochemically stained for CD3+ and CD8+ lymphocytes and quantitatively assessed using QuPath. Levels of inflammation-associated proteins were quantified by multiplexed, enzyme-linked immunosorbent assay panels on matching tumor and tissue samples. Results: Our findings revealed a significant increase in both CD3+ and CD8+ lymphocytes populations in PDAC compared with non-PDAC tissue, except when comparing CD8+ percentages in PDAC versus intraductal papillary mucinous neoplasms (IPMN) (p = 0.5012). Patients with quantitatively assessed CD3+ low tumors (lower 50%) had shorter survival (median 273 days) compared to CD3+ high tumors (upper 50%) with a median overall survival of 642.5 days (p = 0.2184). Patients with quantitatively assessed CD8+ low tumors had significantly shorter survival (median 240 days) compared to CD8+ high tumors with a median overall survival of 1059 days (p = 0.0003). Of 41 proteins assessed in the inflammation assay, higher levels of IL-1B and IL-2 were significantly associated with decreased CD3+ infiltration (r = -0.3704, p = 0.0187, and r = -0.4275, p = 0.0074, respectively). Higher levels of IL-1B were also significantly associated with decreased CD8+ infiltration (r = -0.4299, p = 0.0045), but not IL-2 (r = -0.0078, p = 0.9616). Principal component analysis of the inflammatory analytes showed diverse inflammatory responses in PDAC. Conclusion: In this work, we found a marked heterogeneity in infiltrating CD3+ and CD8+ lymphocytes and individual inflammatory responses in PDAC. Future mechanistic studies should explore personalized therapeutic strategies to target the immune and inflammatory components of the tumor microenvironment. [ABSTRACT FROM AUTHOR]

Background: Undernutrition and malnutrition in children in low- and middle-income countries contribute to high mortality rates. Stunting, a prevalent form of malnutrition, is associated with educational and productivity losses. Environmental enteric dysfunction (EED) and human immunodeficiency virus (HIV) infection worsen these conditions. This study seeks to investigate the presence of enteropathy using EED fecal biomarkers in HIV-infected children who are stable on antiretroviral therapy (ART) across various nutritional statuses. By understanding the interplay between EED, HIV, and nutritional status, this study aims to provide insights that can inform targeted interventions to optimize nutritional outcomes in HIV infected children. Methods/Principal findings: This study evaluated the levels of alpha-1-antitrypsin, calprotectin and myeloperoxidase in frozen fecal samples from 61 HIV infected (mean age 9.16 ±3.08 years) and 31 HIV uninfected (6.65 ±3.41 years) children in Malawi. Anthropometric measurements and clinical data were collected. The height-for-age z-score (-1.66 vs -1.27, p = 0.040) and BMI-for-age z-score (-0.36 vs 0.01, p = 0.037) were lower in HIV infected children. Enzyme-linked immunosorbent assays were used to measure biomarker concentrations. Statistical tests were applied to compare biomarker levels based on HIV status and anthropometric parameters. Myeloperoxidase, alpha-1-antitrypsin, and calprotectin concentrations did not differ between HIV infected and HIV uninfected children of different age groups. In HIV infected children from 5–15 years, there is no difference in biomarker concentration between the stunted and non-stunted groups. Conclusion/Significance: Our study found a higher prevalence of stunting in HIV infected children compared to uninfected children, but no significant differences in biomarker concentrations. This suggests no causal relationship between enteropathy and stunting in HIV infected children. These results contribute to the understanding of growth impairment in HIV infected children and emphasize the need for further research, particularly a longitudinal, biopsy-controlled study. [ABSTRACT FROM AUTHOR]

Detection of infections in wildlife species is increasingly important to reduce the risk of spreading zoonotic and economically important parasites, understand disease epidemiology and promote the conservation of wildlife species. Serological tests are key in disease diagnosis and surveillance by detecting immunoglobulins against infectious agents. However, the need for species-specific reagents has limited the application of serological tests in wildlife species. This study evaluated the serum immunoglobulin-binding capability of polyclonal anti-kangaroo antibody and two non-species-specific reagents, including protein A/G and protein L, with the largest range of Australian marsupial species so far, including 32 species representing three major marsupial orders. Immunoglobulin-binding capability was assessed using immunoblotting, enzyme-linked immunosorbent assay and Western blot techniques. Variation in immunoglobulin-binding capability was observed between the three reagents and across the species tested, both across but also within taxonomic groups. Taxonomic distance was thus not always a good predictor of immunoglobulin-binding affinity, emphasizing the need to validate these reagents for each species separately. However, all three reagents bound with the serum immunoglobulins of most marsupial species tested. The findings of this study provide a valuable reference for species differences in affinity to protein A/G, protein L and anti-kangaroo antibody, assisting in the selection of appropriate reagents and the development of sero-immunological assays in Australian marsupials. [ABSTRACT FROM AUTHOR]

Canine monocytic ehrlichiosis (CME) has been observed to impact renal function. Currently, the recognition of acute kidney injury is through the nonspecific biomarker serum creatinine (sCr). Novel markers of renal injury such as urinary clusterin (uClust) and urinary cystatin B (uCysB) may increase our understanding of the relationship between ehrlichiosis and renal cellular injury. The aim of this study was to evaluate novel renal injury biomarkers in dogs with acute CME. Twenty healthy dogs were enrolled in the control group (CG), and 16 dogs naturally infected with Ehrlichia canis were included in the Ehrlichia Group (EG). All dogs were followed for 45 days. EG dogs were treated with doxycycline twice daily for the first 30 days. Urine and serum were collected at: 0, 0.5, 1, 15, 30, and 45 days after start of treatment. Urine concentrations of uClust and uCysB were determined using a research ELISA immunoassay. A linear mixed model was used to estimate population mean of renal injury markers with patient as the random effect, and day and treatment as fixed effects. EG was observed to have higher uClust values compared to CG (estimated population mean EG: 213 ng/dL vs. CG: 84 ng/dL, P < 0.001). EG was observed to have higher uCysB values compared to CG (estimated population mean EG: 248 ng/dL vs. CG: 38 ng/dL, P < 0.001). Increases in uCysB and uClust suggest the presence of renal injury and a possible mechanism for the observed predisposition to chronic kidney disease in dogs with ehrlichiosis. [ABSTRACT FROM AUTHOR]

Bovine viral diarrhoea, caused by the bovine viral diarrhoea virus (BVDV) in the Pestivirus genus of the Flaviviridae, is one of the most important diseases of cattle world wide causing poor reproductive performance in adult cattle and mucosal disease in calves. In addition it causes immunosuppression and increased susceptibility to other infections, the impact of which is uncertain, particularly in sub-Saharan Africa where animals are exposed to a much wider range and higher intensity of infections compared to Europe. There are no previous estimates of the seroprevalence of BVDV in cattle in Cameroon. This paper describes the serological screening for antibodies to BVDV and antigen of BVDV in a cattle population in the Adamawa Region of Cameroon in 2000. The estimates of herd-level and within herd seroprevalences adjusted for test imperfections were 92% and 30% respectively and 16.5% of herds were classed as having a persistently infected calf (PI) in the herd within the last year based on the "spot" test approach. There was evidence of clustering of herds with PI calves across the north and west of the Region which corresponds with the higher cattle density areas and of self-clearance of infection from herds. A multivariable model was developed for the risk of having a PI calf in the herd; proximity to antelope, owning a goat, mixing with > 10 other herds at grazing and the catchment area of the veterinary centre the herd was registered at were all significant risk factors. Very little is known about BVDV in sub-Saharan Africa and these high seroprevalences suggest that there is a large problem which may be having both direct impacts on fertility and neonate mortality and morbidity and also indirect effects through immunosuppression and susceptibility to other infections. Understanding and accounting for BVDV should be an important component of epidemiological studies of other diseases in sub-Saharan Africa.

A number of different interferon-gamma ELISpot protocols are in use in laboratories studying antigen-specific immune responses. It is therefore unclear how results from different assays compare, and what factors most significantly influence assay outcome. One such difference is that some laboratories use a short in vitro stimulation period of cells before they are transferred to the ELISpot plate; this is commonly done in the case of frozen cells, in order to enhance assay sensitivity. Other differences that may be significant include antibody coating of plates, the use of media with or without serum, the serum source and the number of cells added to the wells. The aim of this paper was to identify which components of the different ELISpot protocols influenced assay sensitivity and inter-laboratory variation. Four laboratories provided protocols for quantifying numbers of interferon-gamma spot forming cells in human peripheral blood mononuclear cells stimulated with Mycobacterium tuberculosis derived antigens. The differences in the protocols were compared directly. We found that several sources of variation in assay protocols can be eliminated, for example by avoiding serum supplementation and using AIM-V serum free medium. In addition, the number of cells added to ELISpot wells should also be standardised. Importantly, delays in peripheral blood mononuclear cell processing before stimulation had a marked effect on the number of detectable spot forming cells; processing delay thus should be minimised as well as standardised. Finally, a pre-stimulation culture period improved the sensitivity of the assay, however this effect may be both antigen and donor dependent. In conclusion, small differences in ELISpot protocols in routine use can affect the results obtained and care should be given to conditions selected for use in a given study. A pre-stimulation step may improve the sensitivity of the assay, particularly when cells have been previously frozen.

Brucellosis is considered by the Food and Agricultural Organisation and the World Health Organisation as one of the most widespread zoonoses in the world. It is a major veterinary public health challenge as animals are almost exclusively the source of infection for people. It is often undiagnosed in both human patients and the animal sources and it is widely acknowledged that the epidemiology of brucellosis in humans and animals is poorly understood, particularly in sub-Saharan Africa. It is therefore important to develop better diagnostic tools in order to improve our understanding of the epidemiology and also for use in the field for disease control and eradication. As with any new diagnostic test, it is essential that it is validated in as many populations as possible in order to characterise its performance and improve the interpretation of its results. This paper describes a comparison between a new lateral flow assasy (LFA) for bovine brucellosis and the widely used cELISA in a no gold standard analysis to estimate test performance in this West African cattle population. A Bayesian formulation of the Hui-Walter latent class model incorporated previous studies' data on sensitivity and specificity of the cELISA. The results indicate that the new LFA is very sensitive (approximately 87%) and highly specific (approximately 97%). The analysis also suggests that the current cut-off of the cELSIA may not be optimal for this cattle population but alternative cut-offs did not significantly change the estimates of the LFA. This study demonstrates the potential usefulness of this simple to use test in field based surveillance and control which could be easily adopted for use in developing countries with only basic laboratory facilities.

In this study, we describe a novel porcine parechovirus-like virus (tentatively named PLV-CHN) from healthy piglets in China using 454 high-throughput sequencing. The complete genome of the virus comprises 6832 bp, encoding a predicted polyprotein of 2132 amino acids that is most similar to Ljungan virus (32% identity). A similar virus that belongs to a novel Picornaviridae genus, named swine pasivirus 1 (SPaV-1), was reported during the preparation of this paper. Sequence analysis revealed that PLV-CHN and SPaV1 shared 82% nucleotide identity and 89% amino acid identity. Further genomic and phylogenetic analyses suggested that both SPaV1 and PLV-CHN shared similar genomic characteristics and belong to the same novel Picornaviridae genus. A total of 36 (20.0%) fecal samples from 180 healthy piglets were positive for PLV-CHN by RT-PCR, while no fecal samples from 100 healthy children and 100 children with diarrhea, and no cerebrospinal fluid samples from 196 children with suspected viral encephalitis, was positive for the virus. However, Western blot and enzyme-linked immunosorbent assays using recombinant PLV-CHN VP1 polypeptide as an antigen showed a high seroprevalence of 63.5% in the healthy population. When grouped by age, the antibody-positivity rates showed that the majority of children under 12 years of age have been infected by the virus. It was suggested that PLV-CHN, SPaV1, or an as-yet-uncharacterized virus can infect humans early in life. Thus, investigation of the role of this novel virus is vital. [ABSTRACT FROM AUTHOR]

Nanobodies (or variable domain of the heavy chain of the heavy-chain antibodies, VHHs) are single-domain antigen-binding fragments derived from camelid heavy chain antibodies. Their comparatively small size, monomeric behavior, high stability, high solubility, and ability to bind epitopes inaccessible to conventional antibodies make them especially suitable for many therapeutic and biotechnological applications. In this paper, for the first time, we created the immunized Camelus Bactrianus VHH yeast two-hybrid (Y2H) library according to the Clontech Mate & Plate library construction system. The transformation efficiency and titer of the VHH Y2H library were 7.26×106 cfu/3 µg and 2×109 cfu/ml, which met the demand for Y2H library screening. Using as an example the porcine circovirus type 2 (PCV2) Cap protein as bait, we screened 21 positive Cap-specific VHH sequences. Among these sequences, 7 of 9 randomly selected clones were strongly positive as indicated by enzyme-linked immunosorbent assay, either using PCV2 viral lysis or purified Cap protein as coated antigen. Additionally, the immunocytochemistry results further indicated that the screened VHHs could specifically detected PCV2 in the infected cells. All this suggests the feasibility of in vivo VHH throughput screening based on Y2H strategy. [ABSTRACT FROM AUTHOR]

Background: Early initiation of antiretroviral therapy reduces risk of transmission to the uninfected partner in HIV discordant couples, but there are relatively little observational data on HIV transmission within couples from non-trial settings. The aims of this paper are to estimate HIV incidence among HIV discordant couples using longstanding observational data from a rural Ugandan population and to identify factors associated with HIV transmission within couples, including the role of HSV-2 infection. Methods: Using existing data collected at population-wide annual serological and behavioural surveys in a rural district in southwest Uganda between 1989 and 2007, HIV discordant partners were identified. Stored serum samples were tested for HSV-2 serostatus using the Kalon ELISA test. HIV seroconversion rates and factors association with HIV seroconversion were analysed using Poisson regression. Results: HIV status of both partners was known in 2465 couples and of these 259 (10.5%) were HIV serodiscordant. At enrolment, HSV-2 prevalence was 87.3% in HIV positive partners and 71.5% in HIV negative partners. Of the 259 discordant couples, 62 converted to HIV (seroconversion rate 7.11/100 PYAR, 95%CI; 5.54, 9.11) with the rate decreasing from 10.89 in 1990–1994 to 4.32 in 2005–2007. Factors independently associated with HIV seroconversion were female sex, non-Muslim religion, greater age difference (man older than woman by more than 15 years), higher viral load in the positive partner and earlier calendar period. HSV-2 was not independently associated with HIV acquisition (HR 1.62, 95%CI; 0.57, 4.55) or transmission (HR 0.61, 95%CI; 0.24, 1.57). No transmissions occurred in the 29 couples where the index partner was on ART during follow up (872 person-years on ART). Discussion: HIV negative partners in serodiscordant couples have a high incidence of HIV if the index partner is not on antiretroviral therapy and should be provided with interventions such as couple counselling, condoms and antiretroviral treatment. [ABSTRACT FROM AUTHOR]

Situated in southeastern Sierra Leone, Kenema Government Hospital (KGH) maintains one of the world’s only Lassa fever isolation wards and was a strategic Ebola virus disease (EVD) treatment facility during the 2014 EVD outbreak. Since 2006, the Viral Hemorrhagic Fever Consortium (VHFC) has carried out research activities at KGH, capturing clinical and laboratory data for suspected cases of Lassa fever. Here we describe the approach, progress, and challenges in designing and maintaining a data capture and management system (DCMS) at KGH to assist infectious disease researchers in building and sustaining DCMS in low-resource environments. Results on screening patterns and case-fatality rates are provided to illustrate the context and scope of the DCMS covered in this study. A medical records system and DCMS was designed and implemented between 2010 and 2016 linking historical and prospective Lassa fever data sources across KGH Lassa fever units and its peripheral health units. Data were captured using a case report form (CRF) system, enzyme-linked immunosorbent assay (ELISA) plate readers, polymerase chain reaction (PCR) machines, blood chemistry analyzers, and data auditing procedures. Between 2008 and 2016, blood samples for 4,229 suspected Lassa fever cases were screened at KGH, ranging from 219 samples in 2008 to a peak of 760 samples in 2011. Lassa fever case-fatality rates before and following the Ebola outbreak were 65.5% (148/226) and 89.5% (17/19), respectively, suggesting that fewer, but more seriously ill subjects with Lassa fever presented to KGH following the 2014 EVD outbreak (p = .040). DCMS challenges included weak specificity of the Lassa fever suspected case definition, limited capture of patient survival outcome data, internet costs, lapses in internet connectivity, low bandwidth, equipment and software maintenance, lack of computer teaching laboratories, and workload fluctuations due to variable screening activity. DCMS are the backbone of international research efforts and additional literature is needed on the topic for establishing benchmarks and driving goal-based approaches for its advancement in developing countries. [ABSTRACT FROM AUTHOR]

Aims: To present a new approach for estimating the "true prevalence" of malaria and apply it to datasets from Peru, Vietnam, and Cambodia. Methods: Bayesian models were developed for estimating both the malaria prevalence using different diagnostic tests (microscopy, PCR & ELISA), without the need of a gold standard, and the tests' characteristics. Several sources of information, i.e. data, expert opinions and other sources of knowledge can be integrated into the model. This approach resulting in an optimal and harmonized estimate of malaria infection prevalence, with no conflict between the different sources of information, was tested on data from Peru, Vietnam and Cambodia. Results: Malaria sero-prevalence was relatively low in all sites, with ELISA showing the highest estimates. The sensitivity of microscopy and ELISA were statistically lower in Vietnam than in the other sites. Similarly, the specificities of microscopy, ELISA and PCR were significantly lower in Vietnam than in the other sites. In Vietnam and Peru, microscopy was closer to the "true" estimate than the other 2 tests while as expected ELISA, with its lower specificity, usually overestimated the prevalence. Conclusions: Bayesian methods are useful for analyzing prevalence results when no gold standard diagnostic test is available. Though some results are expected, e.g. PCR more sensitive than microscopy, a standardized and context-independent quantification of the diagnostic tests' characteristics (sensitivity and specificity) and the underlying malaria prevalence may be useful for comparing different sites. Indeed, the use of a single diagnostic technique could strongly bias the prevalence estimation. This limitation can be circumvented by using a Bayesian framework taking into account the imperfect characteristics of the currently available diagnostic tests. As discussed in the paper, this approach may further support global malaria burden estimation initiatives. [ABSTRACT FROM AUTHOR]

Background: During the last years, several studies have reported the significant relationship between the production of soluble HLA-G molecules (sHLA-G) by 48-72 hours early embryos and an increased implantation rate in IVF protocols. As consequence, the detection of HLA-G modulation was suggested as a marker to identify the best embryos to be transferred. On the opposite, no suitable markers are available for the oocyte selection. Methodology/Principal Findings: The major finding of the present paper is that the release of ICAM-1 might be predictive of oocyte maturation. The results obtained are confirmed using three independent methodologies, such as ELISA, Bio-Plex assay and Western blotting. The sICAM-1 release is very high in immature oocytes, decrease in mature oocytes and become even lower in in vitro fertilized embryos. No significant differences were observed in the levels of sICAM-1 release between immature oocytes with different morphological characteristics. On the contrary, when the mature oocytes were subdivided accordingly to morphological criteria, the mean sICAM-I levels in grade 1 oocytes were significantly decreased when compared to grade 2 and 3 oocytes. Conclusions/Significance: The reduction of the number of fertilized oocytes and transferred embryos represents the main target of assisted reproductive medicine. We propose sICAM-1 as a biochemical marker for oocyte maturation and grading, with a possible interesting rebound in assisted reproduction techniques. [ABSTRACT FROM AUTHOR]

Objectives: To investigate maternal antibody levels to varicella in infants <12 months of age in Ontario, Canada. Study design: In this study, we included specimens from infants <12 months of age, born at ≥37 weeks gestational age, who had sera collected at The Hospital for Sick Children (Toronto, Canada) between 2014–2016. We tested sera using a glycoprotein-based enzyme-linked immunosorbent assay (gpELISA). We measured varicella susceptibility (antibody concentration <150mIU/mL) and mean varicella antibody concentration, and assessed the probability of susceptibility and concentration between one and 11 months of age using multivariable logistic regression and Poisson regression. Results: We found that 32% of 196 included specimens represented infants susceptible to varicella at one month of age, increasing to nearly 80% at three months of age. At six months of age, all infants were susceptible to varicella and the predicted mean varicella antibody concentration declined to 62 mIU/mL (95% confidence interval 40, 84), well below the threshold of protection. Conclusions: We found that varicella maternal antibody levels wane rapidly in infants, leaving most infants susceptible by four months of age. Our findings have implications for the timing of first dose of varicella-containing vaccine, infection control measures, and infant post-exposure prophylaxis recommendations. [ABSTRACT FROM AUTHOR]

Borrelia burgdorferi sensu lato and Rickettsia spp. are worldwide causes of tick-borne infections. We aimed to estimate the seroprevalence of immunoglobulin G (IgG) antibodies against different tick-borne diseases (TBDs) and determine risk factors among Mongolians from 2016 to 2020. Blood samples were obtained from voluntary participants with a history of suspected tick bite who visited our hospital, and IgG antibodies against Rickettsia and Borrelia were detected using enzyme-linked immunosorbent assay (ELISA). The IgG antibody seropositivity rate against Rickettsia was 21.8% (1032/4724), while 3.4% (162/4724) of participants tested positive for serum IgG antibodies against Borrelia by ELISA.Binary logistic regression analysis was performed to evaluate risk factors for tick-borne rickettsiosis (TBR) and tick-borne borreliosis (TBB) using IgG serum sample. Age, occupation, and residence were significantly associated with these diseases; however, sex did not show any significant association. Seroprevalence was significantly higher among herders (40.6%, 95% confidence interval [CI]: 35.5–45.8; odds ratio [OR] 0.61; P < 0.001) and students (32.8%, 95% CI: 30.2–35.4; OR 0.75; P < 0.001) than among individuals with other occupations. The 25–29 age group had a slightly higher seroprevalence (35.1%, 95% CI: 28.1–42.6; OR 0.61; P < 0.006) than those in other age groups. Province was a stronger predictor of TBR than occupation and age group. In univariate subgroup analysis by age group, occupation, and residence were significantly associated with TBR seroprevalence, whereas age and province were associated with TBB seroprevalence. Thus, risk factors for TBD include residence, occupation, and age group. This study was conducted using samples from all Mongolian provinces and the capital city, and the risk factors and prevalence of Rickettsia and Borreliaare highlighted. [ABSTRACT FROM AUTHOR]

Objectives: The study aimed to determine the allergen, endotoxin and β-(1,3)-glucan concentrations at various areas on a university campus of veterinary medicine. Methods: Dust samples were collected four times a year for three years using electrostatic dust collectors (EDC) at 25 different locations on a campus of veterinary medicine and in laboratories of inorganic chemistry as a control area representing animal-free environment. Major animal allergens from dog, cat, horse, cattle and mouse, domestic mite (DM) allergens, and β-(1,3)-glucan were measured using enzyme immunoassays and endotoxin using the limulus amoebocyte lysate (LAL) assay. Seasonal, annual and local influences on exposure levels were analyzed using Bayesian mixed models. Results: With the exception of mouse allergens, all other determinants were found in almost all locations on the campus and in the control area, but in up to 10.000-fold variable concentrations. By far the highest levels of feline, canine, equine and bovine allergens were detected in buildings where the respective species were examined. The highest levels of mouse and DM allergens, β-(1,3)-glucan and endotoxin occurred together and were associated with locations where large animals were present. In buildings without animals, allergen levels were considerably lower but still elevated at several locations compared to the control area, especially for dog and horse allergens, and β-(1,3)-glucan. Significant seasonal effects were observed for dog, cat, horse and DM allergens, and β-(1,3)-glucan. Variations between years were less apparent than between seasons (except for β-(1,3)-glucan). Conclusions: The strongest influencing factor on the concentration of mammalian allergens was the presence of the corresponding animal at the collection site. Seasonal influence on allergen concentrations was observed, while the overall exposure remained constant over the years. At locations with horses, elevated levels of mite allergens, endotoxin, and β-(1,3)-glucan can be expected, probably due to passive transfer from stable environment. [ABSTRACT FROM AUTHOR]

Objective: To identify the most effective fraction of Nanocnide lobata in the treatment of burn and scald injuries and determine its bioactive constituents. Methods: Chemical identification methods were used to analyze solutions extracted from Nanocnide lobata using petroleum ether, ethyl acetate, n-butanol using a variety of color reactions. The chemical constituents of the extracts were identified by ultra-performance liquid chromatography (UPLC)–mass spectrometry (MS). A total of 60 female mice were randomly divided into the following 6 groups: the petroleum ether extract-treated group; the ethyl acetate extract-treated group; the n-butanol extract-treated group; the model group; the control group; and the positive drug group. The burn/scald model was established using Stevenson's method. At 24 hours after modeling, 0.1 g of the corresponding ointment was evenly applied to the wound in each group. Mice in the model group did not undergo treatment, while those in the control group received 0.1 g of Vaseline. Wound characteristics, including color, secretions, hardness, and swelling, were observed and recorded. Photos were taken and the wound area calculated on the 1st, 5th, 8th, 12th, 15th, 18th and 21st days. Hematoxylin-eosin (HE) staining was utilized to observe the wound tissue of mice on the 7th, 14th, and 21st days. An enzyme-linked immunosorbent assay (ELISA) kit was used to measure the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-10, vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-β1. Results: The chemical constituents of Nanocnide lobata mainly include volatile oils, coumarins, and lactones. UPLC–MS analysis revealed 39 main compounds in the Nanocnide lobata extract. Among them, ferulic acid, kaempferitrin, caffeic acid, and salicylic acid have been confirmed to exhibit anti-inflammatory and antioxidant activity related to the treatment of burns and scalds. HE staining revealed a gradual decrease in the number of inflammatory cells and healing of the wounds with increasing time after Nanocnide lobata extract administration. Compared with the model group, the petroleum ether extract-treated group showed significant differences in the levels of TNF-α (161.67±4.93, 106.33±3.21, 77.67±4.04 pg/mL) and IL-10 (291.77±4.93, 185.09±9.54, 141.33±1.53 pg/mL) on the 7th, 14th, and 21st days; a significant difference in the content of TGF-β1 (75.68±3.06 pg/mL) on the 21st day; and a significant difference in the level of VEGF (266.67±4.73, 311.33±10.50 pg/mL) on the 7th and 14th days respectively. Conclusion: Petroleum ether Nanocnide lobata extract and the volatile oil compounds of Nanocnide lobata might be effective drugs in the treatment of burn and scald injuries, as they exhibited a protective effect on burns and scalds by reducing the expression of TNF-α, IL-10 and TGF-β1 and increasing the expression of VEGF. In addition, these compounds may also exert pharmacological effects that promote wound tissue repair, accelerate wound healing, and reduce scar tissue proliferation, inflammation and pain. [ABSTRACT FROM AUTHOR]

Background: Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase, marker and mediator of endothelial dysfunction. Several studies have demonstrated its value in cardiovascular risk stratification and all-cause mortality prediction. The aim was to determine the reference range of plasma ADMA in healthy adults. Methods and results: Taking into account the most widely used ADMA measurement methods, only studies using either high performance liquid chromatography (HPLC) -with fluorescence or mass spectrometric detection-, or enzyme-linked immunosorbent assay (ELISA) to quantify plasma ADMA concentrations were enrolled. 66 studies were included in the quantitative analysis (24 using ELISA and 42 using HPLC) reporting a total number of 5528 non-diabetic, non-hypertensive, non-obese adults without any medication (3178 men and 2350 women, 41.6 ± 16.9 years old). The reference range of ADMA (in μmol/l with 95% confidence interval in parenthesis) was 0.34 (0.29–0.38)– 1.10 (0.85–1.35) with a mean of 0.71 (0.57–0.85) (n = 4093) measured by HPLC and 0.25 (0.18–0.31)– 0.92 (0.76–1.09) with a mean of 0.57 (0.48–0.66) (n = 1435) by ELISA. Conclusions: Numerous publications suggested that asymmetric dimethylarginine is not only an outstanding tool of disease outcome prediction but also a new potential therapeutic target substance; the reference range provided by this meta-analysis can become of great importance and aid to further investigations. However, developing a standard measurement method would be beneficial to facilitate the clinical usage of ADMA. [ABSTRACT FROM AUTHOR]

Background: RTS,S/AS01 induced anti-circumsporozoite protein (CSP) IgG antibodies are associated with the vaccine efficacy. There is currently no international standardisation of the assays used in the measurement of anti-CSP IgG antibody concentrations for use in evaluations of the vaccine's immunogenicity and/or efficacy. Here, we compared the levels of RTS,S/AS01 induced anti-CSP IgG antibodies measured using three different enzyme-Linked ImmunoSorbent Assays (ELISA). Methods: 196 plasma samples were randomly selected from the 447 samples collected during the RTS,S/AS01 phase IIb trial in 2007 from Kenyan children aged between 5–17 months. The vaccine-induced anti-CSP IgG antibodies were then measured using two independently developed ELISA protocols ('Kilifi-RTS,S' and 'Oxford-R21') and compared to the results from the reference 'Ghent-RTS,S' protocol for the same participants. For each pair of protocols, a deming regression model was fitted. Linear equations were then derived to aid in conversions into equivalent ELISA units. The agreement was assessed using Bland and Altman method. Findings: The anti-CSP IgG antibodies measured from the three ELISA protocols were in agreement, and were positively and linearly correlated; 'Oxford' and 'Kilifi' r = 0.93 (95% CI 0.91–0.95), 'Oxford' and 'Ghent' r = 0.94 (95% CI: 0.92–0.96), and 'Kilifi' and 'Ghent' r = 0.97 (95% CI: 0.96–0.98), p<0.0001 for all correlations. Conclusions: With the linearity, agreement and correlations established between the assays, conversion equations can be applied to convert results into equivalent units, enabling comparisons of immunogenicities across different vaccines of the same CSP antigens. This study highlights the need for the international harmonisation of anti-CSP antibody measurements. [ABSTRACT FROM AUTHOR]

In this study, we designed novel truncated Babesia caballi (B. caballi) recombinant proteins from the previously used B. caballi proteins; 134-Kilodalton Protein (rBC134) and Merozoite Rhoptry 48 Protein (rBC48). Then, we evaluated the diagnostic performance of the newly designed proteins when used as a single antigen or when used as cocktail antigen consists of rBC134 full length (rBC134f) + newly designed rBC48 (rBC48t) or newly designed rBC134 (rBC134t) + rBC48t for the detection of B. caballi infection in horse using indirect enzyme-linked immunosorbent assay (iELISA). We used one dose and a half of each antigen in the cocktail formulas. The serum samples were collected from different endemic areas in addition to the sera collected from horses experimentally infected with B. caballi were used in the present study. Cocktail antigen in full dose of (rBC134f + rBC48t) exhibited the highest optical density (OD) values with B. caballi–infected sera and showed the lowest OD values with normal equine sera or B. caballi, and Theileria equi mixed infected sera in comparison with the single antigen. Interestingly, the same cocktail antigen exhibited the highest concordance rate (76.74%) and kappa value (0.79) in the screening of 200 field serum samples collected from five B. caballi endemic countries, including South Africa (n = 40), Ghana (n = 40), Mongolia (n = 40), Thailand (n = 40), and China (n = 40) using iELISA and the results were compared to those of indirect fluorescent antibody test (IFAT) as a reference. Moreover, the identified promising cocktail full dose antigen (rBC134f + rBC48t) showed that it can detect the infection as early as the 4th day post-infection in sera collected from experimentally infected horses. The obtained results revealed the reliability of the rBC134f + rBC48t cocktail antigen when used in full dose for the detection of specific antibodies to B. caballi in horses which will be useful for epidemiological surveys and control of equine babesiosis. [ABSTRACT FROM AUTHOR]

Detection of anti-Strongyloides IgG in urine by enzyme-linked immunosorbent assay (ELISA) for diagnosis of strongyloidiasis reportedly has comparable performance to conventional serum assays. Initial comparisons of urine assays using commercial ELISA kits designated for serology have shown its diagnostic potential but sub-optimal accuracy. In the present study, we optimized urine ELISA protocols based on different antigen types and evaluated their accuracies in determining the epidemiology of strongyloidiasis in Northeast Thailand. Paired urine and fecal samples of 966 individuals from the study community were collected for three consecutive days and tested for strongyloidiasis. We compared three ELISA protocols using different antigens including crude S. stercoralis antigen (Ss-ELISA), crude S. ratti antigen (Sr-ELISA) and recombinant NIE antigen (NIE-ELISA) and fecal examination by agar plate-culture (APCT) technique and formalin-ethyl acetate concentration technique (FECT). The optimized ELISA protocols using three different antigen sources yielded significantly higher prevalence rates of strongyloidiasis (58.9–65.1%) than those by fecal examination methods (19.7%). The prevalence of strongyloidiasis determined by ELISA protocols significantly increased with age (p value < 0.0001) and males had higher prevalence than females (p value < 0.0001). Diagnostic agreements between ELISA protocols were moderate (κ = 0.461–0.586) and the agreement between each ELISA protocol and fecal examinations were slight (κ = 0.139–0.210). The results obtained by urine ELISA protocols using three different antigens showed comparable diagnostic performances, provided further supports for the utility of urine as an alternative clinical specimen for diagnosis of strongyloidiasis. [ABSTRACT FROM AUTHOR]

Background: Obesity is a traditional risk factor for type 2 diabetes mellitus (T2DM). However, recent studies reported that metabolically unhealthy obesity (MUO) exerts a higher risk of developing T2DM than metabolically healthy obesity (MHO) because of its higher state of insulin resistance. This may happen due to metabolic endotoxemia through gut dysbiosis and increased intestinal permeability. Our study aimed to know the association of intestinal permeability using intestinal fatty acid-binding protein (I-FABP) with obesity-related T2DM patients in Indonesia. Methods: This was a cross-sectional study that recruited 63 participants with obesity defined using body mass index (BMI) classification for the Asia-Pacific population (BMI ≥25 kg/m2). All participants were then grouped into T2DM and non-T2DM based on American Diabetes Association (ADA) diagnostic criteria. The I-FABP levels were measured using the enzyme-linked immunosorbent assay method. Results: The I-FABP level of T2DM group was higher compared to non-T2DM group, namely 2.82 (1.23) ng/mL vs. 1.78 (0.81) ng/mL (p<0.001; mean difference 1.033 with 95% CI 0.51–1.55). This difference was not attenuated even after adjustment for age. The fitted regression model using linear regression was: i-FABP = 1.787+1.034*(DM) (R2 = 18.20%, standardized ß = 0.442, p<0.001). Conclusions: This study underscores the association of intestinal permeability with T2DM in people with obesity and supports the evidence of the potential role of intestinal permeability in the pathogenesis of obesity-related T2DM. [ABSTRACT FROM AUTHOR]