ELISA PCR Research Articles

Ultrasensitive and Rapid Visual Detection of Escherichia coli O157:H7 Based on RAA-CRISPR/Cas12a System.

Escherichia coli (E. coli) O157:H7 is a major foodborne and waterborne pathogen that can threaten human health. Due to its high toxicity at low concentrations, it is crucial to establish a time-saving and highly sensitive in situ detection method. Herein, we developed a rapid, ultrasensitive, and visualized method for detecting E. coli O157:H7 based on a combination of Recombinase-Aided Amplification (RAA) and CRISPR/Cas12a technology. The CRISPR/Cas12a-based system was pre-amplified using the RAA method, which showed high sensitivity and enabled detecting as low as ~1 CFU/mL (fluorescence method) and 1 × 102 CFU/mL (lateral flow assay) of E. coli O157:H7, which was much lower than the detection limit of the traditional real-time PCR technology (103 CFU/mL) and ELISA (104~107 CFU/mL). In addition, we demonstrated that this method still has good applicability in practical samples by simulating the detection in real milk and drinking water samples. Importantly, our RAA-CRISPR/Cas12a detection system could complete the overall process (including extraction, amplification, and detection) within 55 min under optimized conditions, which is faster than most other reported sensors, which take several hours to several days. The signal readout could also be visualized by fluorescence generated with a handheld UV lamp or a naked-eye-detected lateral flow assay depending on the DNA reporters used. Because of the advantages of being fast, having high sensitivity, and not requiring sophisticated equipment, this method has a promising application prospect for in situ detection of trace amounts of pathogens.

Prophylactic vs. Therapeutic Effect of Probiotics on the Inflammation Mediated by the NF-κB Pathway in Inflammatory Bowel Conditions.

Probiotic supplements consumed adequately at the proper time can affect health by modulating inflammatory pathways in gastrointestinal epithelial cells and modifying the resultant inflammatory response. The current study applied in vitro models to investigate the effectiveness of probiotics in modulating inflammatory pathways and altering inflammatory gene expression in gastrointestinal epithelial cells, with the ultimate goal of promoting probiotic consumption as a therapeutic and preventive measure for chronic inflammatory bowel conditions. HT-29 cells were treated with Gram-negative bacteria to evaluate the changes in pathways related to inflammation activities before and after treatment with a Lactobacillus spp. cocktail (L. plantarum, L. rhamnosus, L. brevis, and L. ruteri) and a Bifidobacterium spp. cocktail (B. bifidum, B. langum, and B. breve) using the real-time PCR method and ELISA for IL-1β and IL-6 as pro-inflammatory cytokines. The results showed that the expression of NF-κB signaling pathway genes and IL-1β and IL-6 cytokines increased after exposure to Gram-negative components. In contrast, all probiotic combinations significantly decreased the expression of genes and the secretion of cytokines. However, this decrease was significantly smaller in cells that underwent probiotic treatment after inflammation induction. In addition, cocktails containing combined Lactobacillus and Bifidobacterium demonstrated robust anti-inflammatory activity relative to solo cocktails. Our observations confirm that probiotic consumption could positively impact inflammatory conditions and alleviate inflammatory symptoms; they can be particularly effective as a preventive measure. Our study provides preliminary evidence to support the lifetime consumption of probiotics.

Synergistic effect of endurance training and nettle leaf extract on the IDO1-KYN-AHR pathway homeostasis and inhibiting of liver toxicity in rats with STZ-induced diabetes.

Diabetes adversely affects a number of hepatic molecular pathways, including the kynurenine (KYN) pathway. KYN is produced by indoleamine 2,3-dioxygenase (IDO) and activates the aryl hydrocarbon receptor (AHR). This study evaluated the effect of endurance training (EndTr) and nettle leaf extract (NLE) on the IDO1-KYN-AHR pathway in the livers of rats with streptozotocin-induced diabetes. We divided 48 rats into six groups: controls (Ct), treated with EndTr (EndTr), diabetes-induced (D), D treated with NLE (D + NLE), D treated with EndTr (D + EnTr), and D treated with EndTr and NLE (D + EndTr + NLE). EndTr, D + EnTr, and D + EndTr + NLE groups were subjected to training with running on treadmill for 8 weeks, 5 days per week, 25min in first session to 59min at last session with intensity of 55% to 65% VO2max. Using real-time PCR gene (Ahr, Cyp1a1, and Ido1) expressions and ELISA, malondialdehyde (MDA) and protein (IDO1, AHR, and CYP1A1) levels were determined in the liver samples. A significant three-way interaction of exercise, nettle, and diabetes was observed on the all variables (P< 0.001). In particular, significant increases in blood glucose level (BGL), in gene and protein expression, and in MDA and KYN levels were observed in the liver samples of the D group versus the Ct group (P< 0.05). BGL and liver MDA levels were significantly lower in the D + EndTr and D + NLE groups than that in the D group. However, the D + EndTr + NLE group showed a more significant decrease in these factors (P< 0.05). In addition, liver KYN levels were significantly lower in the EndTr group compared with that in the Ct group as well as in the D + EndTr + NLE and D + EndTr groups compared with that in the D groups (P< 0.05). Whereas both the EndTr and D + NLE groups showed lower Ahr expression and AHR level compared with the Ct and D groups, respectively (P< 0.05), the D + EndTr + NLE group showed a higher significant reduction in the AHR level than the D group (P< 0.05). The Cyp1a1 expression and IDO1 level significantly decreased only in the D + EndTr + NLE group compared to that in the D group (P< 0.05). Overall, this study showed that the combination of EndTr and NLE may synergistically restore the imbalanced IDO1-KYN-AHR pathway in diabetic liver.

Insights into Q-markers and molecular mechanism of Sanguisorba saponins in treating ulcerative colitis based on lipid metabolism regulation

BackgroundSanguisorba saponin extract (SSE) is the main active part of Sanguisorba officinalis with various pharmacological activities such as anti-inflammatory, anti-bacterial and anti-oxidant. However, its therapeutic role and underlying mechanisms for ulcerative colitis (UC) still need to be elucidated. PurposeThis study aims to explore the therapeutic effect, effectiveness-material basis-quality markers (Q-markers) and prospective mechanism of function of SSE on UC. MethodsFresh 2.5% dextran sulfate sodium salt (DSS) solution was placed in drinking bottles for 7 days to induce a mouse model of UC. SSE and sulfasalazine (SASP) were supplemented to mice by gavage for consecutive 7 days to investigate the therapeutic role of SSE on UC. Mouse monocyte macrophages (RAW264.7) and human normal colonic epithelial (NCM460) cells were treated with LPS to induce inflammatory responses, followed by pharmacodynamic examination with different concentrations of SSE. Hematoxylin-eosin (HE) and Alcian blue staining were conducted to evaluate the pathological damage of mice colon. Lipidomic technology was conducted to explore the differential lipids closely related to the disease process of UC. Quantitative PCR analysis, immunohistochemistry and ELISA kit were used to measure the expression levels of the corresponding proteins and pro-inflammatory factors. ResultsSSE treatment could effectively reduce the elevated expressions of pro-inflammatory factors in RAW264.7 and NCM460 cells due to LPS stimulation. Intragastric administration of SSE was found to significantly alleviate the symptoms of DSS-induced colon injury and low-polar saponins in SSE. Low polarity saponins, especially ZYS-II, were proved to be the main active substances of SSE in treating UC. In addition, SSE could significantly ameliorate the aberrant lipid metabolism in UC mice. The role of phosphatidylcholine (PC)34:1 in the UC pathogenesis has been fully verified in our previous studies. Herein, SSE-dosing effectively reversed the metabolic disorder of PCs in UC mice, and increased the PC34:1 level to normal via up-regulating the expression of phosphocholine cytidylyltransferase (PCYT1α). ConclusionOur data innovatively revealed that SSE could significantly alleviate the symptoms of UC by reversing the disorder of PC metabolism induced by DSS modeling. SSE was proved for the first time to be a promising and effective candidate for UC treatment.

The effects of Hericium erinaceus extracts on cell viability and telomerase activity in MCF-7 cells

Abstract Objectives This study aims to determine the effects of Hericium erinaceus extracts on cell viability and the effects of H. erinaceus water extract on the telomerase activity of MCF-7 cells. H. erinaceus is an edible mushroom widely used in traditional Chinese medicine. Although its various therapeutic properties, the literature has not yet submitted evidence about H. erinaceus for its effects on the telomerase activity of MCF-7 cells. Methods MCF-7 cells were treated with ethanol, ethanol-water, ether, ethyl acetate, methanol-water, and water extracts to determine the effects on cell viability using the WST8 method. The TeloTAGGG Telomerase PCR ELISA kit was used to assess telomerase activity. Results The water extract was determined to be the most efficient extract to decrease cell viability. The water extract’s half-maximal inhibitory concentration was 250 μg/mL at 72 h. It is found that H. erinaceus has no statistically significant effect compared to positive control on reducing telomerase activity. We found a statistically significant difference in telomerase activity % between H. erinaceus water extracts and negative control (p&lt;0.05). Conclusions Consequently, these differences in telomerase activity are a significant association rather than inferring action. It is considered that water extract shows its cell viability inhibition effects through different mechanisms.

Nickel oxide nanoparticles exposure as a risk factor for male infertility: "In vitro" effects on porcine pre-pubertal Sertoli cells.

Lately, nickel oxide nanoparticles (NiO NPs) have been employed in different industrial and biomedical fields. Several studies have reported that NiO NPs may affect the development of reproductive organs inducing oxidative stress and, resulting in male infertility. We investigated the in vitro effects of NiO NPs on porcine pre-pubertal Sertoli cells (SCs) which undergone acute (24 h) and chronic (from 1 up to 3 weeks) exposure at two subtoxic doses of NiO NPs of 1 μg/ml and 5 μg/ml. After NiO NPs exposure we performed the following analysis: (a) SCs morphological analysis (Light Microscopy); (b) ROS production and oxidative DNA damage, gene expression of antioxidant enzymes (c) SCs functionality (AMH, inhibin B Real-time PCR analysis and ELISA test); (d) apoptosis (WB analysis); (e) pro-inflammatory cytokines (Real-time PCR analysis), and (f) MAPK kinase signaling pathway (WB analysis). We found that the SCs exposed to both subtoxic doses of NiO NPs didn't sustain substantial morphological changes. NiO NPs exposure, at each concentration, reported a marked increase of intracellular ROS at the third week of treatment and DNA damage at all exposure times. We demonstrated, un up-regulation of SOD and HO-1 gene expression, at both concentrations tested. The both subtoxic doses of NiO NPs detected a down-regulation of AMH and inhibin B gene expression and secreted proteins. Only the 5 μg/ml dose induced the activation of caspase-3 at the third week. At the two subtoxic doses of NiO NPs a clear pro-inflammatory response was resulted in an up-regulation of TNF-α and IL-6 in terms of mRNA. Finally, an increased phosphorylation ratio of p-ERK1/2, p-38 and p-AKT was observed up to the third week, at both concentrations. Our results show the negative impact of subtoxic doses NiO NPs chronic exposure on porcine SCs functionality and viability.

Diagnosis of Toxoplasmosis Using Surface Antigen Grade 1 Detection by ELISA, Nano-Gold ELISA, and PCR in Pregnant Women.

The accurate diagnosis of toxoplasmosis has critical importance in pregnant women. Nanotechnology and molecular biology are making possible opportunities for accurate and rapid diagnosis of many infectious diseases. The aim of our study was to compare nano-gold ELISA with ELISA and PCR for diagnosis of toxoplasmosis using Toxoplasma surface antigen grade 1 (SAG1) in pregnant women seeking antenatal care in outpatient clinics. PCR showed the highest diagnostic values than nano-gold ELISA and ELISA regarding sensitivity (97.3% versus 89.2% and 83.8%); specificity (100% versus 94% and 88%); and diagnostic accuracy (98.9% versus 91.95% and 86.2%), respectively. There is no statistical difference between PCR and nanogold ELISA results. Nano-gold ELISA had a significant improvement in diagnosis than the traditional ELISA method. Most likely with the assistance of nanoparticles, more antibodies enter the antigen-antibody complex because of the considerable improvement in the surface area of nano-gold particles. Although PCR had higher diagnostic values than nano ELISA, nano ELISA is cheaper and easier than PCR. We recommend nano-gold ELISA with SAG1 as a promising technique in the diagnosis of toxoplasmosis and survey studies.

Modeling the accuracy of a novel PCR and antibody ELISA for African swine fever virus detection using Bayesian latent class analysis.

Diagnostic test evaluation for African swine fever (ASF) in field settings like Vietnam is critical to understanding test application in intended populations for surveillance and control strategies. Bayesian latent class analysis (BLCA) uses the results of multiple imperfect tests applied to an individual of unknown disease status to estimate the diagnostic sensitivity and specificity of each test, forgoing the need for a reference test. Here, we estimated and compared the diagnostic sensitivity and specificity of a novel indirect ELISA (iELISA) for ASF virus p30 antibody (Innoceleris LLC.) and the VetAlert™ ASF virus DNA Test Kit (qPCR, Tetracore Inc.) in field samples from Vietnam by assuming that disease status 1) is known and 2) is unknown using a BLCA model. In this cross-sectional study, 398 paired, individual swine serum/oral fluid (OF) samples were collected from 30 acutely ASF-affected farms, 37 chronically ASF-affected farms, and 20 ASF-unaffected farms in Vietnam. Samples were tested using both diagnostic assays. Diagnostic sensitivity was calculated assuming samples from ASF-affected farms were true positives and diagnostic sensitivity by assuming samples from unaffected farms were true negatives. ROC curves were plotted and AUC calculated for each test/sample combination. For comparison, a conditionally dependent, four test/sample combination, three population BLCA model was fit. When considering all assumed ASF-affected samples, qPCR sensitivity was higher for serum (65.2%, 95% Confidence Interval [CI] 58.1-71.8) and OF (52%, 95%CI 44.8-59.2) compared to the iELISA (serum: 42.9%, 95%CI 35.9-50.1; OF: 33.3%, 95%CI 26.8-40.4). qPCR-serum had the highest AUC (0.895, 95%CI 0.863-0.928). BLCA estimates were nearly identical to those obtained when assuming disease status and were robust to changes in priors. qPCR sensitivity was considerably higher than ELISA in the acutely-affected population, while ELISA sensitivity was higher in the chronically-affected population. Specificity was nearly perfect for all test/sample types. The effect of disease chronicity on sensitivity and specificity could not be well characterized here due to limited data, but future studies should aim to elucidate these trends to understand the best use of virus and antibody detection methods for ASF. Results presented here will help the design of surveillance and control strategies in Vietnam and other countries affected by ASF.

Amplification-free rapid detection of viral genomes at single molecule level.

Single-molecule analysis of bio-molecules offers key benefits over macroscopic assay methods, as they unlock the ability to quantify various biological phenomena. In particular, micron/nano technologies enable highly sensitive and quantitative bioassays at the single-molecule level, which are widely used for ultra-sensitive biomedical applications, e.g., digital PCR and digital ELISA. To expand the versatility, we recently developed a novel microsystem “SATORI” with emerging CRISPR-Cas technologies that allows an accurate and rapid detection of nucleic-acid at single-molecule level. SATORI with CRISPR-Cas13a detected single-stranded RNA targets with maximal sensitivity of ∼1 aM in <9 min, thereby enabling the rapid detection of viral RNA of SARS-CoV-2. In addition, SATORI with CRISPR-Cas12a enabled single-molecule detection of double-stranded DNA, allowing the rapid detection of viral DNA of monkeypox virus. Collectively, we expect that SATORI will be one of the key technologies for future diagnostics of various infectious diseases.

Molecular-based study of scrub typhus in Kerala, South India from 2014 to 2021: a laboratory-based study.

The online version contains supplementary material available at 10.1007/s00580-023-03443-8.

Investigation of the Epidemiology of Small Ruminant Lentivirus Infections southeast part of the Marmara region of Turkey

Small ruminant lentiviruses (SRLVs) are viral pathogens that are common in goats and sheep, affect production, and cause significant economic losses in small ruminant breeding. Caprine arthritis encephalitis virus (CAEV) and Maedi-Visna virus (MVV) are prototypes of SRLVs. Both of them affect animal health and welfare in sheep and goats and cause progressive and persistent infections in the small ruminant industry. The present study aimed to reveal the epidemiological status of lentivirus infection in the sheep region in Yalova province located in the southeast part of the Marmara region and determine the circulating genotypes by conducting the sequence analysis of the samples detected positive by a molecular method and molecular characterization of the detected field strains. To that end, 231 sheep blood samples were used between May 2016 and April 2018. Based on sampling results of the PCR test and ELISA tests, 5.62% (13/231) and 5.19% (12/231) positivity rates were found in sheep, respectively. According to the ELISA test results, a significant difference was found in terms of age groups (6 months -1 age, 1-3 age, &gt;3 age) (χ2: 6.01; p=0.04). Furthermore, the sequence analysis of the gag gene region detected the existence of the A genotype of small ruminant lentiviruses in sheep. The data obtained from the study revealed a low seroprevalence course of SRLV infection in the study area in the absence of a systematic disease control program.

The Correlation between Increased Expressions of NLRP3 Inflammasome Components in Peripheral Blood Mono-Nuclear Cells and Plaque Vulnerability in Human Carotid Atherosclerosis.

We aimed to determine whether NLRP3 inflammasomes in peripheral blood mononuclear cells (PBMC) were associated with carotid plaque instability in carotid atherosclerosis patients. Consecutive 38 carotid atherosclerosis with vulnerable plaques, 22 carotid atherosclerosis with stable plaques, and 40 healthy subjects with no carotid or coronary artery stenosis were enrolled. They were referred to the Second Hospital of Dalian Medical University from 2013-2019. Carotid plaques were evaluated by modified plaque vulnerability risk score (MPVRS) and pathological assessment. The mRNA and protein expression of NLRP3 inflammasome components in PBMC were determined by quantitative real time PCR and Western blot analysis or ELISA. When consecutive study subjects undergoing carotid endarterectomy were divided into stable (≤4) and unstable (>4) plaque groups according to the MPVRS, the unstable plaque group had significantly raised mRNA and protein expression of NLRP3 inflammasome components in PBMC as compared with the stable plaque group and healthy subject group. Furthermore, subjects with higher NLRP3 protein expression in PBMC had greater incidence of cerebrovascular events. Increased NLRP3 inflammasome components in PBMC is associated with instability of human carotid atherosclerotic plaques, suggesting NLRP3 inflammasome as a potential biomarker for monitoring carotid plaque instability.

Rhamnetin decreases the expression of HMG-CoA reductase gene and increases LDL receptor in HepG2 cells

Context: Rhamnetin is a naturally occurring methylated derivative of quercetin. This flavonoid is abundant in Syzygium aromaticum, Coriandrum sativum Prunus cerasus, and Rhamnus spp. Aims: To evaluate the effects of rhamnetin on HMG-CoA reductase and low-density lipoprotein receptor (LDLR) gene and protein expressions in the HepG2 hepatoma cell line. Methods: The expression of HMG-CoA reductase and LDLR genes and proteins were studied in HepG2 liver cancer cell line by PCR, Western blot, and indirect ELISA, as well as their antioxidant activity. Results: Rhamnetin was non-toxic up to 200 μM on HepG2 at 24, 48, and 72 h. Rhamnetin (25 µM) upregulated LDLR gene expression by 1.66 folds compared to 3.12 folds exerted by the well-known hypocholesterolemic drug simvastatin. Rhamnetin (100 µM) increased the expression of LDLR protein at the cell membrane, while the other concentrations produced no significant change from the control (vehicle-treated). In HepG2 cell lysate, LDLR was increased by 50 µM of rhamnetin. Also, rhamnetin increased SOD activity significantly by 100.98, 86.28, and 100.98% by the concentrations 25, 50, and 100 µM, respectively. Using the same concentrations, rhamnetin reduced H2O2 levels by 50, 67, and 76.34%, respectively. Conclusions: This study demonstrated for the first time that rhamnetin reduced HMG-CoA reductase gene expression and increased LDLR in HepG2 cells.

The Presence of Antineutrophil Cytoplasmic Antibodies and Antiphospholipid Antibodies in Patients with Severe Acute Respiratory Syndrome Coronavirus 2: A Case-Control Study among Sudanese Patients.

Patients infected with COVID-19 are at an increased risk for thrombosis, suggesting a possible role of COVID-19 in the induction of coagulopathy. This study aimed to investigate the presence of prothrombotic antineutrophil cytoplasmic antibodies (ANCA) and antiphospholipid antibodies (aPLs) in the course of COVID-19 infection and to correlate these markers with severity and fatality, suggesting that COVID-19-induced autoimmune thrombosis is a possible axis in the inflammatory circuit of this infection. To investigate this, we conducted a case-control study which included patients with a positive reverse transcription-polymerase chain reaction (RT-PCR) test of COVID-19 and a control group with negative COVID-19 PCR and antibody (IgG-IgM and IgA nucleoprotein) ELISA results. An indirect immunofluorescence assay using granulocyte biochips (Aesku slides from AESKU DIAGNOSTICS, Germany) was used to detect ANCA (IgG), as well as multiplex ELISA for the detection of antiphospholipid antibodies for all patients with COVID-19 and for the control group. The results revealed the detection of antiphospholipid antibodies (IgG) in one patient out of the 45 patients in the case group. 1/45(2.2%) and 7/45(15.6%) tested positive for ANCA. Five were men and two were females, with one case revealed to be positive for both aPL and ANCA. A cytoplasmic reaction on the eosinophil granulocytes was observed in 2 cases; both were positive for ANCA. Other markers (CRP, APTT, PT, INR, ESR, and neutrophil and lymphocyte counts) were included in the study, along with demographic data. No aPL or ANCA reactions were detected for any of the control groups. These findings suggest that aPL and ANCA may be induced during the course of inflammation in COVID-19 and possibly contribute to the disease's severity and mortality.

Leptin-producing monocytes in the airway submucosa may contribute to asthma pathogenesis

BackgroundObesity leads to an increase in the incidence and severity of asthma. Adipokines, such as leptin, secreted by adipocytes induce systemic inflammation, causing airway inflammation. We previously reported that leptin activates both inflammatory and structural cells, including lung fibroblasts. However, little is known about the differential leptin expression and responsiveness to leptin in asthmatic individuals and healthy controls (HC). In this study, we investigated the expression and origin of leptin in asthmatic airways. We also compared the effect of leptin on asthmatic and HC fibroblasts. MethodsLung specimens from asthmatic and non-asthmatic patients were analyzed by immunohistochemical staining using anti-leptin and anti-CD163 antibodies. Leptin mRNA and protein levels in human monocytes were detected by real-time PCR and western blotting and ELISA, respectively. We used flow cytometry to analyze asthmatic and HC lung fibroblasts for leptin receptor (Ob-R) expression. Further, we determined cytokine levels using cytometric bead array and ELISA and intracellular phosphorylation of specific signaling molecules using western blotting. ResultsAsthma specimens displayed accumulation of leptin-positive inflammatory cells, which were also positive for CD163, a high-affinity scavenger receptor expressed by monocytes and macrophages. Leptin expression was observed at both transcript and protein levels in human blood-derived monocytes. No significant differences were observed between asthmatic and HC lung fibroblasts in Ob-R expression, cytokine production, and intracellular phosphorylation of p38 mitogen-activated protein kinase. ConclusionsOur findings reveal similar responsiveness of control and asthmatic fibroblasts to leptin. However, the accumulation of inflammatory leptin-producing monocytes in the airway may contribute to the pathogenesis of asthma.

Circulating Neonatal NAV1.5 (nNAV1.5) Antigen and Anti-nNAV1.5 Antibodies as Potential Biomarkers for Breast Cancer Metastasis

Neonatal Nav1.5 (nNav1.5) has been known to potentiate breast cancer (BCa) metastasis. The detection of anti-nNav1.5 antibodies (anti-nNav1.5-Ab) reflects the immunogenicity of nNav1.5. However, the presences of circulating nNav1.5 antigen and anti-nNav1.5-Ab in the context of BCa metastasis have not been explored yet. Therefore, the study has attempted to conduct such an investigation using both blood samples from 4T1 orthotopic mice and BCa patients. In the preclinical study, forty female BALB/c mice were divided into three groups: 4T1 orthotopic BCa mice (n=17), control mice (n=20) and positive control mice (n=3). After tumour development, the mice were sacrificed to obtain target organs, whole blood, and serum. Histopathology, cytokine analyses, real-time PCR, and indirect ELISA were performed. Histopathology and cytokine analyses showed the establishment of metastasis in 4T1 orthotopic mice. The concentration of vascular endothelial growth factor (VEGF) was significantly higher in the 4T1 orthotopic mice (P&lt;0.0001****). Circulating nNav1.5 antigen and anti-nNav1.5-Ab were detected in 4T1 orthotopic mice, using real-time PCR and indirect ELISA, respectively. Furthermore, there was an inverse relationship between anti-nNav1.5-Ab and the total metastatic foci (P=0.0485*, r=-0.7306). In the clinical study, 32 BCa patients were grouped based on their stages: early-invasive (n=15) and advanced (n=17) stages. Approximately 3 mL of blood was withdrawn, and only indirect ELISA was conducted. The clinical study showed that BCa patients of advanced-stages portrayed higher expression of anti-nNav1.5-Ab compared to early stages of BCa (P =0.0110*). In conclusion, the detection of nNav1.5 antigen and anti-nNav1.5-Ab was consistent with the presence of BCa metastasis.

Evaluation of an in-house PCR for diagnosing scrub typhus along with a preliminary study of genotypic characterization of Orientia tsutshugamushi circulating in Chhattisgarh: A prospective clinico-microbiological study

PurposeScrub typhus, caused by Orientia tsutsugamushi (O. tsutsugamushi) present nonspecific clinical features during manifestation of acute undifferentiated febrile illness (AUFI) to render its early diagnosis difficult. Accordingly, this study was undertaken to assess an in-house groEL PCR versus IgM ELISA for the diagnosis of scrub typhus and to genotypically characterise the randomly selected scrub typhus positive cases. MethodsBlood samples, collected from two hundred twenty one (221) AUFI cases were subjected to groEL PCR and IgM ELISA for diagnosis of scrub typhus. Eleven randomly selected PCR positive cases were processed for DNA sequencing to determine the genetic diversity of O. tsutsugamushi in Chhattisgarh. ResultsScrub typhus prevalence of 35.2% were detected among AUFI cases using both in-house groEL PCR and IgM ELISA. PCR alone showed sensitivity, specificity, positive and negative predictive values of 66.6% (CI: 55.08–76.94), 100% (CI: 90 to 100),100% (CI: 93.15 to 100) and 57.37% (CI: 44.05 to 69.96) while for IgM ELISA, these parameters were 62.8% (CI: 51.13–73.50), 100% (CI: 90 to 100), 100% (CI: 92.75 to 100) and 54.68% (CI: 41.75 to 67.18) respectively. PCR and ELISA could detect scrub typhus in 37.2% and 33.3% cases, when tested alone. groEL PCR detected the O. tsutsugamushi throughout the course of infection. Phylogenetic analysis depicted 5 of 11 positive cases belonged to Kuroki, Japan strain of O. tsutsugamushi, followed by Gilliam and Karp strain in 4 and 2 cases respectively. ConclusionScrub typhus should be considered in differential diagnosis of AUFI. groEL PCR may aid on to IgM ELISA test for optimum laboratory diagnosis of scrub typhus by its implementation especially in seronegative cases. Predominance of Kuroki-like strain followed by Gillian and Karp strains of O. tsutsugamushi in Chhattisgarh confirm variable geographical distribution of O. tsutsugamushi and provide the baseline epidemiological data which will eventually be used to help the researchers for developing better diagnostic tests and vaccine covering the predominant genotypes.

Bayesian estimation of sensitivity and specificity of fecal culture, fecal PCR and serum ELISA for diagnosis of Mycobacterium avium subsp. paratuberculosis infections in sheep

The objective of the present study was to evaluate the diagnostic accuracy of the individual fecal culture (IFC), fecal PCR (FPCR), and serum ELISA for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) infections in sheep from four governorates in Egypt, using a latent class model (LCM) fitted within a Bayesian framework. Furthermore, the within-governorate prevalence of MAP infection in sheep was estimated as a secondary objective. Fecal and blood samples were collected from 370 sheep in four Egyptian governorates. Fecal samples were analyzed by IFC and RT-PCR based on ISMav2 gene, while ELISA was performed on serum samples. The sensitivity (Se) and specificity (Sp) of the three diagnostic tests were estimated using a three-tests-four-populations Bayesian LCM to obtain posterior estimates [medians and 95% Bayesian credible intervals (95% BCI)] for each parameter. The median Se estimates (95% BCI) for IFC, FPCR, and serum ELISA were 31.8% (22.8–41.4), 49.7% (31.8–79.9), and 61.2% (39.8–81.4), respectively. The median Sp estimates (95% BCI) for IFC, FPCR, and serum ELISA were 97.7% (96.1–98.9), 97.7% (95.6–99.5), and 98.4% (96.9–99.3), respectively. The median within-governorate paratuberculosis prevalence (95% BCI) was 5.2% (1.1–13.6), 8.4% (2.9–17.7), 9.4% (3.0–20.7), and 18.2% (10.5–29.5) for the Gharbia, Menoufia, Qalyubia, and Kafr El-Sheikh governorates, respectively. In conclusion, at a ratio of the optical density (OD) sample/OD positive control threshold of > 45%, ELISA showed the highest Se among the three tests and comparable Sp to IFC and FPCR. The test ELISA evaluated in this study is an interesting alternative for detecting MAP in sheep due to its higher Se, lower cost, and shorter turnaround laboratory time compared to IFC and FPCR.

Midazolam impacts acetyl-And butyrylcholinesterase genes: An epigenetic explanation for postoperative delirium?

Midazolam is a widely used short-acting benzodiazepine. However, midazolam is also criticized for its deliriogenic potential. Since delirium is associated with a malfunction of the neurotransmitter acetylcholine, midazolam appears to interfere with its proper metabolism, which can be triggered by epigenetic modifications. Consequently, we tested the hypothesis that midazolam indeed changes the expression and activity of cholinergic genes by acetylcholinesterase assay and qPCR. Furthermore, we investigated the occurrence of changes in the epigenetic landscape by methylation specific PCR, ChiP-Assay and histone ELISA. In an in-vitro model containing SH-SY5Y neuroblastoma cells, U343 glioblastoma cells, and human peripheral blood mononuclear cells, we found that midazolam altered the activity of acetylcholinesterase /buturylcholinesterase (AChE / BChE). Interestingly, the increased expression of the buturylcholinesterase evoked by midazolam was accompanied by a reduced methylation of the BCHE gene and the di-methylation of histone 3 lysine 4 and came along with an increased expression of the lysine specific demethylase KDM1A. Last, inflammatory cytokines were not induced by midazolam. In conclusion, we found a promising mechanistic link between midazolam treatment and delirium, due to a significant disruption in cholinesterase homeostasis. In addition, midazolam seems to provoke profound changes in the epigenetic landscape. Therefore, our results can contribute to a better understanding of the hitherto poorly understood interactions and risk factors of midazolam on delirium.

Evaluation of Cytokine Levels in Human Leptospirosis as Prognostic Indicator

Purpose: Leptospirosis is a tropical zoonotic illness, in which the role of immune response in the pathogenesis is proven but poorly understood. Response of cytokines is said to play a key role in disease progression and pathogenesis. There are proven studies on pro-inflammatory cytokines like TNFα, IL-6 and anti-inflammatory like IL-10 in human leptospirosis however, the role of IL-2, IL-4, IL-15, GCSF and MCP-2 needs more comprehensive studies. Present study was conducted to evaluate the role of IL-2, IL-4, IL-15, GCSF and MCP-2 in human leptospirosis as prognostic indicator. Methods: Blood samples from patients meeting the inclusion criteria for leptospirosis were included in the study. PCR and IgM ELISA were carried out for diagnosis. Serum cytokine levels in Leptospira positive patients and in controls were estimated by ELISA. Statistical analysis was done using IBM SPSS Statistics 20 and Med Calc 16.1. software. Results: Out of 270, 45(16.7%) patients were confirmed as cases of leptospirosis. The mean level of the cytokines (IL-15, MCP-2, G-CSF) differed significantly between the patients and the control group (p &lt; 0.001). GCSF, MCP-2, IL-15 and IL-4 were elevated in most cases. IL-2 level was depressed in 34 out of 45 cases. The AUCs for IL-2, IL-15, MCP-2 and GCSF were 0.906 (95% CI 0.341 to 0.665), 0.929 (95% CI 0.837 to 0.978), 0.909 (95% CI 0.812-0.966) and 0.881 (95% CI 0.777 to 0.948) respectively. On spearman rank correlation, GCSF level showed correlation with MCP-2 (rho = 0.415, p &lt; 0.01). Conclusions: The study provided an understanding of cytokine patterns in leptospirosis, and concluded that IL-15, MCP-2 and GCSF can be used as an effective biomarker for leptospirosis and indicators of disease progressions.