Enzyme-linked Immunosorbent Assay Test Research Articles

DP21 Thymoma and the skin: manifestations of multiorgan autoimmunity

Abstract We present three patients who developed distinct cutaneous manifestations of thymoma: a rare mediastinal tumour. A 25-year-old man was treated for myasthenic crisis. Past medical history included thymectomy in 2016. Admission imaging confirmed recurrence of thymoma. He developed a red–brown macular rash affecting the trunk, back and thighs. Biopsy showed a florid lichenoid dermatitis consistent with graft-versus-host disease (GVHD). After resolution with steroids and mycophenolate mofetil, he subsequently developed a Stevens–Johnson syndrome/toxic epidermal necrolysis-like eruption with erythroderma and epidermal loss. Unfortunately, he developed multiorgan failure and died shortly afterward. A 43-year-old man presented to the emergency dermatology clinic with a 4-month history of a blistering rash affecting the chest and limbs. His previous medical history was significant for thymoma in 2008 and a recurrence in 2013. Skin biopsy demonstrated changes suggestive of bullous pemphigoid, and repeat computed tomography confirmed the recurrence of thymoma. Enzyme-linked immunosorbent assay testing was positive for bullous pemphigoid 180. He has responded well to prednisolone and doxycycline. A 51-year-old woman was reviewed in 2020 and 2021 with pruritic hyperpigmented patches affecting the face, trunk and limbs. Her past medical history included several relapses of thymoma with occurrence in 2002 and relapses in 2007, 2008, 2011, 2015 and 2021. Skin biopsy in June 2021 showed features of cutaneous GVHD. She received ruxolitinib and tapering steroids for thymoma-associated multiorgan autoimmunity (TAMA)-like GVHD and has had a remarkable improvement in her skin disease. Thymoma is described as a rare neoplasm, and up to 34% of thymomas are thought to be accompanied by symptoms of autoimmunity. The most frequently described syndromes include myasthenia gravis, pure red blood cell aplasia and hypogammaglobulinaemia. Other reported presentations include panencephalitis, enterocolitis and autoimmune hepatitis. Thymoma and cutaneous autoimmune presentations have been previously described, often in association with two rare syndromes: TAMA and paraneoplastic autoimmune multiorgan syndrome (PAMS). Both conditions demonstrate variability in clinical phenotype, driven by the activation of humoral and cellular immune responses. Clinically, patients may present with a spectrum of skin disease, including GVHD-like exanthems, lichen planus-like disease and pemphigus-like and pemphigoid-like lesions. Our case series demonstrates the heterogeneous cutaneous manifestations of thymoma-induced autoimmunity. We also describe two extremely rare phenomena accompanying thymomas: TAMA and PAMS. We also suggest that there is much crossover between these syndromes and previous definitions may require further review. This case series also highlights the importance of the wider multidisciplinary team in managing these patients and the complexities of their disease.

Comparison of Three Serologic Tests for the Detection of Anti-Coxiella burnetii Antibodies in Patients with Q Fever.

The performance of a commercial immunofluorescence assay (IFA commercial), an in-house immunofluorescence assay (IFA in-house) and an indirect enzyme-linked immunosorbent assay (ELISA) were evaluated in the detection of antibodies anti-C. burnetii in the serum of Q fever patients and persons without the disease. For the study, seropositive and seronegative samples for Q fever (n = 200) from a serum bank of the Instituto Adolfo Lutz in Brazil were used. Commercial IFA was considered in this study as the gold standard for diagnosing Q fever. The in-house IFA demonstrated good agreement with the commercial test, showing high sensitivity (91%) and specificity (97%) compared to the gold standard, with a Kappa coefficient of 0.8954. The indirect ELISA test showed lower agreement with the gold standard, showing low sensitivity (67%), although the specificity of the technique was high (97%) and the Kappa coefficient was moderate (0.6631). In-house IFA is an excellent alternative for diagnosing Q fever.

Recurrent cardiac hydatid cysts with pericardial protrusion; a rare case presentation of Echinococcus granulosus

Hydatid cyst (HC) is a rare pathology mostly found in less developed, livestock-raising countries. HC can be found anywhere in the body, however it is a rare finding in the heart, and its recurrence in the heart and pericardium is very rare. A 54-year-old woman who had undergone surgery for the removal of HC in another country 11 years before presented with shortness of breath and heaviness in the chest. The examinations and computerized tomography (CT) scan revealed a large mass measuring 60×50 mm in the pericardium cavity originating from the right atrial wall. Infection with Echinococcus granulosus was confirmed with serological enzyme-linked immunosorbent assay (ELISA) test and the patient underwent cardiac surgery. The patient underwent cardiopulmonary bypass, nevertheless did not suffer cardiac arrest, and aortic cross-clamping was not performed for her. The removed mass was filled with daughter cysts. The endocysts were removed and intramural cysts were drained. Following the surgery, the patient received treatment with albendazole for six months. Cardiac hydatid cyst recurrence is a rare but possible incident, and annual follow-ups and echocardiography are recommended.

Gut barrier protein levels in serial blood samples from critically ill trauma patients during and after intensive care unit stay.

In an effort to better manage critically ill patients hospitalised in the intensive care unit (ICU) after experiencing multiple traumas, the present study aimed to assess whether plasma levels of intestinal epithelial cell barrier proteins, including occludin, claudin-1, junctional adhesion molecule (JAM-1), tricellulin and zonulin, could be used as novel biomarkers. Additional potential markers such as intestinal fatty acid-binding protein (I-FABP), D-lactate, lipopolysaccharide (LPS) and citrulline were also evaluated. We also aimed to determine the possible relationships between the clinical, laboratory, and nutritional status of patients and the measured marker levels. Plasma samples from 29 patients (first, second, fifth and tenth days in the ICU and on days 7, 30 and 60 after hospital discharge) and 23 controls were subjected to commercial enzyme-linked immunosorbent assay (ELISA) testing. On first day (admission) and on the second day, plasma I-FABP, D-lactate, citrulline, occludin, claudin-1, tricellulin and zonulin levels were high in trauma patients and positively correlated with lactate, C-reactive protein (CRP), number of days of ICU hospitalisation, Acute Physiology and Chronic Health Evaluation II (APACHE II) score and daily Sequential Organ Failure Assessment (SOFA) scores (P < 0.05-P < 0.01). The results of the present study showed that occludin, claudin-1, tricellulin and zonulin proteins, as well as I-FABP, D-lactate and citrulline, may be used as promising biomarkers for the evaluation of disease severity in critically ill trauma patients, despite the complexity of the analysis of various barrier markers. However, our results should be supported by future studies.

An ELISA Test Able to Predict the Development of Oral Cancer: The Significance of the Interplay between Steroid Receptors and the EGF Receptor for Early Diagnosis.

Oral disorders including non-homogeneous leukoplakia, erythroplakia, erosive lichen planus, and many others can potentially progress to oral squamous cell carcinoma (OSCC). Currently, the late diagnosis of OSCC contributes to high mortality rates, emphasizing the need for specific markers and early intervention. In this study, we present a novel, quick, sensitive, and non-invasive method for the early detection and screening of oral cancer, enabling the qualitative assessment of neoplastic forms even before the onset of symptoms. Our method directly examines the expression of oral cancer biomarkers, such as the epithelial growth factor receptor (EGFR), and steroid receptors, including the androgen receptor (AR) and the estrogen receptor (ER). The crosstalk between sexual hormones and the EGF receptor plays a crucial role in the progression of different types of cancers, including head and neck squamous cell carcinoma. To implement our method, we developed a kit box comprising nine wells or stations, each containing buffers, lysis systems, and dried/lyophilized antibodies stored at room temperature. The kit includes instruments for sample collection and a PVDF strip (Immobilon) with specific primary antibodies immobilized on it. These antibodies capture the target proteins from cytological samples. Additionally, complementary tools are provided to ensure efficient utilization and optimal test performance. The technique can be performed outside the laboratory, either "patient side" with an instant chemocolorimetric response or with a digital reader utilizing the enzyme-linked immunosorbent assay (ELISA) method.

Clinical and Epidemiological Characteristics of 30 Fatal Cases of Crimean-Congo Hemorrhagic Fever in Kabul, Afghanistan: A Retrospective Observational Study.

Crimean-Congo hemorrhagic fever (CCHF) is a zoonotic disease associated with a high fatality rate. CCHF is endemic in Afghanistan, and its morbidity and mortality have increased recently but there is limited data about the characteristics of fatal cases. We aimed to report the clinical and epidemiological features of fatal CCHF cases who were admitted to Kabul Referral Infectious Diseases (Antani) Hospital. This is a retrospective cross-sectional study. The demographic and presenting clinical and laboratory features of 30 fatal CCHF cases diagnosed by reverse transcription polymerase chain reaction (RT-PCR) or enzyme-linked immunosorbent assay (ELISA) tests were collected from the patients' records between March 2021 and March 2023. During the study period, a total of 118 laboratory-confirmed CCHF patients were admitted to Kabul Antani Hospital of whom 30 patients (25 males, 5 females) consequently died, indicating a 25.4% case fatality rate (CFR). The age of the fatal cases ranged from 15 to 62 years and their mean age was 36.6 ± 11.7 years. Concerning occupation, the patients were butchers (23.3%), animal dealers (20%), shepherds (16.6%), housewives (16.6%), farmers (10%), student (3.3%), and others (10%). The clinical symptoms of the patients on admission were fever (100%), generalized body pain (100%), fatigue (90%), bleeding (any type) (86.6%), headache (80%), nausea/vomiting (73.3%), and diarrhea (70%). The initial abnormal laboratory findings were leukopenia (80%), leukocytosis (6.6%), anemia (73.3%), and thrombocytopenia (100%), raised hepatic enzymes (ALT & AST) (96.6%) and prolonged prothrombin time/international normalized ratio (PT/INR) (100%). The hemorrhagic manifestations associated with low platelet and raised PT/INR levels are linked with fatal outcomes. A high index of clinical suspicion is required to recognize the disease at an early stage and to begin the treatment promptly for reducing mortality.

Mechanisms of Wuyao (Linderae Radix) in treating chronic pelvic inflammatory disease based on network pharmacology and animal experiments

ObjectiveTo predict the active components and action targets of Wuyao (Linderae Radix) in the treatment of chronic pelvic inflammatory disease (CPID) based on network pharmacology, explore possible mechanisms of the treatment through animal experiments, and provide a scientific basis for clinical applications of Wuyao (Linderae Radix). MethodsPossible active components and targets of Wuyao (Linderae Radix) in the treatment of CPID were obtained applying network pharmacology and molecular docking technology. CPID rat models were established using the mixed Escherichia coli, Staphylococcus aureus, and Ureaplasma urealyticum plus the performance of mechanical injury. Hematoxylin-eosin (HE) staining was applied to observe the pathological changes in the uterus, fallopian tube, and spleens of rat models. The contents of nitric oxide (NO), superoxide dismutase (SOD), and malondialdehyde (MDA) in the serum of rats were determined with the use of corresponding detection kits. Enzyme-linked immunosorbent assay (ELISA) test was used to measure the expression of interleukin (IL)-6 and IL-10 in the serum of rat models. Flow cytometry was used to determine the percentage of CD4+ and CD8a+ T cells as well as CD4+ CD25+ regulatory T cells (Tregs) in the spleen of rat models. ResultsA total of nine potential active components and four core therapeutic targets related to inflammatory response in Wuyao (Linderae Radix) were obtained. The animal experiments showed that Wuyao (Linderae Radix) markedly inhibited uterus swelling, regulated morphological changes in the fallopian tube and spleen, effectively reduced inflammatory infiltration and injuries in the uterus and fallopian tube, and improved spleen functions in CPID rats. Moreover, Wuyao (Linderae Radix) markedly reduced the levels of NO, IL-6, and MDA, and increased the levels of IL-10 and SOD in the serum of rats. Wuyao (Linderae Radix) also elevated the percentage of CD4+ T cells and the CD4+ T/CD8a+ T cell ratio, reduced the percentage of CD8a+ T cells, and raised the percentage of CD4+ CD25+ Tregs that had been abnormally decreased in rat models (P < 0.05). ConclusionWuyao (Linderae Radix) could have therapeutic effects on CPID rats by relieving oxidative stress, mitigating inflammatory levels, and regulating the immuno-function of T cell subgroups to improve the pathological changes in CPID rats. It is a medicinal herb worth being further explored for its clinical values.

Evaluation of Immunogenicity of Clostridium perfringens Type B Toxoid and Inactivated FMD (O) Virus with Adjuvant (ISA70-MF59).

Foot and mouth disease (FMD) and enterotoxemia are important diseases of hoofed animals. Vaccination against livestock pathogens, especially these two diseases, plays a key role in the prevention and control of these diseases. The use of combined vaccines with the aim of creating a better immune response and producing cheaper vaccines is a great contribution to Vaccine industry. This research aimed to compare the immunogenicity of FMD (O) and Clostridium perfringens type B toxoid along with adjuvant (MF59) and Montanide (ISA70) to create the best immunogenicity. To investigate the immune responses of vaccines, it was injected into an animal model, and the antibody titer was measured by enzyme-linked immunosorbent assay (ELISA) test and VN antibody titer. The results showed that the formulation with MF59 adjuvant brought more stable immunogenicity against FMD and Clostridium perfringens type B, and the length of the immunogenicity period also increased significantly. Therefore, the combined vaccine (Clostridium perfringens + FMD) could play a major role invaccine industry as an alternative vaccine against Clostridium perfringens and FMD in livestock.

Comparative Evaluation of Specific Antibody against Mycobacterium tuberculosis ESAT-6 Recombinant Antigen in Healthy Subject with Positive and Negative Skin Test.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis). The laboratory diagnosis of the disease includes various bacteriologic and immunologic methods. Despite the effectiveness of many of these methods in diagnosing active TB, their high cost and time-consuming nature have led researchers to adopt more accurate and rapid screening methods based on specific antigens for M. tuberculosis. The present study aimed to measure specific antibody serum levels against the early secretory antigenic target 6 kDa (ESAT-6) recombinant protein in healthy people and compare it to TB patients. The target population included 27 TB patients and 87 healthy individuals with no clinical TB symptoms. The healthy population was divided into two groups, including positive purified protein derivative (PPD) and negative PPD (35 and 52 people, respectively), using the Tuberculin skin test. The specific antibody level against the ESAT-6 recombinant antigen and the PPD protein was measured using an indirect Enzyme-Linked Immunosorbent Assay (ELISA) test. The results of the study showed that the majority of the healthy population with no symptoms of clinical TB and having negative skin test results did not have antibodies against the recombinant ESAT-6 (98%) and PPD (96%) antigens. On the other hand, there was a high level of the specific antibody of the ESAT-6 recombinant and PPD antigens in TB patients (77%). It is notable that in people with positive skin test results, the level of the antibody against the ESAT-6 recombinant antigen and PPD antigen was 94%. The results demonstrated that the ELISA method based on the measurement of antibodies against the ESAT-6 recombinant antigen can be a proper diagnostic method for rapid and accurate screening of healthy from infected people.

Acute Phase Proteins in Dogs with Natural Infection by Trypanosoma cruzi.

Acute phase proteins have been used as tools for the diagnosis, monitoring, and prognosis of several diseases in domestic animals. However, the dynamics of these proteins in infection by Trypanosoma cruzi, the causative agent of Chagas disease in dogs, is still unknown. The aim of this study was to determine concentrations of acute phase proteins (C-reactive protein, haptoglobin, ferritin and paraoxonase-1) in dogs in a coastal town of Ecuador, with natural Trypanosoma cruzi infection with or without seroreactivity of Ehrlichia canis, Ehrlichia ewingii, Anaplasma phagocytophilum, Anaplasma platys, Borrelia burgdorferi and Dirofilaria immitis. For the detection of Trypanosoma cruzi serum antibodies, two different antigen-based enzyme-linked immunosorbent assay tests were implemented. For the detection of seroreactivity of Ehrlichia canis, Ehrlichia ewingii, Anaplasma phagocytophilum, Anaplasma platys, Borrelia burgdorferi and Dirofilaria immitis, an IDEXX SNAP® 4Dx® test was used. To determine the concentration of C-reactive protein and ferritin, an immunoturbidimetric assay was used; haptoglobin concentration was measured using a commercial colorimetric method validated in dogs; a spectrophotometric method was used to determine the serum concentration of paraoxonase-1. Results showed a reduction in the serum levels of paraoxonase-1 in Trypanosoma cruzi-seroreactive dogs, either with or without seroreactivity to other vector-borne diseases. A serum ferritin increment was observed in Trypanosoma cruzi-seroreactive dogs with seroreactivity to any other vector-borne diseases. Our findings suggest that paraoxonase-1 levels are reduced in Trypanosoma cruzi-seroreactive dogs without evident clinical signs of Chagas disease, despite their seroreactivity to the other vector-borne diseases studied. These findings could indicate an oxidative stress response in Trypanosoma cruzi-seroreactive dogs with no evident signs of inflammation.

The transcriptional foundations of interferon-λ-mediated endometrial cell to uterine receptivity.

Interferon-λ (IFN-λ) is a novel non-redundant regulator that participates in the fetal-maternal immune interaction, including immune regulation, uterine receptivity, cell migration and adhesion, and endometrium apoptosis. However, the exact transcriptional foundation for endometrial signaling of IFN-λ is not completely understood, and studies regarding IFN-λ to implantation failure in vivo are limited. The gene expression profile of human endometrial Ishikawa cell line treated with IFN-λ or IFN-α (100ng/mL) for 6h was analyzed using RNA-sequencing. Real-time qPCR, western blotting, and enzyme-linked immunosorbent assay (ELISA) tests were used to validate these sequencing data. An in vivo IFN-λ knock-down mouse pregnancy model was performed, and the phenotype analysis and the intrauterine biomarkers detection were applied with the uterus samples. High levels of messenger RNA (mRNA) were detected for genes previously associated with endometrial receptivity, including LIF, AXL, CRYAB, EPHB2, CCL5, and DDX58, following IFN-λ treatment. Moreover, the data indicated IFN-λ reduced pro-inflammatory gene activity compared with IFN-α, including members of the ISG, TNF, SP100 and interleukin genes. The in vivo mouse pregnancy model showed that inhibition of intrauterine IFN-λ results in aberrant epithelial phenotype and significantly decreases the embryo implantation rates and derails normal uterine receptivity. These findings demonstrate the antagonistic and agonistic roles of IFNs in the endometrial cell, suggesting a selective role of IFN-λ in endometrial receptivity and immunological tolerance regulation. Moreover, the findings provide valuable insight into potential biomarkers related to endometrial receptivity and facilitate an understanding of the molecular changes observed during infertility treatment and contraception usage.

Serological Prevalence of Toxoplasma gondii, Neospora caninum, and Sarcoptes scabiei var. suis in Wild Boars (Sus scrofa) Hunted in a Highly Anthropized Area in Italy.

Due to the increasing expansion into urban and rural areas, wild boars represent a potential source of infection with zoonotic and animal-specific parasites for both humans and animals. Therefore, the aim of this study was to investigate the serological prevalence to Toxoplasma gondii, Neospora caninum, and Sarcoptes scabiei var. suis in blood samples from wild boars (Sus scrofa) hunted in an anthropized area in Italy. Enzyme-linked immunosorbent assay (ELISA) tests were used to detect antibodies anti-T. gondii and anti-S. scabiei and an immunofluorescence antibody test (IFAT) for antibodies anti-N. caninum. 81 out of 128 wild boars (P = 63.3%) resulted positive for at least one of the three parasites. 68 of them were seropositive to T. gondii (P = 53.1%) and 14 to N. caninum (P = 10.9%). 9 wild boars resulted seropositive to S. scabiei var. suis (P = 7.0%). Sampling season was the only significant risk factor related to S. scabiei var. suis seroprevalence (OR = 7.8). The high occurrence of T. gondii supports the role of this species as a source of infection for other animals and humans. Furthermore, the serological prevalence of N. caninum and S. scabiei var. suis in wild boars from the study area characterized by the presence of numerous dairy cattle and pig farms is relevant to demonstrate its suitability for the circulation of these parasites both in domestic and wild species.

A Trypan Blue-Based ELISA Practice Test to Evaluate Trainee Performance

Enzyme-linked immunosorbent assay (ELISA) is a valuable tool in various chemical and biological fields for investigating the presence and quantity of proteins of interest in given samples. The high importance of the assay makes it widely used in biological research and hence a crucial skill to master for up-and-coming scientists. A major impediment to teaching the skill to new students in research laboratories is the high cost of commercial ELISA kits. As a result of the cost of the kits, most investigators prefer that more experienced students or technicians carry out the assay to prevent errors, thus depriving less experienced students the chance to actively learn the process. Here, we present a practice ELISA test that can be done in any laboratory using different dilutions of trypan blue as the reagents and samples, thereby creating a product at one-hundredth the cost of the least expensive ELISA kits. Research has continuously shown that active learning tools such as this simulation enhance student engagement and self-efficacy. This practice test also generates a numerical score based on the students’ accuracy in pipetting and attention to detail, allowing students to identify sources of error. Therefore, the practice ELISA experiment acts as a medium to teach proper technique before performing a real ELISA, thus avoiding wasting expensive reagents and limited samples.

BSA Interference in Immunoassays in Individuals with Egg Allergy

Aim: The aim of current study was to determine interference by bovine serum albümine (BSA) as blocking agent in enzyme-linked immunosorbent assay (ELISA) carried out in individuals with egg allergy. Material and Methods: 14 people diagnosed with egg allergy and 7 people without allergy were included. The sample were studied with an indirect ELISA method for egg-white IgG antibody developed in our laboratory. Effect BSA on interference was studied by manipulating antigene coating (none vs. egg white extract), blocking (1% BSA vs. Tween 80), and sample diluent (PBS vs. PBS + 0.5% BSA). Results: In wells that were blocked with 1% BSA without being coated with antigen, positive samples cross-reacted with BSA to give an optical density (OD) of 0.99 ± 0.16, while negative samples gave an OD of 0.08±0.01 (p&amp;lt;0.05). However, when the same samples were diluted with 0.5% BSA, the OD of positive samples decreased (from 0.99±0.16 to 0.08±0.01), and the statistical difference with negative samples disappeared. It was observed that tween, which was used as a blocking and diluting agent, did not cross-react with the samples. Positive samples gave an OD of 0.66±0.07 in antigen (egg white extract) coated and tween-blocked wells, and 1.01±0.11 OD in BSA blocking (p&amp;lt;0.05). When the samples were diluted with 0.5% BSA, positive samples gave 0.18±0.01 OD on the antigen coated plate, while negative samples gave 0.12±0.04 OD (p&amp;lt;0.05). Conclusion: Ovalbumin, which is found in high levels in eggs, has a similar molecular structure to BSA, and some antibodies produced against ovalbumin in people with egg allergy may also cross-react against BSA. Therefore, it was concluded that the use of BSA in both dilution and blocking solution should be avoided if the samples of individuals with egg sensitivity are to be analyzed by ELISA method. It has been observed that Tween can be easily applied as an alternative blocking agent in allergy ELISA tests.

Validation of ELISA Test-system for Trastuzumab (Herceptin, Hertikad) quantitative determination in biological fluids

Relevance. Trastuzumab is the drug of choice for the HER2+ breast cancer treatment. To determine the trastuzumab pharmacodynamics in personalized therapy a validated bioanalytical method for measuring the concentration of the drug in biological fluids is required. The aim: creation and assessment of the suitability (validation) of a test system based on enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of trastuzumab concentration in human serum/plasma. Materials and methods. The presented test system is a classic ELISA kit with a 96-well polystyrene plate, the wells of which are coated with monoclonal antibodies specific to trastuzumab, secondary goat antibodies to the Fc fragment conjugated with horseradish peroxidase (HRP), substrate solution — (3,5,3',5')-tetramethylbenzidine (TMB) and stop solution. Quality control solutions were prepared by diluting known concentrations of trastuzumab in blank serum. Results. In the course of the work the limit of detection (0.84 ng/ml) and the lower limit of quantitative determination (1.41 ng/ml) of trastuzumab in serum/plasma were established and the high selectivity of analyte determination in a multicomponent matrix was proved. The found average values of trastuzumab concentrations did not deviate from the nominal values by more than 14 % in the entire range of determined concentrations, the intraand interseries precision of the test system did not exceed 8%, and the total method error was 20.1 %. The demonstrated dilution linearity allows the assay to be used to analyze a wide range of trastuzumab concentrations in biological samples. The stability of the components of the test system is defined as at least 1 year under storage conditions. Conclusion. The presented ELISA test system complies with international validation requirements and it is suitable for practical use.

106 Serial Evaluation ofA. Marginale Positivity Rate Within a Single Herd of Mature Beef Cows

Abstract Anaplasmosis is an economically devastating disease in cattle that is caused by the rickettsial pathogen Anaplasma marginale. Infected cattle can serve as biological reservoirs for the organism, thus enabling further transmission of the disease to naive individuals via ticks, biting flies and contaminated needles. It is estimated that this disease causes over $300 million in expenses for the U.S. cattle industry annually. As a result, a better understanding of this disease and its prevalence is needed. As part of an ongoing collaborative effort between the University of Arkansas and the Kansas State University (KSU) College of Veterinary Medicine, general surveillance testing of beef cattle herds in Washington county, Arkansas previously indicated a possible 30% positivity rate for A. marginale in 2019. The objective of this study was to utilize both quantitative polymerase chain reaction (qPCR) and competitive enzyme-linked immunosorbent assay (cELISA) tests to determine the whole-herd prevalence of A. marginale within the University of Arkansas Washington county research herd. In the fall of 2020, the entire fall-calving herd (168, apparently healthy, mature beef cows) was sampled and tested. Both whole blood and serum samples were collected from each animal. qPCR testing was completed on each whole blood sample submitted at the KSU College of Veterinary Medicine, while cELISA testing was completed on each serum sample by the University of Arkansas. Of the68 cows tested, 20 cows tested seropositive, with a % Inhibition of 30 or above (mean = 45%). Confirmatory qPCR testing on all seropositive samples, however, indicated that all cows were negative for active infection with A. marginale. Subsequently, in the spring of 2022, all available cows that had previously tested seropositive for A. marginale were retested using the same protocol as previously described, except that all qPCR testing was completed by University of Arkansas Veterinary Entomology Lab. Of the 17 mature cows still in inventory, that had previously tested seropositive for A. marginale, only 7 were still seropositive with a % inhibition of 30 or above (mean = 39%). However, confirmatory qPCR testing on all 7 seropositive samples indicated that all cows were negative for active infections with A. marginale. Because of its lower cost, compared with qPCR testing, ELISA testing is commonly recommended to and utilized by producers and veterinarians as a first-line diagnostic for individual and herd health evaluation and management. Information gathered by this study indicates that cELISA testing alone may not provide an accurate depiction of actual infection rates within a herd, and that further investigation into possible causes of false positivity are required. As part of this study, additional assays investigating the specificity of the commercial Anaplasmosis cELISA and possible cross-reaction with other endemic rickettsial pathogens will be performed.

Comparison of Cytokine Profile between Postmenopausal Women with and Without Osteoporosis - A Case-Control Study.

Chronic low-grade inflammation is involved in the pathogenesis of postmenopausal osteoporosis, but the cytokines implicated remain elusive. This study aimed to compare the difference in cytokine profile between postmenopausal women with and without osteoporosis in Klang Valley, Malaysia. Postmenopausal women with (n = 20) and without osteoporosis (n = 20) were recruited for this study. Their bone health status was determined using dual-energy X-ray absorptiometry. Their fasting blood was collected for proteomic analysis. A protein array was performed for four subjects randomly selected from each group to screen the potential cytokines. Three cytokines at least 20% different between groups and consistently expressed by each subject were selected for validation using enzyme-linked immunosorbent assays (ELISA). The protein array screening demonstrated that platelet-derived growth factor-BB, interleukin- 6 receptor (IL-6R), and tissue inhibitor of metallopeptidase-2 were higher in women with osteoporosis than women without osteoporosis (n = 4 per group), and consistently expressed by all women. Only body mass index (BMI)-adjusted logarithmically transformed IL-6R levels were lower among postmenopausal women with osteoporosis compared to women with normal bone health (p = 0.026) (n = 16 per group) in the ELISA test. IL-6R was lower among postmenopausal women with osteoporosis compared to women with normal bone health after adjusting for BMI. However, a large-scale epidemiological study with proteomic analysis needs to confirm the findings.

Small cell lung cancer: circulating tumor cell lines and expression of mediators of angiogenesis and coagulation.

Coagulation is frequently activated in cancer patients and has been correlated with an unfavorable prognosis. To evaluate whether a putative release of tissue factor (TF) by circulating tumor cells (CTCs) represents a target to impair the dissemination of small cell lung cancer (SCLC), the expression of relevant proteins in a panel of permanent SCLC and SCLC CTC cell lines that have been established at the Medical University of Vienna. Five CTC and SCLC lines were analyzed using a TF enzyme-linked immunosorbent assay (ELISA) tests, RNA sequencing, and western blot arrays covering 55 angiogenic mediators. Furthermore, the influence of topotecan and epirubicin as well as hypoxia-like conditions on the expression of these mediators was investigated. The results demonstrate that the SCLC CTC cell lines express no significant amounts of active TF but thrombospondin-1 (TSP-1), urokinase-type plasminogen activator receptor (uPAR), vascular endothelial-derived growth factor (VEGF) and angiopoietin-2 in two cases. The major difference between the SCLC and SCLC CTC cell lines found was the loss of the expression of angiogenin in the blood-derived CTC lines. Topotecan and epirubicin decreased the expression of VEGF, whereas hypoxia-like conditions upregulated VEGF. Active TF capable of triggering coagulation seems not to be expressed in SCLC CTC cell lines in significant levels and, thus, CTC-derived TF seems dispensable for dissemination. Nevertheless, all CTC lines form large spheroids, termed tumorospheres, which may become trapped in clots of the microvasculature and extravasate in this supportive microenvironment. The contribution of clotting to the protection and dissemination of CTCs in SCLC may be different from other solid tumors such as breast cancer.

Prevalence of hepatitis B infection and its associated factors among pregnant mothers attending antenatal care at public hospitals at Hararghe, Eastern Ethiopia.

Hepatitis B infection is one of the world's most serious public health problems, causing significant morbidity and mortality. More than 2 billion individuals around the world have been infected with the hepatitis B virus (HBV), and approximately 400 million people are chronically infected with the virus, with more than a million dying each year from hepatitis B virus-related liver disease. A newborn infant whose mother is positive for both HBsAg and HBeAg has a 90% chance of developing chronic infection by the age of 6. Its infectivity is a 100 times that of the human immunodeficiency virus, but it receives little attention in public health. Therefore, this study was conducted to assess the prevalence of hepatitis B infection and its associated factors among pregnant mothers attending antenatal care at public hospitals in west Hararghe, eastern Ethiopia 2020. This institution-based cross-sectional study was conducted on 300 pregnant mothers selected by systematic random sampling from September to December 2020. Data were collected by face-to-face interview using a pretested structured questionnaire. A blood sample was collected and tested for hepatitis B surface antigen using the enzyme-linked immunosorbent assay test method. Data were entered into EpiData version 3.1 and exported to Statistical Package for the Social Science version 22 for analysis. Bivariate and multivariable logistic regressions were used to assess the association between outcome and predictor variables. P-value <0.05 was considered to be statistically significant. The overall seroprevalence of hepatitis B virus infection was 8% [95% confidence interval (CI): 5.3-11.0] among pregnant mothers. History of tonsillectomy [adjusted odd ratio (AOR) = 5.7; 95% CI: 1.3-23.9], tattoo (AOR = 4.3; 95% CI: 1.1, 17.0), having multiple sexual partners (AOR = 10.8; 95% CI: 2.5, 45.9), and history of contact with jaundiced patients (AOR = 5.6; 95% CI: 1.2, 25.7) were factors associated with the seroprevalence of hepatitis B virus infection among pregnant mothers. The hepatitis B virus was highly prevalent. A history of tonsillectomy, tattooing, having multiple partners, and contact with jaundiced patients were factors associated with hepatitis B virus infection. To reduce HBV transmissions, the government should increase HBV vaccination coverage. All newborns should receive the hepatitis B vaccine as soon as possible after birth. It is also recommended that all pregnant women have HBsAg testing and antiviral prophylaxis to reduce the risk of transmission from mother to child. Hospitals, districts, regional health bureaus, and medical professionals should also educate pregnant women about hepatitis B virus transmission and prevention, both in the hospital and in the community, with a focus on modifiable risk factors.

Detection of Mycoplasma gallisepticum, Isolation, and Determination of Tylosin Susceptibility of Isolates from Commercial Chickens.

Mycoplasma gallisepticum (MG) is a contagious avian pathogen that causes financial losses to the poultry industry. Isolation of the pathogen is difficult and time-consuming, and therefore, far from a routine method. Serological testing methods to detect antibodies resistant to MG are widely used in routine diagnosis. Tylosin is a class of macrolide antibiotics tremendously administered in veterinary medicine for the treatment of mycoplasmosis and prophylaxis. This study aimed to detect MG by immunoassay testing, culture, and polymerase chain reaction (PCR) in commercial poultry farms and to investigate the tylosin susceptibility of the isolates. To verify the presence of antibodies resistant to MG, 750 blood samples were randomly collected from 38 broiler farms from 2019 to 2022 in Mazandaran and Golestan provinces, Iran, and rapid slide agglutination (RSA) assay was performed. Positive results were analyzed by the enzyme-linked immunosorbent assay (ELISA) for further investigation. Here, 920 swab samples were collected from 38 non-vaccinated commercial farms for culture, and PCR tests were performed for the isolated strains. The activities of tylosin were tested in vitro against these isolates using the broth microdilution method. The lowest antibiotic concentration that resulted in a color change was considered the minimum inhibitory concentration (MIC) value. Twenty-four (63.1%) farms were positive in the RSA test, and 21 (55.2%) farms were positive in the ELISA test. Nine (23.68%) of the farms grew on culture media, and 8 (21.05%) were detected as Gallisepticum species by PCR. The geometric mean of MIC for tylosin was 5.75 µg/ml, MIC50 was 4 µg/ml, and MIC90 was 8 µg/ml. The results indicated that commercial farms were infected with MG. Considering the ability of MG to spread and the probable use of the RSA test as a rapid and cheap method, it can be argued that ELISA and RSA serological tests can be used to find MG in poultry flocks, and the positive result should be confirmed by standard microbiological tests or PCR. It was also found that the isolated parts of MG changed their sensitivity to tylosin, indicating the need for routine testing to optimize treatment dose and efficiency.