Development Of Diagnostic Tests Research Articles

Application two-dimensional gel electrophoresis coupled with LC-MS/MS to identify candidate serodiagnostic antigens for early detection Psoroptes ovis var. cuniculi infection.

Currently, the 'gold standard' for diagnosis of Psoroptes ovis infections is detecting Psoroptes mites or eggs in skin scrapings under microscopy, but it is prone to be mis-diagnosed for detecting early infection of P. ovis. Hence, seeking a reliable diagnostic technique for detecting early-stage mite infections is extremely desirable. Enzyme linked immunosorbent assay (ELISA) has proven to be useful for the diagnosis of early-stage P. ovis infection. Thus, the purpose of this study was to screen serodiagnostic candidate antigens that can detect early P. ovis infection. Psoroptes ovis var. cuniculi wash proteins (PsoWA), which contained an enriched source of secretory and excretory antigens, were separated by two-dimensional gel electrophoresis (2-DE) and screened by immunoblot using sera from rabbits with early-stage Psoroptes infection (1 week and 3 weeks). Immunogenic proteins were submitted for sequencing by liquid chromatography tandem-mass spectrometry (LC-MS/MS) analyses. Three potential diagnostic antigens were identified (PsoSP3, Pso14-3-3(1) and Pso14-3-3(2)) in this study. These were further expressed in E. coli expression system to evaluate the serodiagnostic potential of these recombinant proteins for detecting early-stage P. ovis infection using an indirect ELISA (iELISA). Western blotting showed that 34 protein spots were recognized by rabbit sera of 1 week post-infection (wpi) and 3 wpi. The 2-DE results showed that a total of 199 proteins were detected with molecular weights varying from 20 to 100 kDa and isoelectric point (pI) from 4.1 to 9.3. Among these, 90 proteins were detected both at 1 wpi sera and 3 wpi sera, and the numbers of the specific identified proteins were 27 for 1 wpi sera and 82 for 3wpi sera. Moreover, rPsoSP3 showed better diagnostic efficacy than rPso14-3-3(1) and rPso14-3-3(2) in detecting early-stage P. ovis infection for its higher values of sensitivity, specificity and area under the receiver operating characteristic curve. Our study describes the first immunoproteomic analysis to identify early diagnostic candidate antigens of P. ovis, and the identified antigens of Psoroptes in our study have significant implications for the development of early-stage diagnostic tests. PsoSP3 is a promising early diagnostic antigen for detecting P. ovis var. cuniculi infection.

Distinct pro-inflammatory/pro-angiogenetic signatures distinguish children with Long COVID from controls.

Recent proteomic studies have documented that Long COVID in adults is characterized by a pro-inflammatory signature with thromboinflammation. However, if similar events happen also in children with Long COVID has never been investigated. We performed an extensive protein analysis of blood plasma from pediatric patients younger than 19 years of age Long COVID and a control group of children with acute COVID-19, MIS-C, and healthy controls resulted similar for sex distribution and age. Children were classified as Long COVID if symptoms persisted for at least 8 weeks since the initial infection, negatively impacted daily life and could not be explained otherwise. 112 children were included in the study, including 34 children fulfilling clinical criteria of Long COVID, 32 acute SARS-CoV-2 infection, 27 MIS-C and 19 healthy controls. Compared with controls, pediatric Long COVID was characterized by higher expression of the proinflammatory and pro-angiogenetic set of chemokines CXCL11, CXCL1, CXCL5, CXCL6, CXCL8, TNFSF11, OSM, STAMBP1a. A Machine Learning model based on proteomic profile was able to identify LC with an accuracy of 0.93, specificity of 0.86 and sensitivity of 0.97. Pediatric Long COVID patients have a well distinct blood protein signature marked by increased ongoing general and endothelial inflammation, similarly as happens in adults. Pediatric Long COVID has a distinct blood protein signature marked by increased ongoing general and endothelial inflammation. This is the first study studying and documenting proinflammatory profile in blood samples of children with long COVID. Long COVID was characterized by higher expression of the proinflammatory and pro-angiogenetic set of chemokines CXCL11, CXCL1, CXCL5, CXCL6, CXCL8, TNFSF11, OSM, STAMBP1a. A proteomic profile was able to identify Long COVID with an accuracy of 0.93, specificity of 0.86 and sensitivity of 0.97. These findings may inform development of future diagnostic tests.

P0082 Proteomic profiling of colonic mucosa in patients with ulcerative colitis: detection of molecular patterns of inflammation.

Abstract Background The mechanisms of recurrence and progression of ulcerative colitis (UC) require detailed study. Modern advances in proteomic analysis methods are ideal for studying the molecular characteristics of inflammation in the colonic mucosa (CM) of UC patients. Methods The study was comparative cohort with parallel design and included 88 patients (range from 22 to 35 years, 37 men and 51 women): 53 (60.2%) pancolitis and 35 (39.8%) left-sided UC, mild and moderate activity. The duration of UC was 5.3 years. The control group included 30 healthy individuals. Biosamples of CM in patients with UC in the active stage and in healthy persons were received by ileocolonoscopy with CM biopsy. The separation of individual proteins of CM was based on technologies of IEF, SDS-PAGE, 2DPAGE, by standard sets (MB-HIC C8 Kit, MB-IMAC Cu, MB-Wax Kit, «Bruker», USA). Automated mass spectrometry imaging was performed by MALDI-TOF-MS/MS, Ultraflex II, «Bruker», USA). The partially identified sequences were then submitted to “BLAST protein-protein” and screened against the Homo sapiens Swissprot database to check if this identification matched the MASCOT-identification (Matrix Science, UK). The data of the molecular interactions and functional features of proteins were received with STRING 10 database. Results Based on the data of standard methods of identification of UC and molecular phenotyping of colon mucosa we conducted new prognostic markers, molecular pathways of UC and diagnostic tests in patients with UC. We identified functional groups of peptides and proteins in molecular patterns of CM (fig. 1). SMAD family member 2 (SMAD2) activates the transcription of TFG1β, that leads the development of fibrosis in colon submucosa in UC patients; the stimulation of the expression of apoС-III in affected CM in UC is associated with the activation of the FOXO1 signaling pathway that supports inflammatory processes; caspase 8 protects colonocytes from TNFα-induced cell death through a necroptosis mechanism via the blockade of the RIP3 expression. Significant decrease of the expression of PPARγ promotes the activation of STAT and AP-1 signaling pathways, that promotes the increase of the synthesis of IL-2, 6, 8, 12,TNFα, matrix metalloproteinases, the activity of immune and inflammation processes in colon mucosa. The reduction of expression of β-defensin-1 in cells of colon mucosa are accompanied with increased expression of CCR6, that promotes the formation of inflammatory infiltrates in colonic submucosa in UC.The molecular interactions of β-defensin-1 are presented on figure 2. Conclusion Bioinformatics analysis revealed the presence of the new potentially markers of the progression UC, that’s may provide for the development of novel diagnostic tests for UC.

Transcriptomic identification of genes associated with thrombosis and coagulation in lipopolysaccharide-exposed bovine monocyte-derived macrophages.

The objective of this study was to investigate the differential expression of genes associated with coagulation in bovine monocyte-derived macrophages (MoMΦ) exposed to lipopolysaccharide (LPS) in vitro. We hypothesized that MoMΦ stimulated with LPS would have upregulation of procoagulant genes and downregulation of genes protecting against coagulation. MoMΦ were isolated from Holstein steers and exposed to Escherichia coli-derived LPS or a control for 3 hours. We used transcriptomics (RNA sequencing) to characterize the differential expression of genes associated with coagulation in the LPS-exposed MoMΦ relative to the control group. 1,602 genes were upregulated and 1,209 genes downregulated 3 hours after exposure to LPS. Monocyte-derived macrophages exposed to LPS displayed statistically significant upregulation of 4 proinflammatory genes, 2 anti-inflammatory genes, 8 genes involved in promoting coagulation, and 5 genes considered protective against coagulation. There was significant downregulation of 1 gene involved in the promotion of coagulation. Our results showed increased expression of most genes investigated promoting and protecting against coagulation and increased expression of proinflammatory and anti-inflammatory genes. These findings suggest that MoMΦ exhibit a multifaceted response in the early response to LPS, promoting and protecting against excessive coagulation. This multifaceted response highlights the interplay between different pathways involved in early sepsis. Our data demonstrate the utility of using MoMΦ as a model system to investigate sepsis-associated coagulopathies. These insights into the early transcriptomic changes in response to LPS may help guide future research on the development of treatment modalities or diagnostic tests for patients with sepsis.

Development of a monoclonal antibody specifically recognizing a linear epitope on the E1 protein of Getah virus

Getah virus (GETV) is a mosquito-borne alphavirus that can cause disease outbreaks in domesticated mammals. The E1 protein of GETV plays a crucial role in mediating the fusion of viruses and host cells. In this study, the recombinant GETV E1 protein was expressed and administered to BALB/c mice. Hybridoma cells secreting a monoclonal antibody (mAb) 7D4-2 against E1 protein were subsequently obtained using a cell fusion protocol between SP2/0 cells and splenocytes. The reactivity of mAb 7D4-2 with GETV was confirmed through Western blot analysis and indirect immunofluorescence assay (IFA). A precise B cell linear epitope, 281-DIPDTAF-287, was identified using Western blot analysis with the produced mAb 7D4-2 by constructing and expressing a series of truncated His-fused E1 proteins. Conservation analysis indicated that this epitope is highly conserved in Group III strains of GETV, but exhibits an amino acid difference (T285S) compared to Group I, Group II, and Group IV. Cross-reactivity analysis by Western blot demonstrated that the B-cell epitope containing the mutation could be recognized by mAb 7D4-2. The ability of mAb 7D4-2 to recognize the epitope carrying the T285S mutation was further confirmed using an infectious clone of GETV. This study enhances the understanding of the biological characteristics of the E1 protein and will facilitate the future development of diagnostic tests for GETV.

Exploring Bioinformatics-Driven Approaches for Enhanced Diagnosis of Chlamydia trachomatis Infections: Analysis of Target Proteins

Chlamydia trachomatis, a major cause of sexually transmitted infection, poses a range of symptoms including genital discharge, pain during urination, and abdominal pains in women, and can lead to serious health complications such as pelvic inflammatory diseases, infertility, and ectopic pregnancy if left untreated. The need for rapid and accurate detection is imperative so prompt treatment and control of the disease can be achieved. This study conducted an immunoinformatic analysis of proteins of Chlamydia trachomatis (incA, hctA, ompA, omcB, rpoB, and HSP60) for the development of a lateral flow assay-based diagnostic test. Detailed in silico evaluation of selected proteins from publicly available genomic databases was conducted to evaluate their suitability as targets for lateral flow assay-based detection. The series of tests included antigenicity, toxicity, solubility, physicochemical characteristics and molecular docking of the derived constructs, and protein sequence. Chimeric construct was derived from the prediction of linear B cell epitopes, helper T cell major histocompatibility complex II binding epitopes, and IL4 and IL10 inducers using bioinformatic tools at standard thresholds. With a Ramachandra’s score of 95.4% and Z-score of -5.1, results indicate that the construct efficacy is high in potential to provide extreme specificity and sensitivity for the detection of Chlamydia trachomatis in clinical samples as compared to traditional culture-based methods using nucleic acid amplification, hereby providing a quicker and more accurate diagnostic tool for Chlamydia trachomatis infection. Findings offer valuable data for the development of a rapid and reliable diagnostic point-of-care test kit for Chlamydia trachomatis that allows for drastic reduction in clinical wait time and treatment.

Exploring Bioinformatics-Driven Approaches for Enhanced Diagnosis of Chlamydia trachomatis Infections: Analysis of Target Proteins

Chlamydia trachomatis, a major cause of sexually transmitted infection, poses a range of symptoms including genital discharge, pain during urination, and abdominal pains in women, and can lead to serious health complications such as pelvic inflammatory diseases, infertility, and ectopic pregnancy if left untreated. The need for rapid and accurate detection is imperative so prompt treatment and control of the disease can be achieved. This study conducted an immunoinformatic analysis of proteins of Chlamydia trachomatis (incA, hctA, ompA, omcB, rpoB, and HSP60) for the development of a lateral flow assay-based diagnostic test. Detailed in silico evaluation of selected proteins from publicly available genomic databases was conducted to evaluate their suitability as targets for lateral flow assay-based detection. The series of tests included antigenicity, toxicity, solubility, physicochemical characteristics and molecular docking of the derived constructs, and protein sequence. Chimeric construct was derived from the prediction of linear B cell epitopes, helper T cell major histocompatibility complex II binding epitopes, and IL4 and IL10 inducers using bioinformatic tools at standard thresholds. With a Ramachandra’s score of 95.4% and Z-score of -5.1, results indicate that the construct efficacy is high in potential to provide extreme specificity and sensitivity for the detection of Chlamydia trachomatis in clinical samples as compared to traditional culture-based methods using nucleic acid amplification, hereby providing a quicker and more accurate diagnostic tool for Chlamydia trachomatis infection. Findings offer valuable data for the development of a rapid and reliable diagnostic point-of-care test kit for Chlamydia trachomatis that allows for drastic reduction in clinical wait time and treatment.

Isolation and characterization of DNA aptamers against the HlyE antigen of Salmonella Typhi

Aptamers have emerged as prominent ligands in clinical diagnostics because they provide various advantages over antibodies, such as quicker generation time, reduced manufacturing costs, minimal batch-to-batch variability, greater modifiability, and improved thermal stability. In the present study, we isolated and characterized DNA aptamers that can specifically bind to the hemolysin E (HlyE) antigen of Salmonella Typhi for future development of typhoid diagnostic tests. The DNA aptamers against Salmonella Typhi HlyE were isolated using systematic evolution of ligands by exponential enrichment (SELEX), and their binding affinity and specificity were assessed utilizing enzyme-linked oligonucleotide assay (ELONA). A total of 11 distinct aptamers were identified, and the binding affinities and species selectivities of the three most probable aptamers were determined. Kd values were obtained in the nanomolar range, with the highest affinity of 83.6 nM determined for AptHlyE97. In addition, AptHlyE11, AptHlyE45 and AptHlyE97 clearly distinguished S. Typhi HlyE from other tested bacteria, such as Salmonella Paratyphi A, Salmonella Paratyphi B, Shigella flexneri, Klebsiella pneumonia and Escherichia coli, therefore displaying desirable specificity. These novel aptamers could be used as diagnostic ligands for the future development of inexpensive and effective point-of-care tests for typhoid surveillance, especially in developing countries of the tropics and subtropics.

Rapid generation and characterization of recombinant HCoV-OC43-VR1558 infectious clones expressing reporter Renilla luciferase

Viral infectious clones (ICs) serve as robust platforms for studying viral biology and screening antiviral agents using reverse genetics. However, the molecular profiles and complex limitations of human coronaviruses (HCoVs) pose a challenge to ICs development. In this study, we report a novel platform to develop the ICs for HCoV-OC43-VR1558 using a one-step assembly method in yeast by transformation-associated recombination (TAR) technology. Recombinant HCoV-OC43-VR1558, named as rOC43(1558)-WT, was rapidly generated by TAR. In addition, recombinant HCoV-OC43-VR1558-expressing reporter genes, named as rOC43(1558)-ns2FusionRluc, was also generated based on TAR by inserting the ns2 region of the IC with Renilla luciferase (Rluc). We further characterized their replication through virus titration using 50% tissue culture infective dose (TCID50) and indirect immunofluorescence assay (IFA), luciferase reporter assay, and western blotting (WB) assay. The genetic stability of the recombinant HCoV-OC43 was assessed through viral genome sequencing following passaging in BHK-21 cells. These reporter viruses were validated as screening tools for inhibitors in vitro by evaluating the antiviral activities of remdesivir and chloroquine. The phenotypes of HCoV-OC43-VR1558 and HCoV-OC43-VR759 were compared in vitro and in vivo. The TAR-based one-step assembly of IC was successfully applied, facilitating the rapid generation of recombinant HCoV-OC43 and providing a useful platform for the investigation of biological mechanisms, development of vaccines and diagnostic tests, and screening inhibitors of HCoVs.

The concept of nociplastic pain-where to from here?

Nociplastic pain, a third mechanistic pain descriptor in addition to nociceptive and neuropathic pain, was adopted in 2017 by the International Association for the Study of Pain (IASP). It is defined as "pain that arises from altered nociception" not fully explained by nociceptive or neuropathic pain mechanisms. Peripheral and/or central sensitization, manifesting as allodynia and hyperalgesia, is typically present, although not specific for nociplastic pain. Criteria for possible nociplastic pain manifesting in the musculoskeletal system define a minimum of 4 conditions: (1) pain duration of more than 3 months; (2) regional, multifocal or widespread rather than discrete distribution of pain; (3) pain cannot entirely be explained by nociceptive or neuropathic mechanisms; and (4) clinical signs of pain hypersensitivity present in the region of pain. Educational endeavors and field testing of criteria are needed. Pharmacological treatment guidelines, based on the three pain types, need to be developed. Currently pharmacological treatments of nociplastic pain resemble those of neuropathic; however, opioids should be avoided. A major challenge is to unravel pathophysiological mechanisms driving altered nociception in patients suffering from nociplastic pain. Examples from fibromyalgia would include pathophysiology of the peripheral as well as central nervous system, such as autoreactive antibodies acting at the level of the dorsal root ganglia and aberrant cerebral pain processing, including altered brain network architecture. Understanding pathophysiological mechanisms and their interactions is a prerequisite for the development of diagnostic tests allowing for individualized treatments and development of new strategies for prevention and treatment.

Clinicopathological biomarker patterns, venom detection and venom proteomics in canine Vipera berus envenomation

Vipera berus (V. berus) bites are associated with high morbidity, including kidney injury, in dogs. Although antivenom is often used and perceived effective to treat this type of snakebite, it is costly and associated with adverse events and specific diagnostics for this type of snakebite are lacking. We sought to improve diagnostics in V. berus envenomation by using currently available tools, including evaluating urinary albumin as a biomarker for snakebite-associated kidney injury. Additionally, we planned to adapt a method from human medicine for venom detection in clinical samples from bitten dogs and describe the composition of Norwegian V. berus venom. Serum biochemical analytes and urine albumin (ELISA) were measured in samples collected at 24 hours and two weeks after bite in 29 envenomated dogs. An adapted ELISA was applied to detect venom in urine and plasma collected from 25 cases between presentation and 24 hours after bite, using a commercial antivenom as the capture and detection antibody. Proteomic analysis of venom collected from 11 V. berus was performed using liquid chromatography-tandem mass spectrometry. Elevated serum C-reactive protein, creatine kinase, and aspartate aminotransferase were common for the case group. Although no case dogs showed acute kidney injury with azotemia and/or reduced urine output, elevated urinary albumin concentrations may indicate early or mild kidney injury in some case dogs. The venom ELISA detected positive signals in both plasma and urine for up to 24 hours after bite. However, with false positives detected in plasma, urine seemed to be the most appropriate body fluid for this assay. The venom proteome identified L-amino acid oxidases as the dominant component. In conclusion, serum biochemical and urinary albumin analyses are useful tools for evaluating canine V. berus envenomation. The venom ELISA is proposed as a promising tool for studies of V. berus envenomation and future diagnostic test development. Venom from the studied Norwegian V. berus was shown to differ considerably from previous reports from other countries, implying geographical variation in composition.

Development of Three-Tier Diagnostic Test to Measure Misconceptions of Grade 5 Students about Fractions

This research developed a measuring instrument in the form of a three-tier diagnostic test to measure students' misconceptions in fraction material in grade V. This research aims to determine (1) the validity, reliability, and item characteristics of the three-tier diagnostic test and (2) the level of misconceptions of grade V elementary school students in Pontianak. This research used quantitative approach and test development model by Mardapi, that has nine steps. The subjects were fifth-grade students in Pontianak City, and the developed test instruments focused on addition-subtraction of fractions, each with two sets of questions A and B. The instrument were analyzed for content and construct validity, reliability, and item characteristics based on IRT using Partial Credit Model (PCM). The results show that the developed test instrument demonstrated strong content and construct validity. Reliability analysis shows high reliability, with Cronbach's Alpha values >0,7. Item fit analysis reveal that 91% and 73% were fit, therefore the unfit items were fixed or eliminated. The analysis of misconceptions shows that 39.52% of students do not understand the concept of fractions’ addition and subtraction, and 29.09% experienced misconceptions. Additionally, 12.61% have false positives, and 8.97% have false negatives. Only 6.30% understood the concept correctly, and 3.52% guessed correctly or understood but had doubts. Generally, the final test instrument demonstrated good validity, reliability and item qualities.

A-311 Evaluating the Predictive Value of the sFlt-1/PlGF Ratio in Hypertensive Pregnancies for Preeclampsia

Abstract Background Preeclampsia (PE) is a hypertensive pregnancy disorder affecting around 5% of all pregnancies, and it continues to be a leading cause of maternal and neonatal morbidity and mortality in the United States. The incidence of hypertensive pregnancies is on the rise, attributed to the increasing prevalence of obesity and chronic hypertension. The development of a straightforward diagnostic test to accurately differentiate PE from gestational or chronic hypertension is crucial to avoid unnecessary hospitalizations. Despite the ongoing development of prediction models for PE, conventional clinical and laboratory measures have not been effective in predicting disease progression. Recent research has focused on the ratio of two serum biomarkers: soluble fms-like tyrosine kinase-1 (sFlt-1) and placental growth factor (PlGF), particularly in patients with a clinical suspicion of PE in acute settings. These studies have shown promise, with many indicating that the sFlt-1/PlGF ratio can effectively predict the presence or absence of PE. This study was conducted to assess the predictive value of the sFlt-1/PlGF ratio in pregnancies complicated by hypertension, aiming to contribute to the early detection and management of PE. Methods Pregnant individuals with hypertensive disorders at or above the age of 18 receiving prenatal care at NewYork-Presbysterian-Weill Cornell Hospital were enrolled in this study. We measured the levels of sFlt-1 and PlGF using fully automated Elecsys immunoassays (Roche Diagnostics). Subsequently, a prospective analysis of the serum sFlt-1/PlGF ratio was performed. A receiver operating characteristic (ROC) curve was used to calculate the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the ratio in predicting PE. To assess the prognostic accuracy of the sFlt-1/PlGF ratio, patients were randomly assigned to a development and a validation cohort at a ratio of 30:70. Results We collected a total of 137 serum samples of hypertensive pregnancies, with all ≥20 weeks of gestational age (GA). Of these, 47 (34.3%) developed PE within 2 weeks. The samples were divided into two cohorts: 98 were included in the development cohort, and 39 in the validation cohort. Within these groups, 34 (34.7%) of the development cohort and 13 (33.3%) of the validation cohort developed PE within 2 weeks, respectively. In the development cohort, the median sFlt-1/PlGF ratio was 85 (Interquartile range, [IQR], 44-164) among pregnant women who developed PE within 2 weeks. This was significantly higher compared to a median ratio of 8 (IQR, 3-35) observed in those who did not develop PE (p<0.001). The area under the ROC curve was 0.87, indicating good discriminative ability. An optimized cutoff ratio of 38 was determined, based on the max of combined sensitivity and specificity. When applied into the validation cohort, this cutoff yields a 90% (95% confidence interval [CI], 70-97) NPV and 58% (95%CI, 37-76) PPV with an 85% sensitivity (95%CI, 58-96) and a 69% specificity (95%CI, 50-83). Conclusions In women with a hypertensive disorder of pregnancy presenting GA≥20 weeks of gestation, the measurement of serum sFlt-1/PlGF ratio demonstrated clinical value in predicting the development of PE.

A New Clinical Test to Evaluate Intrinsic Foot Muscle Function.

Intrinsic foot muscle weakness has been implicated in a wide range of forefoot conditions and is typically measured by the paper grip test. The proposed "knuckle test" is intended to evaluate intrinsic foot muscle function, in which active flexion of toes makes the metatarsal heads ("knuckles") prominent in the forefoot. This study seeks to compare prevalence of negative knuckle test results and intrinsic muscle weakness in patients, primarily by association with a positive paper grip test result and secondarily with presence of toe deformities. Ninety-eight patients, 55 with toe deformity and 43 without, were evaluated for ability to flex their toes to the extent of knuckle prominence (knuckle test) and grip paper on the ground with their toes (paper grip test). Variables were compared between those with positive and negative knuckle test results. A negative knuckle test result is significantly associated with the inability to grip a paper strip (P < .05). Given that the paper grip test outcomes are a well-established sign of intrinsic muscle weakness, the significant association between knuckle and paper grip test outcomes substantiates the use of the knuckle test to detect intrinsic muscle weakness. The knuckle test is a potentially quick, simple, and cost-effective clinical maneuver to indicate intrinsic foot muscle weakness in a patient. Level III, diagnostic test development.

The epithelial era of asthma research: knowledge gaps and future direction for patient care.

The Epithelial Science Expert Group convened on 18-19 October 2023, in Naples, Italy, to discuss the current understanding of the fundamental role of the airway epithelium in asthma and other respiratory diseases and to explore the future direction of patient care. This review summarises the key concepts and research questions that were raised. As an introduction to the epithelial era of research, the evolution of asthma management throughout the ages was discussed and the role of the epithelium as an immune-functioning organ was elucidated. The role of the bronchial epithelial cells in lower airway diseases beyond severe asthma was considered, as well as the role of the epithelium in upper airway diseases such as chronic rhinosinusitis. The biology and application of biomarkers in patient care was also discussed. The Epithelial Science Expert Group also explored future research needs by identifying the current knowledge and research gaps in asthma management and ranking them by priority. It was identified that there is a need to define and support early assessment of asthma to characterise patients at high risk of severe asthma. Furthermore, a better understanding of asthma progression is required. The development of new treatments and diagnostic tests as well as the identification of new biomarkers will also be required to address the current unmet needs. Finally, an increased understanding of epithelial dysfunction will determine if we can alter disease progression and achieve clinical remission.

Limitations and perspectives of the novel salivary test for endometriosis: an open web-based survey study of German gynecologic healthcare providers.

The description of a salivary miRNA signature for endometriosis has led to the development of a non-invasive diagnostic test. Current healthcare provider practices regarding the test remain uncaptured. The application of this test in practice was examined in a web-based survey, with the aim to provide their opinions on it. We conducted an open web-based survey study between November 2023 and January 2024. Members of the German society of gynecologic endoscopy (Arbeitsgemeinschaft gynäkologische Endoskopie, AGE), society of endometriosis (Arbeitsgemeinschaft Endometriose, AGEM), and the endometriosis research foundation (Stiftung Endometriose Forschung, SEF) were contacted per e-mail twice. Participants' data were anonymized. Differences in responses based on self-reported expertise in the field (basic knowledge, specialized knowledge, expert) were assessed using the χ2-test or Fisher's exact test. Statistical significance was set as p < 0.05. In total 141 of 190 respondents completely responded to the survey (> 75% of the questions of the survey). Twenty-one physicians reported having experience with the test, while most participants had at least specialized knowledge on the field (112/141). In terms of specific questions, more than 90% found the costs high; almost 85% did not believe that the test replaces standard diagnostic tools (histology, clinical examination, and sonography). Eighty-six providers supported the use of the test in adolescents. Gynecologists with basic knowledge had a more positive attitude compared with more experienced ones in terms of usefulness (Fisher's exact test, p < 0.001). Significant differences were demonstrated between expertise groups regarding (not only) applicability in adolescents (Fisher's exact test, p = 0.004), and using the test for screening purposes (χ2-test, p = 0.002). Despite the promising benefits of a salivary test for endometriosis, German healthcare providers would not change current practices. Nevertheless, less experienced colleagues were more positive towards the test.

Abstract B023: Unravelling fibroinflammatory and immune signatures for pancreatic cancer early detection

Abstract Pancreatic cancer is associated with late-stage diagnosis and poor patient prognosis. The disease is characterised by chronic dysregulated immunity and inflammation. Moreover, chronic pancreatitis is linked with an increased risk of pancreatic cancer. Tissue remodelling during disease progression involves a complex network of cells and a crosstalk between cancer cells, stromal components and immune cells, but it remains poorly explored. Thus, to improve the outcome of patients, there is a need for better understanding of biological changes associated with the initiation and progression of pancreatic cancer at the molecular and spatial levels. Using samples obtained from our Accelerated Diagnosis of neuroEndocrine and Pancreatic TumourS (ADEPTS) biobank, we have evaluated blood samples for the discovery of non- invasive biomarkers to discriminate patients with pancreatic cancer from symptomatic patients without cancer. In this pilot study, the transcriptome of peripheral blood mononuclear cells (PBMCs), isolated using a ficoll gradient was explored to identify PDAC-associated mRNA signatures. Moreover, the Olink® Explore Inflammation and Oncology panels were performed in a cohort of 40 patients with early stage (I-II) PDAC, 40 chronic pancreatitis samples and 40 control patients with benign symptomatic upper GI diseases. To validate and enrich these results, a panel of inflammatory and immune markers including immunoglobulins, cytokines and chemokines was also run using multiplex techniques. Symptoms observed in our cohort were also analysed together with clinical (e.g diabetic status, biochemistry profile) and demographic data (age, sex, ethnicity, post code etc). We have identified mRNA transcriptome signatures and differentially expressed proteins associated with inflammatory and immunological processes in PDAC compared to benign symptomatic samples. Our results offer potential diagnostic utility for early-stage disease. Additionally, our unique cohort of patient samples comprising of those with confirmed PDAC and those presenting to clinic with benign disease and similar non-specific symptoms, allows for the development of non-invasive diagnostic tests with better translational applicability. Future work will involve the validation of these findings in a larger patient cohort and the use of NanoString GeoMx® Digital Spatial Profiler to perform whole transcriptome and proteome profiling in human chronic pancreatic and pancreatic cancer tissue samples. Citation Format: Kyra Fraser, Maria Rosado, Stephen P Pereira, Pilar Acedo. Unravelling fibroinflammatory and immune signatures for pancreatic cancer early detection [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B023.

Diagnostic Performance of Thyroid FNA: Experience of Belarusian National Referral Thyroid Cancer Center.

A fine-needle aspiration (FNA) biopsy is a basic procedure to verify the morphology of thyroid nodules. However, significant variations in the diagnostic performance of FNA, as well as fluctuations in the rate of malignancy (ROM) in different categories of the Bethesda System for Reporting Thyroid Cytopathology, have been discovered in many studies. The development and employment of additional molecular tests in daily clinical practice require an understanding of the prevalence and structure of thyroid malignancy in each category of the Bethesda system. Current research is a continuous retrospective cohort study of the results of the examination and treatment of 1652 patients with thyroid tumors who have undergone primary surgery in 2021 at a national referral thyroid cancer center. FNA diagnostic performance was studied by comparing cytological diagnoses with histological outcomes. The authors evaluated ROM in each Bethesda category and identified the tumors representing a diagnostic pitfall. The overall ROM was 72.4%. When considering ROM in each Bethesda category, it was 16.7% in Bethesda I, 10.1% in Bethesda II, 23.0% in Bethesda IV, 74.8% in Bethesda V, 99.4% in Bethesda VI. The false-positive rate was 65.0%, false negative-0.67%. Follicular and oncocytic adenomas, as well as the follicular variant of papillary thyroid cancer, were the most troublesome entities for patients' management. ROM was comparable to the values implied by the Bethesda system. Follicular tumors were the main source of high false-positive rates. This necessitates the refinement of existing tests and the development of new diagnostic tests to overcome the abovementioned problems.

387. VALIDATION OF PROMARKERESO, A DIAGNOSTIC BLOOD TEST TO IDENTIFY ESOPHAGEAL ADENOCARCINOMA

Abstract Background Esophageal adenocarcinoma (EAC) has become the predominant type of esophageal cancer in Western countries. Early diagnosis and intervention of EAC significantly reduces the disease mortality. A previous study identified a panel of novel serum glycoprotein biomarkers that were associated with EAC, enabling the development of a prototype diagnostic blood test, PromarkerEso. The current study aimed to validate PromarkerEso in an independent cohort of patients with EAC. Methods Blood samples from an independent cohort of 165 participants (n=99 negative controls, n=66 diagnosed with EAC) were analyzed. Five candidate biomarkers were measured after lectin pulldown using a targeted mass spectrometry assay. Model performance was assessed by the area under the receiver operating characteristics curve (AUC-ROC) with sensitivity and specificity determined. Results After adjusting for clinical variables, diagnostic models showed good discrimination to distinguish participants with EAC from negative control participants. Conclusions This study validates a panel of serum glycoprotein biomarkers for the diagnosis of EAC. These biomarkers have potential utility as a simple blood test, PromarkerEso, that could be used for screening EAC in a large population.

Purification and characterization of soluble recombinant Crimean-Congo hemorrhagic fever virus glycoprotein Gc expressed in mammalian 293F cells

BackgroundCrimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonotic disease that presents with severe hemorrhagic manifestations and is associated with significant fatality rates. The causative agent, Crimean-Congo Hemorrhagic Fever Virus (CCHFV), is a high-priority pathogen identified by the World Health Organization with no approved vaccine or specific treatment available. In addition, there is a critical need for enhanced diagnostic tools to improve public health awareness, prevention measures, and disease control strategies.MethodsWe designed plasmids to enable the purification of soluble CCHFV glycoprotein Gc expressed in mammalian 293 F cells, followed by purification using affinity and size exclusion chromatography. The purified antigen was analyzed by SDS-PAGE and Western blotting to confirm its reactivity to antibodies from CCHF survivors. Additionally, an in-house indirect ELISA was developed using the purified Gc as a coating antigen.ResultsThe optimized expression system successfully produced soluble and pure Gc antigen after affinity chromatography. The protein showed specific reactivity with CCHFV-positive serum antibodies in Western blot analysis. The indirect ELISA assay demonstrated high efficacy in distinguishing between CCHFV-positive and -negative serum samples, indicating its potential as a valuable diagnostic tool. Size exclusion chromatography further confirmed the presence of aggregates in our protein preparation.ConclusionsThe purified Gc antigen shows promise for developing direct diagnostic assays for CCHFV. The antigen’s suitability for subunit vaccine development and its application as bait for monoclonal antibody isolation from survivors could be investigated further. This work lays the foundation for future research into the development of rapid diagnostic tests for field deployment.