Clinical Samples Research Articles

Psychometric Validation of the New Misophonia Screening List-Child and Youth and AMISOS-Y (Dutch Child- and Parent-Report Versions) for Assessing Misophonia in Youth.

Misophonia is a recently identified disorder of decreased sound tolerance that often originates in childhood. Currently, there is a lack of validated questionnaires for screening and assessing misophonia severity in children/adolescents. This paper presents an iterative validation process of two innovative (parallel child-/parent-reported) questionnaires: the Misophonia Screening List-Child and Youth for screening, and the Amsterdam Misophonia Scale-Youth (AMISOS-Y) for assessing misophonia severity in youth. After instrument refinement, we performed ROC curve, reliability, and principal component analyses, and assessed concurrent, convergent and divergent validity, on a combined sample (aged 8-18; clinical sample N = 94 youth, 95 parents; control group screening N = 197 youth, 56 parents; control group AMISOS-Y N = 192 youth, 55 parents). Both questionnaires were unidimensional and displayed excellent psychometric properties (α = 0.95-0.96). Future replication studies are needed in community and clinical samples to contribute to a unified diagnostic framework. Trial registered 09/2021: NL-OMON20775.

Antibiotic resistance and serotype distribution of Shigella strains in Bangladesh over the period of 2014-2022: evidence from a nationwide hospital-based surveillance for cholera and other diarrheal diseases.

This nationwide study in Bangladesh assessed Shigella infections from 2014 to 2022 from clinical samples. S. flexneri was predominant, with concerning antibiotic resistance, notably to ciprofloxacin and nalidixic acid in over 96% of isolates. This emphasizes the urgency of prudent antibiotic use and improved hygiene. The findings provide crucial antimicrobial resistance patterns of Shigella species, highlighting the need for ongoing resistance monitoring and potentially informing future vaccine trials.

Rapid Dynamic Surface-Enhanced Raman Spectroscopy Detection of Amoxicillin-Mediated Morphological Changes in a Pathogen for Diagnosis of Clinical Urine Samples.

The swift and stable detection of pathogens in urine samples holds significant implications for the immediate clinical diagnosis and treatment of urinary tract infections (UTIs). In this study, we propose a detection strategy utilizing a hybrid substrate composed of graphene oxide (GO) and silver nanoparticles (Ag NPs) for the detection of pathogens subjected to amoxicillin-mediated (amo-mediated) treatment. This strategy employs dynamic surface-enhanced Raman spectroscopy (D-SERS) for stable and rapid detection, capturing signal variations induced by amo-mediated changes in pathogen morphology. During the 5 min D-SERS detection time window, stable SERS signals were detected for three types of pathogens and four types of pathogens were successfully distinguished using principal component analysis (PCA). In comparison to conventional nanosubstrates, the GO/Ag NP hybrid substrate exhibits outstanding stability and enhancement effects. This approach enables the dual detection of the pathogen cell structure and metabolites, facilitating specific identification of pathogens in the urinary tract, with a detection limit for Escherichia coli reaching 1 × 104 colony-forming units (CFU)/mL, meeting the clinical microbiology laboratory diagnostic standards for UTIs (105 CFU/mL). Testing of 188 clinically collected urine samples using this strategy yielded a sensitivity (SENS) of 86.4% and a specificity (SPC) of 89.7%. This introduces a novel method for diagnosing UTIs, offering broad applications in the field of clinical pathogen detection.

Distinction of clinical features and microbiological methods between Chlamydia psittaci and Legionella pneumophila pneumonia confirmed by metagenomic next-generation sequencing

Objectives Detection and diagnosis of Chlamydia psittaci (C. psittaci) pneumonia is often overlooked due to conventional methods limitations and similarity to other atypical community acquired pneumonia (CAP). Using mNGS, we aimed to distinguish psittacosis from legionellosis for early C. psittaci pneumonia diagnosis and better prognosis. Methods Thirty-seven patients diagnosed with atypical CAP were enrolled in this retrospective study, including 14 C. psittaci pneumonia and 23 Legionella pneumophila (L. pneumophila) pneumonia. We collected and compared baseline, lab results, radiology imaging, conventional microbiological methods and more importantly, mNGS results of clinical samples, as well as the treatments and prognosis between psittacosis and legionellosis. Results Patients with C. psittaci and L. pneumophila had similar symptoms and were presented with high levels of inflammatory markers. However, patients with C. psittaci pneumonia were more likely to have exposure to birds or parrots [11 (78.6%) vs. 2 (8.7%), p < 0.001], had higher proportions of fever and chill (p = 0.015 and 0.035), higher levels of hemoglobin and albumin (p = 0.002 and 0.018) compared with those with L. pneumophila. Of 14 C. psittaci patients, only one had positive IgM antibody, with no positive cultures. Early identification of pathogens by mNGS method contributed to timely antibiotics’ adjustment and better outcomes then, yet with similar hospital mortality between two groups [7.1% (1/14) vs. 34.8% (8/23), p = 0.112]. Conclusion Early mNGS detection of atypical pathogens in multiple samples improves on traditional methods, promptly adjust empirical antimicrobial treatment to pathogen-targeted antibiotics, further improve prognosis.

Grazing and food addiction: Associations between varied patterns of overconsumption and addictive-like eating.

Food addiction (FA) research has primarily concentrated on binge eating as a symptom of this condition. However, FA may encompass various overeating behaviours, including compulsive grazing - the repetitive consumption of small amounts of food with loss of control. This study extends our previous research by including a clinical sample to investigate whether compulsive grazing exists in a population with higher prevalence and severity of FA. It also examines whether weight or sex moderates the severity or frequency of FA. Adults between the ages of 20 and 50 years with obesity were recruited from a weight-management clinic (n=437). Participants completed an online questionnaire to assess various eating behaviours (binge eating, compulsive grazing, reward-based eating) and related personality measures (impulsivity, addictive personality traits). Multiple and logistic regression modelling were used with the Yale Food Addiction Scale symptom count and diagnosis as the dependent variables, respectively. A chi-square test of independence and an analysis of variance were used to determine sex differences. Compulsive grazing was a significant factor in FA diagnosis and symptom severity, confirming earlier work. Binge eating was a significant factor in FA symptom severity. Additionally, females from a weight-management clinic sample were more likely to receive an FA diagnosis than females from the general population. No sex differences were found in males or in the symptom score option for FA. These findings suggest that various compulsive overeating patterns encompass FA. Individuals with obesity and co-morbid FA may require specialized treatments, which may benefit from sex-specific differentiations.

A Systematic Review of Parent Socialization of Negative Affect in Clinical Child Samples: Relations to Youth Emotion Regulation Abilities.

Emotion-focused parenting practices, known as parent emotion socialization, play a crucial role in shaping youth's emotion regulation (ER) abilities. The impact of parent emotion socialization behaviors (ESB) on youth ER has been studied extensively in community samples. However, research on these relations in clinical samples is more limited, albeit growing. The current systematic review sought to evaluate the existing literature examining parent ESB of negative affect in various clinical child and adolescent samples. A literature search was conducted in April 2023, resulting in 1153 abstracts being reviewed. Two reviewers independently screened the titles and abstracts to identify relevant papers, with 152 articles being independently reviewed in full, of which 26 articles met inclusion criteria and are included in the current review. Studies (16 cross-sectional, 6 longitudinal, 4 intervention) utilized multi-methods of assessing youth ER, including questionnaires, physiological, and observational measures in a range of clinical samples, including youth diagnosed with internalizing, externalizing, and neurodevelopmental disorders, as well as youth at-risk for clinical diagnoses such as physically abused children. Overall, results support significant, negative associations between parent ESB practices and youth emotion dysregulation (rs = .22-.35) and negativity/lability (rs = .19-.60), and positive associations with youth ER abilities (rs = .18-.76). Some studies highlighted varying impacts of parental ESB on children with versus without clinical disorders/symptomology, with effects being more pronounced within clinical populations. Implications for these findings and future research directions are discussed, including the significance of focusing on parent ESB in intervention work with clinical child populations.

TC-Sniffer: A Transformer-CNN Bibranch Framework Leveraging Auxiliary VOCs for Few-Shot UBC Diagnosis via Electronic Noses.

Utilizing electronic noses (e-noses) with pattern recognition algorithms offers a promising noninvasive method for the early detection of urinary bladder cancer (UBC). However, limited clinical samples often hinder existing artificial intelligence (AI)-assisted diagnosis. This paper proposes TC-Sniffer, a novel bibranch framework for few-shot UBC diagnosis, leveraging easily obtainable UBC-related volatile organic components (VOCs) as auxiliary classification categories. These VOCs are biomarkers of UBC, helping the model learn more UBC-specific features, reducing overfitting in small sample scenarios, and reflecting the imbalanced distribution of clinical samples. TC-Sniffer employs intensity-based augmentation to address small sample size issues and focal loss to alleviate model bias due to the class imbalance caused by auxiliary VOCs. The architecture combines transformers and temporal convolutional neural networks to capture long- and short-range dependencies, achieving comprehensive representation learning. Additionally, feature-level constraints further enhance the learning of distinctive features for each class. Experimental results using e-nose data collected from a custom-designed sensor array show that TC-Sniffer significantly surpasses existing approaches, achieving a mean accuracy of 92.95% with only five UBC training samples. Moreover, the fine-grained classification results show that the model can distinguish between nonmuscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC), both of which are subtypes of UBC. The superior performance of TC-Sniffer highlights its potential for timely and accurate cancer diagnosis in challenging clinical settings.

Analytical and clinical evaluation of a duplex RT-qPCR assay for the detection and identification of o’nyong-nyong and chikungunya virus

The mosquito-borne alphavirus o’nyong-nyong virus (ONNV) has proven its potential to cause major human outbreaks. On the African continent, ONNV causes unspecific febrile illness and co-circulates with the close relative chikungunya virus (CHIKV). The true scale of ONNV burden is poorly understood in Africa, because of the scarce availability of molecular in-house and commercial assays, strong cross-reactivity between ONNV and CHIKV in serological assays and a lack of surveillance. We designed a new RT-qPCR assay targeting the E1 gene for the detection of ONNV that can be used in monoplex or in duplex format, combined with a previously published CHIKV monoplex assay targeting the nsp1. The lower limit of detection with 95% positivity rate was determined to be <10 RNA copies/µL in monoplex and duplex format for both ONNV and CHIKV. Both monoplex assays and the duplex assay proved to be linear within the tested range of approximately 108 to 102 RNA copies/µl, and showed 100% specificity against a wide panel of arbovirus supernatants as well as several other pathogens in clinical samples. Testing of CHIKV positive serum and ONNV spiked plasma samples confirmed the suitability of the assays in a clinical setting. The new assays provide a robust tool for molecular ONNV as well as ONNV/CHIKV simultaneous detection and may contribute to clarify the true burden of the two viruses, to improve arbovirus surveillance and to strengthen epidemics preparedness in Africa.

Abstract A036: Next gen liquid biopsy: Comprehensive analysis from a single tube of blood

Abstract Introduction There remains a largely untapped potential for utilizing liquid biopsy as a non- invasive, real-time window into tumor biology. In this study, we report on the use of the high- multiplex protein quantitation capability of the OrionTM spatial biology platform (RareCyte®, Inc) and cell picking capability of the CyteFinder® rare cell platform, to perform a comprehensive liquid biopsy analysis of whole blood that provides a deeper understanding of samples. This novel technology was used to characterize circulating tumor cells (CTCs), perform immune profiling of white blood cells (WBCs), and perform targeted mutation profiling of CTCs and cell-free DNA (cfDNA). All of these analytes can be measured from one 7.5 mL draw of blood. This array of tests was applied to cancer samples to demonstrate clinical feasibility. MethodsBlood samples were collected into AccuCyte® blood collection tubes. Buffy coats were processed to microscope slides using AccuCyte® blood processing kits. Slides were fixed and stained with an 18-plex immuno-oncology panel and imaged using the Orion Spatial Biology platform to evaluate protein biomarker expression on CTCs and to profile WBCs. Individual CTCs and WBCs from clinical samples were isolated using the CytePicker® for downstream molecular analysis. cfDNA was isolated from plasma collected during the AccuCyte process. Picked CTCs, WBCs, and cfDNA were sequenced using the CleanPlex® OncoZoom® Cancer Hotspot Kit. ResultsCancer patient blood was tested with a high-plex immunofluorescence assay using the Orion spatial biology system. CTCs were identified and phenotypically characterized in multiple cancer types. On the same slides, immunophenotyping of WBCs was performed. T- cell, B-cell, and macrophage populations were quantified. Additional sub-populations of T-cells (regulatory and memory) and proliferating cells (Ki67+) were enumerated. Expression of checkpoint-specific markers PD-L1 and PD-1 was measured on CTCs and WBCs. Sequencing results from individual picked CTCs showed evidence of minor clones representative of tumor heterogeneity that were not detectable in the cfDNA analysis. Conclusions This novel application of a high-plex spatial biology imaging system to liquid biopsies, in conjunction with the proven capabilities of rare cell detection and genomic analysis of single cells and cfDNA, has the potential to revolutionize liquid biopsy testing. From a single blood sample, this approach allows one to ask important questions about the complex interactions between the immune system and the tumor and to follow these interactions longitudinally in real time over the course of the disease. The goal is to leverage this unprecedented depth of analysis to improve patient outcomes and better understand the biological complexity of disease. Future directions of this work are to study rare types of WBCs that indicate immune activation and response to the tumor, and the detection and longitudinal monitoring of cell therapies such as CAR T cells after infusion. Citation Format: Arturo B Ramirez, Jon Ladd, Erin Bayer, Brock Bartels, Rachel Ponting, Arista Tischner. Next gen liquid biopsy: Comprehensive analysis from a single tube of blood [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A036.

Identification and characterization of novel ferroptosis-related genes in acute myocardial infarction.

Acute myocardial infarction (AMI) is a leading cause of death and morbidity worldwide. Ferroptosis, a form of regulated cell death, plays a critical role in modulating immune functions during AMI. This study aimed to identify ferroptosis-related hub genes that could serve as potential therapeutic targets in the progression of AMI. Bioinformatics was used to identify overlapping genes associated with ferroptosis and the infiltration of 22 immune cells by Cell-type Identification by Estimating Relative Subsets of RNA Transcript (CIBERSORT) analysis. The expression of ferroptosis-related genes in AMI was validated across independent datasets, clinical samples, and in vitro cellular experiments. The predictive value for heart failure was evaluated in the first dimension of principal component analysis (PCA) using receiver operating characteristic (ROC) analysis. The study identified 11 key ferroptosis-related genes significantly correlated with immune cell abundance. CIBERSORT analysis highlighted immune dysregulation in AMI. JDP2, DUSP1, TLR4, NFS1, and SLC1A5 were identified as potential biomarkers for AMI progression. Additionally, JDP2, DUSP1, and DDIT4 demonstrated strong predictive value for long-term heart failure. This study highlights the potential association of ferroptosis-related genes with the pathogenesis of AMI, suggesting a role in the molecular mechanisms that may underlie acute coronary events.

NOP10 predicts poor prognosis and promotes pancreatic cancer progression.

Telomere shortening and RNA pseudo-uridylation are common features of tumors. NOP10 is a member of the H/ACA snoRNP family, essential for maintaining telomerase activity and RNA pseudouridylation. NOP10 has been indicated to be substantially expressed in tumors such as breast and lung cancers and is associated with poor prognosis. Currently, no investigation exists on NOP10 in pancreatic cancer (PC). This is the first investigation to elucidate the impact on tumorigenesis and prognostic value of NOP10 in pancreatic adenocarcinoma (PAAD). NOP10 expression and its survival prognostic significance were analyzed via clinical PAAD data from the TCGA database and NOP10 expression in other tumors from the GEPIA database. Furthermore, the NOP10 expression and survival prognosis in clinical samples were validated by qRT-PCR. In-vitro experiments were carried out to elucidate the impact of NOP10 on the biological function of PC cells. It was revealed that NOP10 expression was increased in PC tissues than in the normal pancreatic tissues. High NOP10 expression was markedly linked with poorer prognosis. NOP10 may be involved in focal adhesion, channel activity, cAMP signaling pathway, the interaction of neuroactive ligand-receptor, and cell adhesion molecules cams. NOP10 was associated with the tumour immune microenvironment and drug sensitivity. Down-regulation of NOP10 expression suppressed PC cells' ability to proliferate, migrate, and invade. This investigation elucidated the prognostic and predictive importance of NOP10 in PAAD and revealed that NOP10 is associated with poor prognostic features, survival prognosis and TIME. Knockdown of NOP10 inhibits the progression of PAAD.

Single-Molecule Detection of Serum MicroRNAs for Medulloblastoma with Biphasic Sandwich Hybridization-Assisted Plasmonic Resonant Scattering Imaging.

MicroRNA (miRNA) dysregulation is closely related to the occurrence and progression of medulloblastoma (MB). However, the full potential of serum circulating miRNAs in MB diagnosis is restricted by their ultralow abundance in peripheral blood due to blood-brain barrier. Here, we report the direct preamplification-free detection of aberrant expression of oncogenic miRNAs in serum from MB patients by proposing a simple yet robust single-molecule assay that combines biphasic sandwich hybridization in nucleic acids and the dark-field single-particle plasmonic imaging (B2S2PI). In this strategy, signal DNA was prehybridized with target miRNA in homogeneous solution to form sDNA-RNA complexes. Then the captured DNA strands with rationally adjusted surface densities could efficiently capture the sDNA-RNA complexes to generate a well-separated DNA-RNA sandwich structure. The combination of homogeneous and heterogeneous reactions enabled interface-mediated hybridization reactions to maintain molecular stability with fewer bases, making it suitable for the direct amplification-free assays of short miRNA targets. Labeling the DNA-RNA hybrids with plasmatic gold nanotags allowed nondestructive recognition and imaging of individual miRNA targets under mild conditions with high signal-to-noise ratio. By digitally counting and analyzing the bright plasmonic resonant scattering spots, B2S2PI enabled both the measurement of a low femtomolar concentration of circulating miRNA-21 in 5 μL sample volume within a turnaround of 2 h and the discrimination of single base mismatches. Moreover, B2S2PI was universal for detecting miRNAs with different sequences and secondary structures. Further analysis of clinical serum samples revealed that B2S2PI was capable of accurately distinguishing MB patients from noncancer controls with an area under the curve (AUC) of 0.99, which was superior to that of qRT-PCR. B2S2PI holds promise as a novel alternative means for single-molecule miRNA assay and sheds light on the circulating nucleic acid-based liquid biopsy of intracranial malignant tumors.

Conducting polymer functionalized Cu-metal organic framework-based electrochemical immunosensor for rapid and sensitive quantitation of Escherichia coli O157:H7.

Escherichia coli (E. coli) O157:H7 is an important food-borne pathogen that can cause hemorrhagic diarrhea and enteritis in humans and animals. Realizing the rapid quantitation of E. coli O157:H7 is of great significance for the guarantee of food safety and disease control. In this study, an electrochemical immunosensing technique based on a functionalized composite of Cu-metal organic framework (Cu-MOF) and poly (3,4-ethylenedioxythiophene)-poly (styrenesulfonate) (PEDOT:PSS) is developed, achieving rapid and sensitive quantitation of E. coli O157:H7 in food and clinical feces samples. The organic functionalization of Cu-MOF significantly improves the interface conductivity to facilitate electron transfer and provides the sulfonic groups (-SO3H) to conjugate bio-recognizing elements for target determination. The immunosensor delivers a linear detection range of 3 × 102 ~ 3 × 108cfu/mL, a low limit of detection (LOD) of 7.4cfu/mL, and a short analysistime of 40min. In addition, it does not show any cross-reactivity with other common pathogens and exhibits high repeatability with relative standard deviations (RSDs) all lower than 2.09%, providing a promising approach for warranting food safety and control of E. coli O157:H7 disease.

Multi-center study on the application potential of Siaα-2,6Gal in early and differential diagnosis of lung cancer.

This study aimed to investigate the application potential of the abnormal glycan structure Siaα-2,6Gal in the early and differential diagnosis of lung cancer. Clinical data and serum samples from 730 patients and 120 healthy individuals participating in clinical trials on Siaα-2,6Gal were collected at three medical centers between January 2022 and June 2023. The levels of Siaα-2,6Gal, carcinoembryonic antigen (CEA), cytokeratin 19 fragment antigen (CYFRA21-1), squamous cell carcinoma antigen (SCC), neuron-specific enolase (NSE), and pro-gastrin-releasing peptide (ProGRP) in serum were measured. The application potentials of these markers in the early and differential diagnosis, classification, and staging of lung cancer were explored. (1) Serum Siaα-2,6Gal levels in the lung cancer group were 2,606 (1,970-3,458) U/mL, significantly higher than those in the benign lung disease, miscellaneous malignant tumor, miscellaneous benign disease, and healthy individual groups at 1,359 (950-1,528), 1,252 (903-1,532), 1,196 (850-1,490), and 1,210 (1,100-1,287) U/mL (P < 0.0001). (2) Serum Siaα-2,6Gal levels in the early-stage lung cancer (stages 0-II) group were 2,576 (1,929-3,338) U/mL, significantly higher than those in the benign pulmonary nodule group at 1,419 (1,105-1,820) U/mL (P < 0.0001). (3) Receiver operating characteristic curves showed that Siaα-2,6Gal had a high diagnostic efficiency for lung cancer (area under the curve (AUC) = 0.9217), significantly superior to CEA, CYFRA21-1, SCC, NSE, and ProGRP (AUCs of 0.6618, 0.6605, 0.5783, 0.5985, and 0.6381). Siaα-2,6Gal is a promising biomarker for lung cancer diagnosis and may offer superior differential diagnosis of early-stage lung cancer from benign pulmonary nodules compared to traditional tumor markers.

Periodontitis promotes tumor growth and immune evasion via PD-1/PD-L1.

Our study investigated the role of experimental periodontitis on tumor growth, local and systemic immunosuppressive status, and programmed death receptor 1 (PD-1) / programmed death ligand 1 (PD-L1) expression in oral squamous cell carcinoma (OSCC) and prostate cancer. Mouse oral or prostate cancer xenograft models were divided into control, periodontitis and periodontitis + anti-PD-1 groups. Tumor volume and weight were recorded and the levels of relevant immune-suppressive cells and T cells were detected by flow cytometry or immunofluorescence. THP-1 cells were stimulated using conditioned media of LPS-stimulated Cal-27 cells and PD-L1 expression was measured by quantitative real-time PCR, western blotting and immunofluorescence. Tumor specimens from OSCC patients with or without periodontitis were also collected for immunofluorescence. Periodontitis significantly promoted tumor volume and weight. Compared to the control, the proportions of tumor-associated macrophages (TAMs), regulatory T cells (Tregs), PD-L1+TAMs and PD-1+CD8+T cells increased, while CD8+T cells decreased in the periodontitis group. Immunofluorescence demonstrated that there was an increase in PD-L1+TAMs and PD-1+CD8+T cells, but a decrease in IFN-γ+CD8+T cells in both xenografts and clinical OSCC samples with periodontitis. In vitro, LPS-stimulated Cal-27 cells had a stronger potential to induce PD-L1 expression in macrophages compared with unstimulated Cal-27 cells. And the promoting effect of periodontitis on tumor growth and immune evasion was significantly attenuated after anti-PD-1 therapy. Periodontitis may facilitate tumor growth and immune escape evidenced by the increased immune-suppressive cells and the decreased functional T cells, via enhancing PD-1/PD-L1 expression in the tumor microenvironment.

Abstract A040: Using mitochondrial abundance in tumor-derived extracellular vesicles for pancreatic cancer screening

Abstract Pancreatic cancer, marked by a high mortality rate and rising incidence, often goes undetected until advanced stages, complicating treatment. Tumor-derived extracellular vesicles (EVs) have emerged as promising cancer biomarkers due to their cargo from parent cells. However, current detection methods are often time-consuming, expensive, and complex. To overcome these challenges, we developed a screening assay for pancreatic cancer that uses the mitochondrial DNA to nuclear DNA ratio in tumor-derived EVs as a distinguishing feature. This assay combines immunoprecipitation with qPCR quantification for direct detection of tumor-derived EVs from serum, utilizing DNA-isolation-free techniques and duplexing probes for qPCR, thus saving at least 3 hours. Clinical sample testing demonstrated the assay's potential as a translational tool for cancer screening, showing a weak correlation with prognosis biomarkers and sufficient discriminatory power between healthy controls, pancreatitis, and pancreatic cancer cases. Citation Format: Dali Sun. Using mitochondrial abundance in tumor-derived extracellular vesicles for pancreatic cancer screening [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A040.

Prognostic role of Androgen Receptor splice variant 7 (AR-V7) in the pathogenesis of breast cancer.

The Androgen Receptor (AR) has emerged as an endocrine therapy target in Breast Cancer, exhibiting up to 80% expression in clinical cases. AR-V7, a constitutively activated splice variant of AR with a truncated ligand-binding domain (LBD), demonstrates ligand-independent transcriptional activity and resistance to nonsteroidal antiandrogens like Bicalutamide or Enzalutamide, targeting the LBD. In metastatic prostate cancer, elevated AR-V7 levels lead to therapeutic resistance and increased metastasis. In this study, we evaluated the expression of AR and AR-V7 in cell lines and a cohort of 89 patients undergoing surgical intervention for treatment-naïve breast cancer. Further clinicopathological correlations and survival analysis were performed to evaluate the relationship between the AR and AR-V7 expression and clinical outcomes. AR-V7/AR-FL ratio was elevated in the TNBC cell line and downregulation of AR-FL upon AR antagonists' treatment led to a compensatory increase in AR-V7. Clinical samples showed significantly elevated expression of AR and AR-V7 in tumors compared to control cases. Further clinicopathological correlation revealed aggressive clinical traits, higher pathological grades, and poor survival with AR-V7 expression. Our study unravels AR-V7 as a marker for poor clinical outcomes, predicting breast cancer aggressiveness, and encourages consideration of AR-V7 as a probable target for therapeutic intervention.

A Novel High-Throughput Sample-in-Result-Out Device for the Rapid Detection of Viral Nucleic Acids

Clustered regularly interspaced short palindromic repeats (CRISPR) molecular diagnostic technology is one of the most reliable diagnostic tools for infectious diseases due to its short reaction time, high sensitivity, and excellent specificity. However, compared with fluorescent polymerase chain reaction (PCR) technology, CRISPR molecular diagnostic technology lacks high-throughput automated instrumentation and standardized detection reagents for high sensitivity, limiting its large-scale clinical application. In this study, a high-throughput automated device was developed by combining reagent lyophilization, extraction-free technology, and a one-pot consumable system. This innovative approach enabled the rapid sample-in-result-out detection of 48 samples in 25 min and demonstrated high sensitivity and specificity for the qualitative analysis of clinical samples. The obtained results show that the detection limit of the designed system for African swine fever virus (ASFV) is 0.5 copies/μL. As a proof concept, a single-tube dual-target nucleic acid detection method was developed, achieving a detection limit of 5 copies/μL for the ORF1ab and N genes of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) within 45 min. The method is highly specific, reliable, and stable, providing a feasible solution for the clinical application of CRISPR nucleic acid detection technology.

Specific detection of duck adeno-associated virus using a TaqMan-based real-time PCR assay

Duck adeno-associated Virus (DAAV) is a novel pathogen that was recently discovered in ducks. To establish a molecular detection assay for DAAV for further epidemiological investigation and pathogenic mechanism. Here, we designed specific primers and probes according to the sequence characteristics of the newly discovered DAAV and then established a TaqMan real-time PCR method (TaqMan-qPCR) for the detection of DAAV. Our data showed that the established TaqMan-qPCR for detecting DAAV had high sensitivity, with the lowest detection limit of 29.1 copies/μL. No cross reaction was found with duck circovirus (DuCV), H9N2 subtype avian influenza virus (AIV), avian Tembusu virus (ATmV). duck hepatitis A virus 1 and 3 (DHAV-1 and DHAV-3), duck adenovirus A (DAdV-A), duck adenovirus 3 (DAdV-3), or duck enteritis virus (DEV). The repeatability was excellent, with the coefficients of variation of repeated intragroup and intergroup tests ranging from 0.12–0.21% and 0.62–1.42%, respectively. Seventy-eight clinical samples collected from diseased or deceased ducklings were tested. The results showed that the DAAV positive rate was 21.79%, and a triple infection (DAAV+MDPV+GPV) was found. These data provide technical support for further molecular epidemiological surveillance and pathogenic mechanism studies of DAAV infection.

Species Identification and Genotyping of Cutaneous Leishmaniasis in Clinical Samples Based on ITS1-PCR- Sequencing in Southeast Iran

Background: Cutaneous leishmaniasis (CL) is one of the most common parasitic diseases in many regions of Iran. It has a major role in deprived societies. We aimed to identify Leishmania species based on molecular as ITS1-rDNA-PCR internal transcribed spacer 1 (ITS1) region, microscopy, and culture techniques in diagnosing cutaneous leishmaniasis. Methods: From April 2018 to May 2020, we conducted a cross-sectional study involving 32 patients with suspected CL lesions in Sistan and Baluchistan Province, located in southeast Iran. Samples were subjected to microscopic examination, culture, and PCR amplification targeting the internal transcribed spacer 1 (ITS1) region. DNA sequencing was performed on PCR-positive samples for species identification and phylogenetic analysis. Results: PCR demonstrated superior sensitivity (93.75%, 30/32) compared to culture (56.25%, 18/32) and microscopic examination (53.1%, 17/32). Molecular analysis revealed that L. major was the predominant causative agent of CL in the study area, with L. tropica occurring less frequently. Sequencing and phylogenetic analysis of the ITS1 region showed high intraspecies similarity among L. tropica isolates, while L. major isolates exhibited greater genetic diversity. Conclusion: This study shows the co-existence of L. major and L. tropica in Mirjaveh, southeast Iran, with L. major as the primary cause. While L. tropica isolates displayed high genetic similarity, L. major samples were more diverse, indicating different epidemiological patterns. These findings highlight the importance of molecular methods for accurately identifying Leishmania species and understanding CL epidemiology in the region