Clinical Samples Research Articles

P-157. Molecular epidemiology of carbapenemase genes among carbapenem-resistant Klebsiella pneumoniae (CR-Kp) from the Philippines: implications for surveillance and treatment

Abstract Background In the Philippines, the proportion of carbapenem-resistant Klebsiella pneumoniae (CR-Kp) has doubled in the past decade and now exceeds 15%. However, the prevalence of different carbapenemase genes remains unknown. Unlike the United States and China where blaKPC predominates, blaOXA-48-like serine carbapenemases and metallo-β-lactamases (MBL) blaNDM, -IMP, and -VIM frequently occur among CR-Kp in the Asia-Pacific region. In order to better inform empiric treatment strategies and assist with infection prevention, we investigated the carbapenemases present in CR-Kp from the Philippine General Hospital (PGH), a 1,500-bed tertiary care national referral center in Manila, Philippines.Table 1.Carbapenemase genes detected with Xpert® Carba-R among carbapenem-resistant Klebsiella pneumoniae isolates from the Philippine General Hospital in Manila, Philippines. Methods CR-Kp from clinical samples were identified in the clinical microbiology laboratory at PGH using the VITEK® 2 platform (bioMerieux, USA). These isolates were collected and underwent rapid molecular testing with Xpert® Carba-R (Cepheid, USA) at the central laboratory of the Philippine National Institutes of Health. Molecular test results were typically available within 24 hours from the time of identification. These results were relayed to the treating physicians to guide clinical decision-making. Results A total of 81 CR-Kp isolates were collected from February to May 2024. Isolates were obtained from respiratory (n=44, 54.3%), blood (n=24, 29.6%), urine (n=9, 11.1%), and tissue/wound (n=4, 4.9%) samples. Table 1 summarizes the carbapenemase genes detected with Xpert® Carba-R among CR-Kp isolates from PGH, including multiple genes present in some samples. Conclusion Among CR-Kp from PGH, blaNDM was the predominant carbapenemase, followed by blaOXA-48-like and blaKPC genes. A third of the isolates co-harbored blaNDM and a serine carbapenemase, usually blaOXA-48-like. These findings suggest that there is molecular heterogeneity and likely plasmid spread among CR-Kp in the Philippines. These findings also underscore the importance of molecular surveillance programs that can rapidly characterize resistant strains, highlight the emergence of novel genotypes, and inform empiric and definitive therapy for CR-Kp. Disclosures Federico Perez, MD, MS, Merck: Grant/Research Support David Paterson, Infectious Diseases Physician, AMR Action Fund: Board Member|Aurobac: Advisor/Consultant|bioMerieux: Grant/Research Support|bioMerieux: Honoraria|CARB-X: Advisor/Consultant|Entasis: Expert Testimony|Menarini: Honoraria|Merck: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support|Shionogi: Honoraria

The Role of APOA1-AS in Colorectal Cancer: Investigating Its Association with Malignant Biological Behaviors.

Colorectal cancer (CRC) is a common malignant tumor associated with high morbidity and mortality. Long non-coding RNAs (lncRNAs) play crucial roles in cancer development and progression. This study aimed to explore the role of lncRNA APOA1-AS in colorectal cancer and elucidate its underlying mechanisms. Clinical samples were collected, and high-throughput sequencing was performed to identify differentially expressed lncRNAs in colorectal cancer. Among these, the key lncRNA APOA1-AS was selected for further investigation. The expression of APOA1-AS in colorectal cancer tissues and cells was evaluated. The effects of APOA1-AS on cell proliferation, migration, invasion, and apoptosis were assessed through knockdown and overexpression of APOA1-AS in SW620 and RKO cells. Additionally, the relationship between APOA1-AS and the malignant biological behaviors of colorectal cancer cells was also investigated. Furthermore, the involvement of APOA1-AS in glucose metabolism reprogramming and the cGMP-PKG signaling pathway was analyzed. A total of 2,985 differentially expressed lncRNAs were identified in colorectal cancer, including APOA1-AS, which showed the most significant upregulation. APOA1-AS expression was significantly higher in colorectal cancer tissues compared to normal tissues. Overexpression of APOA1-AS promoted cell proliferation, migration, and invasion while inhibiting apoptosis in SW620 and RKO cells. Furthermore, APOA1-AS was found to regulate glucose metabolism reprogramming, enhance tumor malignant biological behaviors and facilitate tumor cell drug resistance through the cGMP-PKG signaling pathway. Our study demonstrates that APOA1-AS is a potential key regulator in colorectal cancer development and progression. It functions via glucose metabolism reprogramming and the cGMP-PKG signaling pathway, offering a novel therapeutic target for colorectal cancer.

P-2087. Clinical Application of Broad-Range PCR in Pediatric Patients

Abstract Background Broad-range PCR (BRPCR) and next-generation sequencing (NGS) have the potential to be useful adjunctive tools in diagnosing the infectious etiology of a wide range of pediatric infections. We aimed to assess the clinical utility and application of BRPCR and NGS in children with suspected infections. Methods A retrospective chart review was performed on pediatric patients with clinical samples sent for BRPCR (University of Washington Molecular Microbiology Laboratory, Seattle, WA, USA) between January 2023 and December 2023. Data was extracted from the electronic medical record, and descriptive statistics were performed. Results 51 patients had 55 samples sent for BRPCR. The demographics and sample information are listed in Table 1. A total of 18 (35.3%) patients had an organism identified on culture or BRPCR that was contributory to the clinical picture. Of those, 4 (7.3%) were identified on culture but not on BRPCR, and 6 (11%) were identified on BRPCR but not on culture. 4 (7.3%) cases identified the same organism on culture and BRPCR, while 4 (7.3%) cases identified different organisms on culture and concomitant BRPCR. In 6 (11.8%) cases, antibiotics were de-escalated, and in 7 (13.7%) cases, antibiotics were discontinued. Conclusion BRPCR can be a valuable adjunct to identifying causative organisms in children with suspected infections and can help tailor antibiotic management. Our results highlight the potential impact of BRPCR results to reduce broad-spectrum antibiotic use. Further studies are needed to guide the optimal use of BRPCR and determine its clinical impact on management of pediatric infections. Disclosures All Authors: No reported disclosures

P-2142. Cross-Reactivity of Fungal Enzyme Immunoassays (EIA): Implications for Clinical Diagnosis in Patients with Autoimmune Disease

Abstract Background The immune system exhibits remarkable discriminatory power, distinguishing between self and non-self to mount protective responses against infections. In clinical diagnostics targeting dimorphic fungi, this immunological discrimination is leveraged to detect specific antibodies indicative of disease states. However, reliance on immune modulation for diagnostic purposes presents inherent challenges due to the intricate nature of antigen-antibody interactions. This complexity is particularly pronounced in patients with a history of autoimmune disorders, where the risk of false positive results in immunoassays is heightened due to the dysregulated immune response characteristic of autoimmune conditions. Methods We assessed the potential for cross-reactivity of serum samples from patients with autoimmune disease in dimorphic fungal Enzyme Immunoassays (EIAs) by analyzing a cohort of clinical serum samples sourced from the Florida region, which is non-endemic for dimorphic fungal diseases. Samples were commercially sourced as remnant-banked serum samples (Medix Biochemica, Florida, USA). 25 serum samples obtained from distinct patients were subjected to testing using Coccidioides, Blastomyces, and Histoplasma EIA (MiraVista Diagnostics, Indiana, USA). This ncluded 5 individuals devoid of autoimmune disorders, cohorts of 5 patients each diagnosed with Rheumatoid Arthritis and Multiple Sclerosis, and 10 patients with Systemic Lupus Erythematosus. Samples were tested in triplicate. Results Preliminary findings from our study unveiled nuanced levels of cross-reactivity across the dimorphic fungal EIAs utilized for the three autoimmune disorders examined. Depending on the specific dimorphic fungal EIA assay and autoimmune disorder, approximately 10–30% of serum samples from patients tested intermediate or weakly positive in the respective fungal EIAs. All control patient samples tested negative. Conclusion These preliminary findings underscore the complexity of diagnosing dimorphic fungal infections, highlighting the significance of the patient's immune status as a confounding factor. Clinical practitioners should consider the possibility of autoimmunity when encountering weakly positive or intermediate dimorphic fungal EIA results. Disclosures Arryn Craney, PhD D(ABMM), MiraVista Diagnostics: Employee; Salary Kendra Carlson, n/a, MiraVista Diagnostics: Employee; Salary Kyla Seyfried, n/a, MiraVista Diagnostics: Employee; Salary John Witt, n/a, MiraVista Diagnostics: Employee; Salary Andrew Hanzlicek, DVM, MiraVista Diagnostics: Employee; Salary Nicolas Barros, MD, MiraVista Diagnostics: Advisor/Consultant Lawrence J. Wheat, MD, MiraVista Diagnostics: Employee; Salary|MiraVista Diagnostics: Ownership Interest

P-108. Clinical Evaluation of a Sequencing-based Diagnostic for Bacterial and Fungal ID & AST Directly from Patient Blood Samples

Abstract Background Early pathogen ID and targeted treatment are key to reducing bloodstream infection (BSI) morbidity and mortality. Current diagnostics rely on culture which takes 1-2 days for ID and longer for antimicrobial susceptibility testing (AST) results, or molecular assays with limited panels, few resistance markers, and high false positive rates. We report interim results of a first-in-kind comprehensive ID and predictive AST assay directly from patient blood samples. The system can deliver results in ∼8 hrs, uses ultra-high enrichment of microbial DNA directly from blood, whole-genome sequencing, and a predictive AST machine-learning algorithm to identify a broad range of species and pathogen/drug combinations. Methods We enrolled subjects with suspicion of BSI from 3 EDs and 1 ICU/inpatient in 4 Boston area hospitals in 2 IRB approved observational studies. We collected whole blood in SPS vacutainers and 10mL were processed at Day Zero Diagnostics (DZD) and sequenced on an Oxford Nanopore platform. Sequencing data were analyzed by Keynome® algorithms to determine pathogen ID and predict AST profiles. Tables 1 & 2 list current on-panel pathogens and drug models tested. Performance was compared to hospital microbiology lab phenotypic ID/AST results from blood cultures collected within 0-24 hours of the research draw. Results Species-level ID results in 225 subjects (6525 calls) demonstrated 80.0% sensitivity, 99.9% specificity, 64% PPV, and 99.9% agreement with clinical culture. Eight distinct species were identified among the 20 (8.9%) clinical culture positive samples with on-panel organisms. Thirteen samples had sufficient genome coverage for on-panel AST predictions demonstrating 92.3% categoric agreement with phenotypic AST. Conclusion Our data suggests the DZD diagnostic system can provide accurate ID and AST results directly from blood in patients with suspected BSI. To our knowledge these results are the first demonstration of whole genome recovery and comprehensive ID & AST directly from patient blood samples. This assay has the potential to revolutionize speed to diagnosis of BSI, thus facilitating targeted therapy, improved outcomes, and reduced development of antimicrobial resistance. Disclosures Michael R. Filbin, MD, Day Zero Diagnostics: Grant/Research Support|Quidel: Grant/Research Support Peter Hou, MD, Center for Disease Control: Grant/Research Support|Day Zero Diagnostics: Grant/Research Support|iDoc Telehealth Solutions: Ownership Interest Michael Donnino, MD, Day Zero Diagnostics: Grant/Research Support Archana Asundi, MD, Day Zero Diagnostics: Grant/Research Support|Gilead Sciences: Grant/Research Support|GSK/ViiV Healthcare: Grant/Research Support|Theratechnologies: Grant/Research Support Zoe H. Rogers, MPH, Day Zero Diagnostics: employment Emma Briars, PhD, Day Zero Diagnostics: employment Alison Gassett, MPH, Day Zero Diagnostics: employment Alexander Reidel, BS, Day Zero Diagnostics: employment Alexis Campbell, MA, Day Zero Diagnostics: Grant/Research Support Jason Wittenbach, PhD, Day Zero Diagnostics: employment Nicole Billings, PhD, Day Zero Diagnostics: employment

P-2129. Analytical Validation of a Next-Generation Sequencing Assay for the Detection of Microbial Pathogens in Human Plasma

Abstract Background Fungal and acid-fast bacteria (AFB) infections can be a serious complication for immunosuppressed patients. Identification of the pathogen involved in invasive fungal/AFB infections (IFIs) can allow for reduced time to initiation of the appropriate targeted therapy through elimination of ineffective therapies. Current fungal/AFB diagnostics suffer from low sensitivity/specificity, invasiveness (e.g., biopsy or antigen), have long turnaround times (e.g., culture), and/or require an a priori hypothesis regarding the causative pathogen (e.g., PCR based methods). To overcome shortcomings in fungal/AFB diagnostics, an unbiased next-generation sequencing (NGS) approach can be utilized to characterize pathogen cfDNA present in a patient blood sample. Methods Sequencing of IFIs through hybrid capture has the potential to detect a broad range of pathogens yet can still specifically target the most clinically relevant. Compared to standard whole-genome sequencing methods, hybrid capture followed by NGS can be more specific and sensitive due to an enrichment step using probes specific to cfDNA targets of interest. This additional enrichment step can result in more sequencing reads being dedicated to cfDNA derived from pathogens of interest rather than to cfDNA derived from non-relevant organisms. Here is described a hybrid capture assay that uses >30,000 probes to detect >600 Fungal and select AFB species in plasma. Results Cutoffs for target coverage and kmers/million were established through ROC analysis of 170 contrived plasma samples resulting in an analytical specificity of ∼96% and a sensitivity of ∼1,200 molecules/mL. 98% of the 40 contrived samples used for accuracy were correctly identified while 100% intra- and 93%-inter assay precision was achieved. In silico validation demonstrated the assay’s ability to detect closely related species unable to be tested at the bench. Several species have been detected in the initial clinical samples received thus far, however, all have been below the reportable cutoff values. Conclusion The analytical validation described here demonstrates the analytical performance of hybrid capture in human plasma. Disclosures Curtis Bacon, PhD, Eurofins Viracor: employee Ellis Bixler, n/a, Eurofins Viracor: Employee Scott Cowden, n/a, Eurofins Viracor: Employee Jamie Nutt, n/a, Eurofins Viracor: Employee Steve Kleiboeker, PhD, Eurofins: employee|Eurofins: Stocks/Bonds (Public Company)

Dual-OR Logic-Gated Lateral Flow Strip Assay Based on Colorimetric-Fluorescence Dual Indication for Screening of HPV16/18 in Multiple Scenarios.

The incidence of cervical cancer continues to rise in underdeveloped regions due to low human papillomavirus (HPV) vaccination rates and inadequate screening systems. To achieve convenient, rapid, and accurate detection of HPV, we developed a three-wire lateral flow strip assay system based on dual-OR logic gates for rapid and simultaneous detection of HPV subtypes 16 and 18 in a single test. The system combines three-branch-catalytic hairpin assembly (TCHA)-mediated signal amplification with simple OR logic gate-based signal output to improve detection rates while enabling HPV 16/18 subtype identification. The detection limit of the method was calculated to be 10 aM for the selected target sequences. Meanwhile, the method showed excellent specificity with no false-positive output in real-world detection. The sensitivity of the colorimetric test strips exceeded 90%, while the sensitivity of the fluorescence-based test strips surpassed 95% in detecting clinical samples, demonstrating a high degree of concordance with the results obtained from the real-time quantitative polymerase chain reaction (qPCR). This method provides a simple and easy method for the rapid screening of HPV.

P-2294. Listeriosis in cancer patients: Uncommon and protean

Abstract Background Historically cancer patients (pts) are considered to be at an increased risk of Listeria monocytogenes infection (listeriosis, LT), especially those with ongoing chemotherapy or receipt of glucocorticoids (GCs). However, the features of this uncommon infection have not been described in contemporary cohorts of cancer pts. Methods We performed a 12-year retrospective chart review of cancer pts with LT as confirmed by positive culture from blood or other sterile clinical samples at MD Anderson Cancer Center (2011-2023). Results We identified 20 cancer pts with LT. Twelve (60%) had an underlying hematologic malignancy (Figure 1). Lymphopenia < 1000/µL (17, 85%, severe < 200/µL in 20%) and monocytopenia < 1000/µL (18, 90%, severe < 200/µL in 35%) were common at diagnosis, as well as chemotherapy (13, 65%) and GC use (11, 55%) within 30 days prior to LT diagnosis. Most (15 pts) were < 65 years old. Fever was seen in 10 pts. Vomiting/diarrhea were common (50%), while 45% of pts had headache or altered mental status. LT most often presented with bacteremia (17, 85%) and gastroenteritis (9, 45%) (Figure 2). 7/9 pts with gastroenteritis were bacteremic. Central nervous system (CNS) manifestations (meningitis, brain abscess) were documented in 2 pts who had a lumbar puncture. Focal non-CNS infections were seen in 30% of pts in unusual sites: cellulitis, abscess, endocarditis, cholangitis, urinary tract infection, and peritonitis. Of interest, 11pts (55%) had low SOFA score at presentation and were given antibiotics only after blood cultures were positive for Listeria. In fact, most pts (17, 85%) either did not receive empiric (n=11) or had inappropriate empiric antibiotics (n=6) until LT diagnosis. The 30-day mortality from LT diagnosis was 25% and correlated with high SOFA score ≥ 5 (5/8 pts, P = 0.0005). Delayed appropriate antibiotics were not significantly associated with increased 30-day mortality in this small cohort (Table 1). Conclusion Although uncommon, LT in cancer pts has pleotropic manifestations and variable morbidity, from non-bacteremic gastroenteritis to septicemia, CNS and non-CNS focal infections. Consider LT in ill cancer pts with lymphopenia/monocytopenia and recent chemotherapy or GCs, especially if they have GI symptoms, even in the absence of fever or CNS involvement. Disclosures Sebastian Wurster, MD, MSc, Astellas Pharma: Grant/Research Support|Gilead Sciences: Grant/Research Support Dimitrios P. Kontoyiannis, MD, AbbVie: Advisor/Consultant|Astellas Pharma: Advisor/Consultant|Astellas Pharma: Grant/Research Support|Astellas Pharma: Honoraria|Cidara Therapeutics: Advisor/Consultant|Gilead Sciences: Advisor/Consultant|Gilead Sciences: Grant/Research Support|Gilead Sciences: Honoraria|Knight: Advisor/Consultant|Merck: Advisor/Consultant|Scynexis: Advisor/Consultant

The psychometric properties of the child anxiety and depression life interference scale (CADLIS)

ObjectiveLife interference is a key diagnostic feature for anxiety and depressive disorders. Measures focusing on life interference caused by anxiety and depressive disorders in children and adolescents have received minimal attention. This study evaluated the psychometric properties of the Child Anxiety and Depression Life Interference Scale (CADLIS), a brief child (CADLIS-C) and parent-report (CADLIS-P) measure designed to assess life interference from anxiety and depressive disorders in both the child and parent’s life.MethodA total of 672 parents of children aged 4–18 years completed the CADLIS-P, and 627 children aged 7–18 years completed the CADLIS-C.ResultsThe proposed two-factor CADLIS-C model was not supported, instead, due to high inter-factor covariance a one-factor model of life interference was proposed. The one-factor model demonstrated better model fit. The proposed three-factor model for the CADLIS-P was not supported, instead, an exploratory factor analysis found a two-factor model differentiating life interference into child and parent life interference a better model fit. The CADLIS demonstrated excellent internal consistency, good convergent and divergent validity, interrater correlations and was able to differentiate between children with and without clinical levels of anxiety and depressive symptoms.LimitationsLimitations of the study included the sample population which consisted of a small clinical sample, an over-representation of high-income families and the use of a panel provider.ConclusionsOverall, the CADLIS demonstrated sound psychometric properties. The CADLIS is a reliable measure that demonstrates evidence of convergent validity for the assessment of life interference associated with anxiety and depressive symptoms in children.

An Immune Subtype Classification System Enables the Development of Strategies to Predict and Enhance Immunotherapy Responses in Colorectal Cancer.

Colorectal cancer (CRC) is the second leading cause of cancer-related mortality globally. While immunotherapeutic approaches are effective in a subset of CRC patients, the majority of CRC cases receive limited benefits from immunotherapy. This study developed an immune subtype classification system based on diverse immune cells and pathways. A model constructed through machine learning based on immune subtypes could accurately predict the sensitivity of CRC patients to immunotherapy. Validation of this model across public datasets and clinical samples confirmed its high precision and reliability. Furthermore, drug screening based on the immune subtypes identified the IGF1R inhibitor I-OMe-AG-538 (AG-538) as a potent enhancer of antitumor immunity. Mechanistic investigations revealed that AG-538 induced reactive oxygen species (ROS)-dependent DNA damage and downregulated the expression of multiple repair genes, triggering cGAS/STING-mediated type I IFN signaling within tumor cells. This signaling cascade increased tumor immunogenicity and refined the tumor immune microenvironment, thereby enhancing efficacy of immune checkpoint blockade treatment. In summary, these findings present a predictive model for immune response and highlight the potential of AG-538 combined with anti-PD1 antibodies as a chemoimmunotherapeutic strategy.

P-1302. Alterations in the population structure and antimicrobial resistance of Salmonella enterica before and during the COVID-19 pandemic (2018 and 2020) in New York State

Abstract Background The COVID-19 pandemic impacted many core activities to combat antimicrobial resistance (AMR). Concurrently, there has been increased AMR, particularly in hospital-acquired infections. However, there is little data on AMR surveillance of community-acquired infections, such as foodborne infections, during this period. An estimated 212,500 resistant Salmonella enterica infections occur yearly. Here, we aim to elucidate the impact of the COVID-19 pandemic on the population dynamics of S. enterica lineages and the genetic determinants of AMR.Figure 1:Phylogenetic relationships, pan-genomic, lineage and antimicrobial resistance characteristics of [i]S. enterica[i] genomes before (2018, n=1,212) and during the COVID-19 pandemic (2020, n=975)A. A midpoint-rooted maximum likelihood phylogenetic tree based on 608,598 core-gene single nucleotide polymorphisms. Branch lengths are not displayed. Outer rings show Serotype, Sequence Type, and Region. Branches are colored by sampling year. A map displays the region associated with the source county of the isolate within New York State. B. Displays the Sequence Types as the proportion of the total population. Chi-squared p-values are displayed. C. Serotype as proportions of the yearly population. D. Resistance mechanism as a proportion of the yearly population Benjamini-Hochberg corrected p-values are displayed. Methods We leveraged 2,187 publicly available sequences derived from S. enterica isolates from human clinical samples across 56 counties in New York, USA, before the pandemic (2018, n=1,212 genomes) and during the pandemic (2020, n=975 genomes). Results A midpoint-rooted maximum likelihood phylogeny generated from 608,598 single-nucleotide polymorphisms from 3,482 core genes revealed the intermingling of isolates regardless of year and county of isolation. We did not find significant differences in the diversity (measured using the Simpson diversity index) of sequence types (ST) and serotypes between the two time periods. However, some STs and serotypes exhibited significant changes in the proportion of the annual population. We observed an increase in ST33 and a reduction in ST19, ST34, and low-frequency STs. There was a marked increase in serotypes Hadar, Newport, and Thompson and a decrease in serotypes Saintpaul and the low-frequency serotypes. We also measured the number of genomes carrying specific AMR genes as a proportion of the annual population per year. We found a significant change in the number of genomes carrying in aph(3”)-Ib and aph(6”)-Id (aminoglycoside resistance), tetC (tetracycline), and the fluoroquinolone resistance point mutation (gyrA_D87Y). Conclusion Our findings revealed the bacterial features, such as locally circulating lineages, mutations, and mobile antimicrobial resistance genes, that were altered before and during the COVID-19 pandemic. Together, our data show that major public health emergencies such as the COVID-19 pandemic can inadvertently alter the dynamics of other pathogen populations, including foodborne bacteria, which may have consequences for public health and treatment efforts. Disclosures All Authors: No reported disclosures

P-2226. Clinical Validation and Performance of Molecular Syphilis Diagnosis by Loop-Mediated Isothermal Amplification

Abstract Background Syphilis cases continue to increase rapidly in the US. Molecular detection of the pathogen, Treponema pallidum (TP) may speed turnaround time (TAT) and resolve ambiguous results from current diagnostics methods, which require stepwise serologic testing. Here we describe validation and clinical performance of a loop-mediated isothermal amplification (LAMP) assay for TP. Methods Candidate LAMP primers targeting the 23S rRNA locus of TP were evaluated and the best performing set was used for clinical validation per Clinical Laboratory Standards Institute guidelines. We determined the sensitivity, specificity, precision, accuracy, and reproducibility of the TP-LAMP assay in fresh and formalin-fixed tissue, body fluids, and whole blood using contrived specimens spiked with intact treponemes and residual clinical specimens. For whole blood, automated nucleic acid extraction was performed from 1mL ETDA-anticoagulated sample. Results The 95% limit of detection was determined to be 7-10 genomes/reaction by testing remnant DNA eluates spiked with TP gDNA. Assay specificity was evaluated by testing synthetic DNA targets representing non-cultivable targets and residual clinical samples containing DNA from 114 clinically relevant microbes, including non-TP spirochetes, bacteria, fungi, and viruses. TP-LAMP detected 100% of whole blood samples spiked with 120-125 organisms/mL; 88% of samples with 85 organisms/mL; and 80% of samples spiked with 60 organisms/mL. From 09/2022 - 07/2023 TP-LAMP had 110 results in non-blood samples and the mean TAT was 4.7 days. Orthogonal diagnostic results were available for a subset of 34 samples. In this group the assay was 100% sensitive and specific with 7 positive tests, comprising 2 cases of primary and 5 cases of secondary syphilis. Three tissue specimens with negative TP-LAMP results had false-positive immunohistochemical TP stains due to cross-reactivity with commensal treponemes. Conclusion This assay represents a novel, clinically available method for sensitive and specific syphilis diagnosis. Additionally, the assay can resolve ambiguous findings in tissue. Applications in blood may aid in diagnosis of early or latent disease and resolution of unclear serologic test results. Disclosures Stephen J. Salipante, M.D. Ph.D., Anavasi: Grant/Research Support Joshua Lieberman, MD, PhD, Anavasi: Grant/Research Support

P-1838. The human Herpesvirus dUTPases of EBV and HSV-1 may contribute to neurological disorders by altering the integrity of the blood brain barrier and promoting a pro-inflammatory microenvironment

Abstract Background The global prevalence of Herpes Simplex Virus 1 (HSV-1) and Epstein-Barr Virus (EBV) ranges from 60% to >90%. Some herpesviruses have been linked to neurological diseases including Alzheimer’s disease (AD) and one common feature to these pathologies is disruption of the blood-brain barrier (BBB). The BBB is a key structure responsible for protecting the brain from toxic substances due to its high impermeability and providing energy which is critical for proper brain function. While disruption of the BBB is a hallmark of numerous neurological disorders, the mechanisms underlying herpesvirus-induced BBB dysfunction remain elusive. Herpesvirus dUTPases encoded by EBV and HSV-1 alter blood brain barrier integrity Stress, aging as well as other environmental and genetic factors can reactivate human herpesviruses to undergo lytic or abortive-lytic replication, which results in the release of viral dUTPases as a free protein or in exosomes. dUTPase-containing exosomes can travel to the brain via the bloodstream and target cells at the blood brain barrier, triggering the release of pro-inflammatory cytokines and disturbing the structural and functional properties of cell-cell contact molecules. Altogether, the herpesviruses dUTPases can contribute to neurological disorders by increasing blood brain barrier permeability and promoting neuroinflammation. Methods In this study we investigated whether a viral protein of EBV and HSV-1 called dUTPase can alter BBB integrity using AD clinical samples, in vitro and 3D perfused hiPSC-derived BBB/NVU models, in combination with a multi-system analysis approach that included, trans-endothelial electrical resistance (TEER), confocal imaging, ELISA, proteome array and qRT-PCR/RNAseq. Results Exposure of 3D BBB to HSV-1 or EBV dUTPases resulted in significant BBB permeability, reduced TEER, and loss of functional tight junction protein (TJP; ZO-1 and Claudin-5) structural and functional properties. Both dUTPases significantly increased the expression and/or secretion of IL-1β, IL-6 and TNF-α in immortalized human cerebral microvascular endothelial cells, astrocytes and pericytes. A modest but significant increase in IL-1β and IL-6 mRNA was also observed in dUTPase-treated astrocytes. These cytokines are elevated in patients with AD and are known to disrupt BBB integrity and induce neuroinflammation. Conclusion In conclusion, this study provides compelling evidence that EBV/HSV-1 dUTPases have the potential to disrupt the BBB and foster a pro-inflammatory environment in the brain. Thus, it highlights a novel mechanism linking common herpesviruses to brain pathologies. Disclosures All Authors: No reported disclosures

YAP/TEAD4/SP1-induced VISTA expression as a tumor cell-intrinsic mechanism of immunosuppression in colorectal cancer.

Hyperactivation of the YAP/TEAD transcriptional complex in cancers facilitates the development of an immunosuppressive tumor microenvironment. Herein, we observed that the transcription factor SP1 physically interacts with and stabilizes the YAP/TEAD complex at regulatory genomic loci in colorectal cancer (CRC). In response to serum stimulation, PKCζ (protein kinase C ζ) was found to phosphorylate SP1 and enhance its interaction with TEAD4. As a result, SP1 enhanced the transcriptional activity of YAP/TEAD and coregulated the expression of a group of YAP/TEAD target genes. The immune checkpoint V-domain Ig suppressor of T-cell activation (VISTA) was identified as a direct target of the SP1-YAP/TEAD4 complex and found to be widely expressed in CRC cells. Importantly, YAP-induced VISTA upregulation in human CRC cells was found to strongly suppress the antitumor function of CD8+ T cells. Consistently, elevated VISTA expression was found to be correlated with hyperactivation of the SP1-YAP/TEAD axis and associated with poor prognosis of CRC patients. In addition, we found by serendipity that enzymatic deglycosylation significantly improved the anti-VISTA antibody signal intensity, resulting in more accurate detection of VISTA in clinical tumor samples. Overall, our study identified SP1 as a positive modulator of YAP/TEAD for the transcriptional regulation of VISTA and developed a protein deglycosylation strategy to better detect VISTA expression in clinical samples. These findings revealed a new tumor cell-intrinsic mechanism of YAP/TAZ-mediated cancer immune evasion.

Drug-induced differential culturability in diverse strains of Mycobacterium tuberculosis

Under stress, bacteria display selective growth propensities on solid and liquid media. For Mycobacterium tuberculosis (Mtb), differentially culturable bacteria have been found in tuberculosis (TB) patient sputa. We hypothesized that antibiotic treatment can induce selective culturability in Mtb. We investigated the effect of exposure to TB drugs on Mtb culturability using clinical samples from an ongoing TB patient cohort and by conducting several in vitro experiments with a diverse set of Mtb strains. In patients, serial sputa were more likely to generate Mtb-positive cultures in liquid as opposed to solid medium, with this liquid culture bias extending up to 5 months post diagnosis. Experimentally, there was a disparity between bacterial time to positivity (TTP) and colony forming units (CFUs) when Mtb was exposed to isoniazid (INH) and rifampicin (RIF) alone or in combination. Cultures recovered from RIF treatment yielded more CFUs on agar plates, but INH-treated cultures had a faster TTP in liquid. Follow up experiments using a fluorescently labelled laboratory strain of Mtb revealed that CFUs of INH-treated bacteria were too low, thus over-estimating the killing effect of the treatment. Here we provide evidence that drug exposure affects culturability on solid medium in diverse Mtb strains, which has implications for treatment monitoring and drug-pathogen interaction studies.

CXCR4 promotes tumor stemness maintenance and CDK4/6 inhibitors resistance in ER-positive breast cancer

BackgroundCDK4/6 inhibitors have significantly improved the survival of patients with HR-positive/HER2-negative breast cancer, becoming a first-line treatment option. However, the development of resistance to these inhibitors is inevitable. To address this challenge, novel strategies are required to overcome resistance, necessitating a deeper understanding of its mechanisms. Recent research has identified several dysregulated genes in CDK4/6 inhibitors-resistant breast cancer, but the underlying mechanism is complex due to tumor heterogeneity and warrants further investigation.MethodsRNA sequencing and KEGG pathway analysis was carried out to identify the mainly dysregulated genes in CDK4/6 inhibitors-resistant breast cancer cells. The effects of CXCR4 knockdown and overexpression via siRNAs and plasmids transfection were examined by mammosphere formation, RT-qPCR, flow cytometry, MTT and colony formation assays. The regulation mechanisms were analyzed by RT-qPCR, western blotting and immunofluorescence experiments. Mouse xenografts were used to analyze the role of CXCR4 in regulation palbociclib sensitivity in vivo. Additionally, we collected the clinical samples and performed immunohistochemistry to analyze the clinical significance of CXCR4.ResultsIn our study, we focused on cancer stem cells, a critical contributor to cancer metastasis and therapy resistance, and detected an upregulation of stemness in our established palbociclib-resistant ER-positive breast cancer cells. Additionally, our research pinpointed CXCR4 as a pivotal gene responsible for maintaining cancer stemness and promoting palbociclib resistance. Mechanistically, CXCR4 activates the WNT5A/β-catenin signaling pathway by enhancing the expression of WNT5A and β-catenin, facilitating the nuclear translocation of β-catenin protein. Targeting CXCR4 using siRNAs or small molecular inhibitors effectively reduces cancer stemness and reverses palbociclib resistance both in vitro and in vivo. Clinical sample analysis further underscores the overactivation of the CXCR4/WNT5A/β-catenin axis in palbociclib-resistant breast cancer, suggesting CXCR4 as a potential biomarker for predicting resistance to CDK4/6 inhibitors.ConclusionsCollectively, our study demonstrates that CXCR4 overexpression plays a vital role in maintaining breast cancer stemness and promoting resistance to CDK4/6 inhibitors through the activation of the WNT5A/β-catenin pathway. Targeting CXCR4 may offer a promising therapeutic approach for advanced CDK4/6 inhibitor-resistant ER-positive breast cancer.

Farnesoid X Receptor Attenuates the Tumorigenicity of Liver Cancer Stem Cells by Inhibiting STAT3 Phosphorylation

The Farnesoid X receptor (FXR) has recently been identified as being closely associated with the progression of primary hepatocellular carcinoma. Cancer stem cells (CSCs) play a crucial role in tumor initiation, progression, invasion, metastasis, recurrence, and drug resistance. The elucidation of the role and regulatory mechanism of FXR in CSCs is therefore deemed significant. Here, bioinformatics analysis has revealed a downregulation of FXR in hepatocellular carcinoma (HCC), which showed a negative correlation with HCC malignancy. This result was further confirmed through clinical sample analysis. Subsequently, CSCs were isolated from HCC cell lines and exhibited a significant decrease in the expression of FXR. The activation of FXR resulted in a remarkable inhibition of the proliferation, invasion, and tumorigenicity of CSCs. Furthermore, activated FXR prominently upregulated the expression of SOCS3 while suppressing STAT3 phosphorylation in CSCs. To further investigate this discovery, we established a DEN-induced HCC model in mice and observed that FXR-deficient mice exhibited heightened susceptibility to HCC. This was accompanied by decreased expression levels of SOCS3 and elevated expression and phosphorylation levels of STAT3, as well as significantly enhanced HCC CSCs markers and stemness-related genes expression in DEN-induced HCC tissues of FXR-deficient mice. Additionally, we also found a significant upregulation of CSCs markers and stemness-related genes within HCC clinical samples. Based on these findings, we postulated that targeted regulation of SOCS3 by FXR inhibits STAT3 phosphorylation, thereby exerting an inhibitory effect on CSCs.

Development and utilization of a multiplex PCR assay for detecting three feline enteroviruses.

Feline diarrhea is a common digestive tract disease in clinical practice, with watery feces as the main clinical manifestation. There are numerous pathogenic factors causing feline diarrhea, among which viral infections are prevalent, and feline panleukopenia virus (FPV) is the most common pathogen. In recent years, a variety of novel viruses have been detected in the intestines of cats with diarrhea. For example, feline kobuvirus (FKoV) and feline norovirus (FNoV) have been identified. These viruses may have a direct relationship with feline diarrhea or the connection has yet to be discovered. However, with the continuous emergence of these novel viruses and the frequent contact between pet cats and humans, it is prone to large-scale epidemics and outbreaks of viruses. Therefore, developing an accurate, rapid, and simple method to detect novel enteric viruses is of great significance for the early warning of emerging feline enteric viral infectious diseases. A detailed comparison of the genome sequences of the three aforementioned feline enteroviruses was conducted. Subsequently, three pairs of specific primers were designed by selecting the conserved gene regions, and the single and multiplex PCR amplification reaction systems as well as reaction conditions were repeatedly optimized. The target fragment sizes detected by the multiplex PCR method were 650bp for FPV, 500bp for FKoV, and 340bp for FNoV. Sensitivity tests demonstrated that the lower detection limit was one-tenth of that of single PCR. Meanwhile, the detection results for feline calicivirus (FCV), feline herpesvirus (FHV), and feline coronavirus (FCoV) were all negative. Testing of a total of 209 clinical samples from various regions in Shandong Province revealed that the detection rates of the three viruses were 13.4%, 4.8%, and 3.8%, respectively, and mixed infections were present. In this study, an epidemiological investigation of the three feline enteroviruses was performed, and a sensitive, specific, and reproducible multiplex PCR assay was developed, which can be utilized for the detection of clinical samples.

Thin Layer Chromatography Goes Ultrasmall to Assay Sphingosine Kinase Activation in Single Primary Leukemic Cells.

Cell-to-cell heterogeneity in lipid signaling underlies variations in response and recurrence for many cancers, including leukemias. A highly parallel, miniaturized thin-layer chromatographic platform capable of assaying single cells was developed. Ultrasmall volumes (50 pL) of standard fluorescent lipids were separated with excellent repeatability, reproducibility, and limits of detection. Sphingosine-cyanine 5 (Sph-Cy5) was loaded into cells, and the single-cell contents were separated to identify Sph-Cy5 and two metabolites, Sph-1-phosphate-Cy5 (S1P-Cy5) and hexadecanoic acid Cy5 (HA-Cy5). In leukemic cells, the CD34+ blast cells demonstrated significantly greater conversion of Sph-Cy5 to its phosphorylated form compared to that of the CD34- cells. After treatment with a sphingosine kinase (SphK) inhibitor, the level of formation of S1P-Cy5 remained significantly greater for the inhibited CD34+ cells relative to that of the inhibited CD34- cells. Over 1200 single cells were rapidly assayed using 8 chips within 4 h. Sphingosine kinase activity in the CD34+ blast cells of 3 patients with acute myeloid leukemia was assayed with and without inhibitors. The patient cells displayed intertumor and intratumor heterogeneity, and subsets of cells with distinct enzymatic activities and products, highlighting the diversity of the cells within a clinical sample and between patients.

The role of CLDN11 in promotion of granulosa cell proliferation in polycystic ovary syndrome via activation of the PI3K-AKT signalling pathway

Polycystic ovary syndrome (PCOS) is a complex gynecological endocrinological condition that significantly impacts women’s fertility during their reproductive lifespan. The causes of PCOS are multifaceted, and its pathogenesis is not yet clear. This study established a rat model of PCOS and, in conjunction with clinical samples and database data, analysed the role of claudin 11 (CLDN11) in follicular granulosa cells (GCs) in regulating the proliferation of GCs. Our findings revealed a notable decrease in the protein expression of CLDN11 within the follicular GCs of individuals with PCOS. In vitro rat cell experiments revealed that interference with CLDN11 significantly inhibited viability and increased the apoptosis of GCs. Additional research has illuminated the mechanism by which CLDN11 regulates the expression levels of CCND1 and PCNA through the PI3K/AKT signalling pathway, significantly influencing the proliferation of rat follicular GCs. Furthermore, overexpression of CLDN11 via an adeno-associated virus (AAV) vector was found to reverse the PCOS-like phenotype induced in rats by letrozole. Our findings suggest that CLDN11 stimulates the proliferation of these cells by activating the PI3K/AKT pathway, thereby increasing the expression of CCND1 and PCNA. These discoveries underscore the critical function of CLDN11 in regulating the functionality of follicular GCs, which offers novel insights into the fundamental mechanisms governing PCOS.