bluuebunny/crossref_metadata_2025_split huggingface dataset:
B-200 Expediting Identification of Occult Sepsis with a Novel Diagnostic for Patients Presenting to the ED with Possible Infection
Authors: Jagneaux T , Grantham A , Richard K , Thomas C , D’Antonio C , Laperouse M , Scoggins R , O’Neal H 
Published: October 2024

Abstract

Background
In August 2023 Our Lady of the Lake Regional Medical Center implemented a process for sepsis care based on a novel sepsis diagnostic (IntelliSep) in the Emergency Department (ED). As a component of our Sepsis Learning Health Program, we continually evaluate this process.

Methods
A nurse-driven protocol allows for IntelliSep ordering with triage assessment. Dependent upon bed availability and assessment for clinical stability by the triage staff, patients may be referred to the waiting room after blood draw. An IntelliSep Band 3 result is a critical value, initiating a sepsis pathway. Patients in waiting room at the time of a Band 3 result are immediately placed in an ED bed. We evaluated this process for efficacy and efficiency through review of the medical record.

Results
Between 01-Sep-2023 and 07-Feb-2024, we performed a total of 2322 IntelliSep tests, with 247 (10.6%) resulting prior to bed assignment, consisting of 124 (50.2%) Band 1, 73 (29.6%) Band 2, and 50 (20.2%) Band 3. The median time to bed assignment (TTB) for these patients was 104 min (Q1-Q3 71-180). TTB for Band 3 (median 73 min, Q1-Q3 58-102) was significantly lower than both Band 1 (146 min, Q1-Q3 86-201 min) and Band 2 (98 min, Q1-Q3 70-174 min), p &lt; 0.0001 and 0.01 respectively. Providers admitted 45 (90%) of Band 3 patients and 62 (50%) of Band 1 patients (p &lt; 0.0001). Discharge diagnosis included infection in 47 (94%) and sepsis in 27 (54%) of Band 3 patients, and infection in 686 (54.8%) and sepsis in 2 (1.6%) of Band 1 patients (p &lt; 0.0001 for infection and sepsis).

Conclusions
An IntelliSep-based process for sepsis diagnosis, implemented at triage, can expedite identification and treatment of patients presenting to the ED with occult sepsis who appear clinically stable by triage staff.
B-204 A Real-time Probe-based PCR Assay for Detection of Pneumocystis Pneumonia
Authors: Zhou Z , Li S , Jiang D , Wang Z , Wang Y , Zhang Y , Wang H , Su Y 
Published: October 2024

Abstract

Background
Pneumocystis jirovecii is an atypical opportunistic fungus with lung tropism and worldwide distribution that continues to be one of the major opportunistic pathogens causing severe Pneumocystis pneumonia (PcP) in individuals with acquired immune deficiency syndrome (AIDS) and patients with immunosuppression due to other causes. Widespread prophylaxis and treatment for P. jirovecii with sulfa drugs have decreased the incidence of PcP, but concerns have been raised about the possible emergence of P. jirovecii isolates resistant to these drugs. Point mutations in this gene have been associated with prior exposure to sulfa drugs in other microorganisms, the polymorphic positions 165 and 171, which lead to an amino acid change at positions 55 (Thr55Ala) and 57 (Pro57Ser) of the polypeptide chain, respectively. Fluorescent PCR technology can detect the nucleic acid fragments of P. jirovecii in patients' serum, alveolar perfusion fluid (BALF) and tissue samples, and further identify drug-resistant mutations, which is of great significance for early clinical and accurate initiation of antifungal therapy. MycoMDx Pneumocystis Jirovecii PCR Assay is the use of PCR-fluorescence probe method, combined with other methods to achieve the auxiliary diagnosis of P. jirovecii infection and resistance to sulfonamides (Fig. 1).

Methods
In this study, we did a performance evaluation of the MycoMDx Pneumocystis Jirovecii PCR Assay (CE-approved), including diagnostic sensitivity and specificity, the limit of detection (LoD), precision and stability. The above PCR assay reagents were developed and produced by Dynamiker Biotechnology (Tianjin) Co., Ltd.

Results
The sensitivity and specificity of the PCR assay reagents were above 90%, the LoD was 1000 copies/mL, and the precision and stability were in accordance with the requirements (CV≤5%).

Conclusions
The MycoMDx Pneumocystis Jirovecii PCR Assay has great clinical value by providing a rapid and reliable test to aid in early diagnosis.
B-205 Performance of a Novel Fluorogenic Assay for Detection of Carbapenemase-producing Enterbacteriaceae
Authors: Zhou Z , Li S , Jiang D , Wang Z , Wang Y , Zhang Y , Wang H , Su Y 
Published: October 2024

Abstract

Background
In recent years, under the selective challenge of antibiotics, the variety and number of drugresistant pathogenic microorganisms have increased significantly, which brings great challenges to clinical diagnosis and treatment, especially the infection caused by carbapenem resistant Enterobacteriaceae (CRE). Carbapenemases production is the main mechanism of drug resistance of Enterobacteriaceae to carbapenems. Drug resistance of Carbapenems can be caused by three mechanisms, resulting in the production of five major Carbapenemases. These are Klebsiella pneumoniae enzyme (KPC), New Delhi metal β-lactamase (NDM), carbapenem hydrolyzed oxalase (OXA-48 like), integrin-encoded metal β- lactamase (VIM) and IMP (Imipenemase). The real-time fluorescence quantitative PCR (qPCR) method based on molecular beacons was combined with the melting curve analysis to identify five drug resistance genes simultaneously by a single PCR reaction, with rapid detection, high sensitivity and strong specificity. Based on this principle, we developed the novel fluorogenic assay for rapid detection of Carbapenemases in multidrug-resistant Enterobacteriaceae (Dynamiker Biotechnology (Tianjin) Co., Ltd.).

Methods
We evaluated the performance of the novel fluorogenic assay for rapid detection of Carbapenemases in multidrug-resistant Enterobacteriaceae, including the limit of detection (LoD) and cross-reactivity, and compared it with the lateral flow immunochromatography assay (LFA).

Results
The LoD ranged from 75-450 CFU/mL for the five carbapenemase genes. The analytical specificity for target genes was 100%, as assessed with a panel of 15 pathogens, which indicated no cross-reactions. Comparison of qPCR and LFA results from twenty-three CRE clinical isolates with characterized carbapenemase content demonstrated a complete agreement (Table 1).

Conclusions
The novel fluorogenic assay for rapid detection of Carbapenemases in multidrug-resistant Enterobacteriaceae is an accurate and rapid method to identify KPC, NDM, VIM, IMP and OXA-48-like carbapenemases in the clinical microbiology laboratory, which can guide infection control programs to limit the spread of these organisms.
B-207 Clinical Evaluation of ExiStation™ FA 96 RV for the detection of human respiratory viruses in human nasopharyngeal swab specimen
Authors: Lee J , Kim J , Lee C , Park H 
Published: October 2024

Abstract

Background
There has been growing demand for fully automated multiplex testing of respiratory viruses in molecular diagnostics following the COVID-19 pandemic. ExiStation™ FA 96 RV developed by BIONEER is a multiplex real-time RT-PCR assay. It is designed for the simultaneous detection and differentiation of SARS-CoV-2, Influenza A, Influenza B, and RSV RNA in human nasopharyngeal swab specimens from individuals showing signs and symptoms of respiratory viral infection. ExiStation™ FA 96 automates the entire process from RNA extraction to real-time RT-PCR for up to 96 samples at once within 100 minutes. Moreover, the system is equipped to decap sample tubes, eliminating the need for handling sample transport media. In this study, the clinical performance of the ExiStation™ FA 96 RV assay was evaluated using nasopharyngeal swab samples.

Methods
Nasopharyngeal swab samples, totaling 859 samples, are collected according to the procedures by the clinical institution in France and preserved in Virus transport media. Once anonymized and randomly allocated, the swab transport media tube, without capping procedure, are directly placed onto the ExiStation™ FA 96. Subsequently, both extraction and real-time RT-PCR are automatically processed. Clinical sensitivity, specificity, and agreement are analyzed by comparing results with confirmatory reagents SARS-CoV-2 RT-qPCR Reagent Kit, PKAmp™ Respiratory SARS-CoV-2 RT-PCR Panel Kit, and Bosphore SARS-CoV-2/Flu/RSV Panel Kit, as well as comparator reagent Allplex™ Respiratory Panel 1 and Allplex™ SARS-CoV-2 Assay.

Results
The comparison between confirmatory reagents and the ExiStation™ FA 96 RV resulted in a clinical sensitivity of at least 96.4% (164/164 for SARS-CoV-2, 64/65 for Influenza A, 74/75 for Influenza B, and 53/55 for RSV) and a clinical specificity of 100% (500/500 for all targets). When compared with the comparator reagent, the overall agreement and Cohen's kappa value were both determined to be above 99.1% and 0.989, respectively (664/664 for SARS-CoV-2, 564/565 for Influenza A, 573/575 for Influenza B, and 550/555 for RSV). Additionally, any discrepant samples identified in comparison with the comparator reagent were verified through sequencing analysis. Upon sequencing analysis on a total of 8 samples and aligning the confirmed results with the analysis content, no alterations observed in the analysis of clinical sensitivity and specificity.

Conclusions
The evaluation of the ExiStation™ FA 96 RV assay showed outstanding clinical performance in detecting four respiratory viruses, SARS-CoV-2, Influenza A, Influenza B, and RSV, with high sensitivity and specificity. This study supports that the ExiStation™ FA 96 RV assay is a reliable tool for simultaneous molecular diagnostics of various respiratory infections in clinical laboratories.
B-208 Room Temperature Stable PCR Reagents Preparation Using Capillary-Mediated Vitrification Process
Authors: Renu S , Peris-Taverner Y , Sharpe J , Amle S , Bronsart L , Mohanty P , Shank-Retzlaff M 
Published: October 2024

Abstract

Background
Polymerase chain reaction (PCR) is a sensitive, and most used molecular technique for the disease diagnosis. Reagents used for PCR is available as a one-step and ready-to-use PCR Master Mix, which contains Taq DNA polymerase, dNTPs, MgC12, PCR reaction buffer. However, to preserve bioactivity, PCR reagents must be stored and distributed as frozen. Desiccation of PCR reagents can avoid cold storage and improper handling of wet reagents and its associated errors. We discovered capillary-mediated vitrification (CMV) processes can stabilize different biomolecules and allow them to be stored at ambient temperature. Here, we hypothesized that the CMV process can preserve and improve PCR reagents thermal stability, which will reduce PCR processing time and improve the testing throughput.

Methods
The CMV process was performed by mixing PCR Master Mix individual or all reagents such as Taq DNA polymerase or Hot Start Taq DNA polymerase, dNTPs, 16S rRNA primers, MgC12, and PCR reaction buffer with an excipient buffer and adding the mixture to a porous scaffold. The loaded scaffolds were placed into our vitrification chamber and dried for 30 min. After drying, the CMV-stabilized sample was tested or packed in Mylar pouches and were stored at ambient and elevated temperatures for 42 days. On the day of testing, the stabilized samples were eluted with nuclease-free water and E. coli genomic DNA was added, and the 50 uL reaction was tested by conventional PCR. The PCR product was visualized under agarose gel electrophoresis.

Results
To assess whether the CMV process maintains PCR reagents activity and not interfering with the assay, the individual PCR reagents and as a Master mix are preserved. The CMV stabilized polymerase enzyme, primer, or dNTPs amplified DNA similar to frozen reagents and produced comparable PCR products. Notably, CMV stabilized PCR Master Mix which contains either Taq DNA polymerase or Hot Start Taq DNA polymerase enzyme, dNTPs, primers, MgC12, and reaction buffer yielded intact amplified PCR product, which is comparable to frozen reagents. Likewise, a high intense PCR product was observed in the stabilized PCR Master Mix reagents when stored at room and elevated temperatures for 42 days.

Conclusions
The study results indicate that the CMV process stabilized and improved the PCR Master Mix reagents activity. The study suggests that the CMV technology has the potential to enable ambient storage and shipping of PCR reagents for molecular diagnostic and other related applications.
B-224 Rapid, Extraction-Free, and Portable Molecular Detection of HIV-1 and HCV Directly From Whole Blood
Authors: Michalak K , Tang D , Nguyen K , Lacap P , Kim J , McLaren P J 
Published: October 2024

Abstract

Background
Early diagnosis of human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV) is key to preventing viral transmission and improving linkage to treatment. Isothermal amplification methods, such as reverse transcription looped-mediated amplification (RT-LAMP), have enabled molecular diagnostics to expand beyond centralized laboratories. Despite the advances in molecular point-of-care (POC) testing, many of these tests are costly and still depend on a reliable power-source, specialized equipment, and cold-chain storage, making them impractical for resource-limited settings. To overcome these barriers, there is a need for innovative molecular diagnostics for blood-borne pathogens. Here, we propose a simple, extraction-free, portable workflow for detecting HIV-1 and HCV from whole blood within 40 minutes.

Methods
We developed singleplex RT-LAMP-based tests using modified published primer sets. Contrived whole blood samples containing HIV-1 or HCV virions were diluted in equal parts water and loaded directly into optimized RT-LAMP master mixes. To mitigate cold-chain storage dependence, RT-LAMP reactions were performed using a lyophilized master mix. The reactions were heated for 30 minutes using a hand-held, battery-powered heating device for simultaneous virion lysis and amplification. For simple visual detection, amplification was coupled with a lateral flow-based dipstick test. The analytical sensitivity of each detection test was evaluated using contrived whole blood samples ranging in HIV-1 and HCV 1a viral loads. Additionally, HCV detection was evaluated on genotypes 1b, 2b, and 3a. For result confirmation, a custom CRISPR-Cas12a-based assay was used to verify the presence or absence of pathogen-specific amplicons following amplification.

Results
The diagnostic workflow can be completed within 40 minutes, including less than 10 minutes of hands-on time. At optimal conditions, the HIV-1 and HCV 1a singleplex tests had lower limits of detections of 4.89 log10 cp/mL and 3.77 log10 IU/mL, respectively. Evaluation of the published literature showed that these viral loads are within the ranges observed during acute HIV-1 and HCV infection. HCV detection was also observed for genotypes 1b and 3a, albeit at a lower sensitivity than genotype 1a. No detection was observed for genotype 2b, presumably due to the lower viral loads of the samples. No cross-reactivity between the two tests was observed.

Conclusions
Our proposed workflow allows for rapid detection of blood-borne pathogens without the need for complex equipment or cold-chain storage, showing potential for application in resource-limited settings. Additional validation within a clinical setting using fingerstick blood remains to be tested.
B-243 Validating a highly automated, rapid turnaround urine biomarker assay for diagnosis of acute interstitial nephritis
Authors: Obeid W , Moledina D G , Parikh C R 
Published: October 2024

Abstract

Background
Urinary tumor necrosis factor alpha (TNF-α) and chemokine C-X-C motif ligand 9 (CXCL9) have been identified as potential diagnostic biomarkers for acute interstitial nephritis (AIN). Although validated on standard methods like ELISA and mesoscale discovery (MSD), these platforms have a turnaround time of greater than 4hr. Ella, is a new platform which takes around 90 minutes and is more suited to be used for diagnosis of AIN in hospital setting. However, validation data for urine TNF-α and CXCL-9 in Ella assays are currently unavailable.

Methods
Five samples were selected for dilution-linearity and spike-recovery analysis. At the time of collection, samples were centrifuged at 2,000xg and 4°C for 10 min and the supernatant was aliquoted and stored at -80°C until assayed. We investigated dilution linearity by performing serial dilutions at three levels (Neat, 1:2, 1:4) assayed in duplicate. We performed spike and recovery experiment at three different concentration levels (high, medium, and assay diluent) by adding 5% spike volume of mixed TNF-α and CXCL-9 calibrator or assay diluent to the samples. We defined acceptance when recovery was between 80% - 120%. Intraassay %CV was calculated by averaging %CV of duplicate runs across all experiments. Inter-assay %CV was calculated by averaging %CV three levels of QC over three days. Samples were analyzed on MSD for method comparison and correlation coefficients (Pearson and Spearman) were calculated between the two measurements.

Results
Average recoveries and MSD comparison for TNF-α and CXCL9 are in the table below. Diluting the urine samples 4-fold yielded acceptable recoveries between 80% - 120%. Comparison between MSD and Ella yielded strong correlation for CXCL9 and moderate correlation for TNF-α.

Conclusions
The Ella platform can be used to measure urinary TNF-α and CXCL9 with good precision and acceptable recoveries at 1:4 dilution. Ella measurements compare favorably with those of MSD.
B-244 Analytical Performance and Method Comparison Evaluation of a New High Throughput Fully Automated Plasma GFAP Immunoassay
Authors: Vucetic Z , Szabo M , Salvati M , Schlichtmann B , Patzlaff J , Unruh D , Curtis K , Mediger L , Nichkova-Doseva M 
Published: October 2024

Abstract

Background
Glial Fibrillary Acidic Protein (GFAP) levels are shown to be an indicator of neurologic injury in conditions like Traumatic Brain Injury (TBI) and stroke and as a marker of disease progression in neuromuscular disorders such as multiple sclerosis. Plasma GFAP is also implicated in detecting AD pathology, correlating to the clinical stage of the disease. The performance characteristics of the plasma GFAP immunoassay currently being developed on Beckman Coulter Access 2 and DxI 9000 analyzers are described.

Methods
The prototype GFAP assay is a one-step sandwich assay utilizing an anti-GFAP mouse monoclonal (MAb) antibody/alkaline phosphatase conjugate and paramagnetic particles coated with a complementary anti-GFAP mouse MAb. Sample and reactants are incubated and washed, and then a chemiluminescent substrate is added. The light generated is directly proportional to the GFAP concentration in the sample. The assay time to the first result is ∼30 minutes. Analytical performance evaluation and method comparison across different platforms was performed. 43 EDTA plasma samples, consisting of 19 Alzheimer’s Disease (AD) patient samples and 24 age-matched healthy normal samples, were evaluated.

Results
The prototype GFAP assay has a targeted analytical measuring range of 1.0 pg/ml to approximately 700.0 pg/ml (up to 10,000 pg/ml). All normal samples were quantified with 90% measuring below 20.0 pg/ml and a low dose coefficient of variation &lt;4.0%. A comparison of the prototype GFAP assay on DxI 9000 and Access 2 analyzers shows a Passing-Bablok slope of 0.94 (R=0.992) and an intercept of 0.37 pg/ml. Concordance analysis shows excellent agreement between the Access 2 and DxI 9000 assays. The dose ratio of the median of AD over the median of normal samples for Access 2 and DxI 9000 is 2.31 and 2.41, respectively, with AD patient median dose values of 14.3 pg/ml and 14.6 pg/mL, respectively, and normal sample median dose values of 6.2 pg/ml and 6.0 pg/mL, respectively.

Conclusions
The prototype GFAP assay provides fast, highly sensitive, and precise results in an automated immunoassay on the Beckman Coulter Access 2 and DxI 9000 platforms. The prototype plasma GFAP assay holds promise for diagnosing and monitoring various neurodegenerative diseases, including AD.
B-246 Pab to mab conversion via mass spectrometry: direct antibody discovery from serum
Authors: Crha J , Han X , Le Bihan T , Nunez De Villavicencio Diaz T , Ma B 
Published: October 2024

Abstract

Background
Navigating the intricacies of discovering unique and functional antibody binders for in vitro diagnostic development comes with several technological challenges, such as limited diversity of antibody repertoires, functional screening challenges, and the absence of suitable in vitro models. A proteomics-driven strategy to antibody discovery is a promising solution to overcome these obstacles by facilitating polyclonal to monoclonal antibody conversion.
De novo polyclonal antibody sequencing utilizes mass spectrometry and machine-learning driven bioinformatics to obtain complete antibody sequences from immunoserum or purified protein samples. This approach investigates the entire circulating antibody repertoire at the protein level, thereby broadening the spectrum of potential candidates. It further facilitates functional selection by enabling antibody purification and enrichment techniques. This capability facilitates the identification of antibodies possessing unique functions, crucial for subsequent downstream development.
We demonstrate the application and de novo polyclonal antibody sequencing using mass spectrometry and machine learning bioinformatics to enable the next generation of antibody discovery.

Methods
In this study, we employed a novel mass spectrometry-based approach, REpAb, to de novo sequence polyclonal antibodies directly from the immunized serum of animals or human patients. REpAb integrates mass spectrometry with machine learning-based bioinformatics to de novo sequence and assemble monoclonal antibodies from a polyclonal mixture. Protein lysates underwent analysis using an Orbitrap EclipseTM Series instrument (ThermoFisher Scientific, CA, US) coupled with the LC Evosep One (Evosep, Denmark).

Results
De novo polyclonal sequencing with REpAb yielded unique antibody binders not found in splenocytes or peripheral blood mononuclear cells (PBMCs). De novo antibody sequencing-discovered antibody sequences from naturally exposed humans showed unrestricted germline usage, compared to restricted germline used in antigen-immunized animals. Additionally, a proteomics-based approach for antibody discovery enables functional antibody selection through standard protein A/G purification, followed by negative and positive selection enrichment strategies to isolate antibody clones specific to the region of interest on the antigen.

Conclusions
Sequencing antibodies at the protein level directly reflects the circulating antibody repertoires, which expands the antibody diversity from the BCR repertoire. Proteomic-based antibody discovery empowers the functional selection of antibodies via meticulously designed enrichment strategies. This approach allows for the identification of a diverse pool of candidates possessing specific functions, such as anti-idiotypic features.