bluuebunny/crossref_metadata_2025_split huggingface dataset:
B-206 The Determination of Vanillylmandelic Acid (VMA), Homovanillic Acid (HVA) and 5-Hydroxyindolacetic Acid (5-HIAA) by LC-Ms/MS Assay in Urine Samples for Diagnosis of Neuroendocrynes Tumors
Authors: Junior L F , Paulo B F , Zauli D A 
Published: September 2023

Abstract

Background
The catecholamines dopamine, norepinephrine and epinephrine, as well the neurotransmitter serotonin are biogenic amines that were formed by enzymatic descarboxylation of aminoacids in the body. They realize an important function in central nervous system. Vanillylmandelic acid (VMA) and Homovanillic Acid (HVA) are degradation products of the catecholamines, and 5-Hydroxyindolacetic Acid (5-HIAA) is a serotonine degradation product. They are important to the diagnosis of neuroendocrynes tumors, that produce catecholamines and/or serotonine in excess, resulting in elevated levels of VMA, HVA, and/or 5-HIAA in 24 h collection urine.

Objective
This study aimed to validate a simple LC-MS/MS method for quantification of VMA, HVA and 5-HIAA in isolated and 24 h urine samples for the diagnosis of neuroendocrines tumors.

Methods
Using a deep-well microtiter plate, 20 µL of isolated or 24 h urine was diluted in 540 µL of 0.05% of formic acid solution. Then, 20 µL of an internal standard solution containing stable isotopes of the analytes were added (VMA-D3, HVA-13C6,18O and 5-HIAA-13C6). The plate was sealed, homogenized for 30 s at 2500 rpm, and centrifuged for 15 min at 4000 rpm and ambient temperature. Then, 5.0 µL of the supernatant was injected into the LC-MS/MS system. Chromatographic separation was performed on the Waters ACQUITY UPLC system equipped with Kinetex XB-C18 1.7 µm X 50 mm X 2.1 mm column (Phenomenex), and methanol and formic acid 0.2% solution as mobile phase in 3.5 min of gradient mode separation. The detection was performed on a Waters XEVO TQ-S Micro mass spectrometer with electrospray ionization (ESI+). The current method was validated for carryover, linearity, precision, recovery, and limit of quantification, and it was evaluated by comparing the analysis in the developed method and a commercial kit by HCPL-ECD, of 15 real samples from volunteers in the laboratory.

Results
The method was successfully validated for all the parameters. The linearity was between 0.5–100.0 mg/L. The precision was less than 6.3%, the recovery was between 85.4%–103.4% and the quantification limit was 0.5 mg/L. The comparison with the reference method showed good coefficient correlations (R2 &gt; 0.92) for the three analytes.

Conclusion
We validate a simple LC-MS/MS method to quantify three metabolites to help the diagnosis of neuroendocrynes tumors. The method represents a simplification of the extraction compared with the commercial kit employing HPLC-ECD, and an important reduction of the per sample production cost, without loss of quality.
B-217 The study of association of FokI Polymorphism of Vitamin D Receptor (VDR) Gene, Vitamin D and Parathyroid Hormone Levels in Stage 4–5 Chronic Kidney Disease Patients: A Cross-sectional Study
Authors: Yadav R , Naagar S , Datta S K , Ahirwar A K , Mahajan S , Chopra P , Arulselvi S 
Published: September 2023

Abstract

Background
Chronic Kidney Disease (CKD) is an important public health problem globally and almost 35% patients of CKD have deficiency in vitamin D which progresses to 80% in stage 4–5 CKD patients. Active vitamin D binds to Vitamin D Receptor (VDR) to regulate target gene transcription. Various genetic variations (Single Nucleotide Polymorphism) such as FokI polymorphism, which is a T/C transition polymorphism (ACG to ATG) may affect the VDR gene expression and thus may play an important role in pathogenesis of CKD. The aim of the study is to study association between FokI Polymorphism of Vitamin D Receptor (VDR) Gene, serum Vitamin D and PTH levels in Stage 4–5 CKD patients.

Methods
A total of 150 patients, aged 25–60 years, attending the Department of Nephrology AIIMS New Delhi with chronic kidney disease (CKD) stage 4–5, were enrolled for the study. FokI polymorphism was analyzed by using polymerase chain reaction-restriction fragment length Polymorphism (PCR-RFLP) in study subjects. Following DNA isolation and PCR amplification, restriction digestion was done using FokI restriction endonuclease and visualised on 1.5 % agarose gel electrophoresis. The prevalence of different genotypes and allelic frequency distribution was compared between CKD patients and healthy controls (HC). Levels of PTH, Vitamin D were estimated by eCLIA.

Results
No significant differences in genotype and in allelic frequencies between CKD patients and HC were observed as shown in Table 1. No significant differences in biochemical parameters based on genotypic variations in CKD patients and in HC were observed.

Conclusion
The present study reveals no association of VDR FokI polymorphism with CKD. Furthermore, no significant differences in biochemical parameters were observed in FF, Ff and ff genotypic subgroups in CKD or HC groups. Further analysis revealed no significant differences in biochemical parameters based on genotypic variations in CKD patients and in HC. Table 1.Genotype frequency and allelic frequency of VDR FOK1 gene polymorphism in CKD patients and HC.Genotype(FOK1)Genotype frequency (%)P-valueX2 (chi2)Allelic frequencyCKD (n = 150)HC (n = 150)Total study group (n = 300)0.7700.5220CKD (n = 150)HC (n = 150)FF53.33 (n = 80)57.33 (n = 86)55.33 (n = 166)FfFfFf41.33 (n = 62)37.33 (n = 56)39.33 (n = 118)0.740.260.760.24ff5.33 (n = 8)5.33 (n = 8)5.33 (n = 16)
B-226 Clinical Evaluation of ElsiQ™ STI-12 Detection Kit 3/4 for Detection of Human Sexual Transmitted Infection Pathogens in Human Vaginal Swab and/or Urine Specimens
Authors: Ahn J , Kim D , Lee W , Yoon S , Cho S 
Published: September 2023

Abstract

Background
Nucleic acid amplification tests are a highly sensitive method for detecting sexual transmitted infection (STI) pathogens, and multiplex molecular assays are even more advantageous in screening and diagnosing multiple STI pathogens simultaneously. The ElsiQ™ STI-12 Detection Kit 3/4 (ElsiQ) is in vitro diagnostic marked multiplex real-time PCR assay that targets 6 STI pathogens, including human herpes simplex virus 1 (HSV1), human herpes simplex virus 2 (HSV2), Gardnerella vaginalis (GV), Treponema pallidum (TP), Candida albicans (CA), and Ureaplasma parvum (UP) in human vaginal swab and/or urine specimens.

Methods
In this retrospective study, 575 clinical specimens suspected of having STI pathogen were enrolled; 280 genital swabs and 295 urine samples from April to July 2021. The comparison tests were performed using both the ElsiQ and PANA RealTyper™ STD Kit (PANA). Clinical sensitivity and clinical specificity were evaluated by comparing the patient’s clinical features.

Results
The comparison results between ElsiQ and PANA were all consistency in 5 strains (HSV1, HSV2, GV, TP, UP), and 96.4% positive percent agreement value with 0.97 kappa value in CA. (Table 1). The clinical sensitivity and clinical specificity showed 100% in 5 strains (HSV1, HSV2, GV, TP, UP). Only CA has 97.0% sensitivity, and 100% specificity.

Conclusion
The ElsiQ assay demonstrated excellent analytical performance for detecting six STD pathogens, with high sensitivity and specificity. The results of this study support the use of the ElsiQ assay as a reliable and efficient tool for the simultaneous diagnosis of multiple STIs.
B-230 Monitoring SARS-CoV-2 Subvariants for Evaluation of the Diagnostic Kit’s Annealing Site Using Nanopore Sequencing
Authors: Kim Y , Noh T , Lee S , Kim Y , Prakash D , Lee K 
Published: September 2023

Abstract

Background
Currently, various diagnostic kits are being developed to aid in the rapid diagnosis or detection of SARS-CoV-2, but viruses continue to evolve through mutations resulting in phenotypic changes such as increased infectivity and pathogenicity, or vaccine evasion. Over time, such alterations in sequence may also occur at the annealing sites of the primers and probes in the diagnostic kit, leading to false-negative results. To prevent this, a continuous mutation monitoring is required. The authors performed SARS-CoV-2 Whole Genome Sequencing (WGS) and then compared and monitored the difference between actual experimental and public data from the National Center for Biotechnology Information (NCBI) datasets for each mutant strain.

Methods
WGS was performed by using GridION (Oxford Nanopore Technologies, UK) on 1500 nasopharyngeal swab SARS-CoV-2 positive samples requested for testing at SCL from January 2020 to December 2022. Experimental data were aligned through epi2me and analyzed using the Nextclade platform (Fig. 1A). For public data, 119 sequences reported in Korea (Fig. 1B) were retrieved from the NCBI database, reanalyzed using Nextclade platform, and compared.

Results
In numerous samples tested, mutation, insertion, and deletion were found to occur over time. In particular, in the case of Omicron subvariants, a common Gap (region 1; Gap: 11288–11296, region 2; Gap: 28362–28370) were identified in orf1ab (RdRP), the target gene for the majority of existing diagnostic kits, and in N gene (Fig. 1). Therefore, while designing a diagnostic kit, this region should not be included in the annealing site.

Conclusion
In order to maintain an effective diagnosis and quarantine system for COVID-19, mutations affecting the annealing site of primers and probes must be identified through a continuous genome monitoring. The results obtained from the continuous genome monitoring can be utilized to upgrade the existing commercially available kits as well as to develop new kits.
B-236 Portable 2 Hour Sample-to-Answer Multi-pathogen Phenotypic Antibiotic Susceptibility Test (AST)
Authors: Roegner M E 
Published: September 2023

Abstract

Background
Antimicrobial Resistance is a rapidly growing issue worldwide, arising from the overuse and misuse of antibiotics. The strongest tool the medical community has available to combat AMR is rapid, correct application of antibiotics to identified bacterial infections. Clinics and physician offices seek on-site rapid solutions to speed the pathway to timely and appropriate antibiotic therapies. GeneCapture is prototyping a rapid, sample-to-answer product to provide antibiotic susceptibility results directly from infected urine or wound samples. The test provides a susceptible vs non-susceptible call in under two hours and can be applied to samples with single or mixed pathogens. With support from the Defense Health Agency, a portable table-top instrument and disposable cartridge are being engineered to run this test at the point of care, allowing same-day prescriptions. The process is currently operational on the lab bench.

Methods
Fluorescent labeling of bacterial species, followed by optical counting, determines population growth with/without a suite of antibiotics. An on-board algorithm instantly converts raw cell numbers into a differential call of Susceptible or Non-susceptible for each bug/drug pair. A disposable cartridge with on-board antibiotics has been designed and is being finalized for fabrication. The optical counting is operating in a desktop device.

Results
Testing to date includes 124 strains from 7 of the most common Urinary Tract Infection and wound pathogens against a panel of six antibiotics. A total of 541 antibiotic/bacterial strain pairs in single or multiple pathogen antibiotic susceptibility testing (AST), are producing an initial 88.7% agreement to gold standard culture results. These results are all generated in two hours or less of assay time and represent a strong preliminary data set.

Conclusion
This 2-hour portable, novel, phenotypic AST profiling will produce informed and effective treatment options for medics, clinics and physician offices long before existing AST methods could return results.
B-250 Simultaneous Detection of Twelve Pathogens of Sexually Transmitted Infections Using an Innovative All-in-One PCR Instrument
Authors: Nam S , Lee J , Song M , Heo H , Jeong Y , Kim D , Park H 
Published: September 2023

Abstract

Background
Every day, nearly 1 million people are infected with sexually transmitted infections (STI) that are transmitted through sexual contact. Early diagnosis and treatment of STI are critical in reducing their transmission, and PCR is the most sensitive method among diagnostic tests. To address this need, we have developed a new STI Real-Time PCR-based diagnostic kit, which is capable of detecting twelve STI pathogens simultaneously in up to 94 specimens at once. To evaluate the analytical performance, we used the ExistationTM FA System, which automatically performs from decapping primary sample tubes followed by nucleic acid extraction to PCR analysis within 2 h. Twelve (12) pathogens of STI are described below: Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Ureaplasma urealyticum (UU), Mycoplasma genitalium (MG), Trichomonas vaginalis (TV), Mycoplasma hominis (MH), Herpes simplex virus type1 (HSV1), Herpes simplex virus type2 (HSV2), Ureaplasma parvum (UP), Treponema pallidum (TP), Candida albicans (CA), Gardnerella vaginalis (GV), from male urine and female vaginal swabs.

Methods
The analytical performance evaluation was performed using the new STI diagnostic kit to detect twelve pathogens of STI. The analysis was performed using culture fluids (CT, NG, UU, MG, TV, MH, HSV1, HSV2, UP, CA, GV; ATCC, USA) and TP genomic DNA (Vircell, Spain), which were spiked in urine and vaginal swab media. The limit of detection (LoD) was determined by testing multiple replicates of serial dilutions of the spiked sample around the expected LoD. The LoD was defined as the lowest dilution at which the assay detected ≥95% of 24 replicates. To evaluate precision, each sample was tested 2 times a day in duplicate per run for 5 days. To assess cross-reactivity, twenty-two bacteria and viruses related to STI were used.

Results
The LoD was determined 60.26 IFU/mL, 35.48 cfu/mL, 467.74 copy/mL, 45.71 cell/mL, 19.5 cells/mL, 316.23 copy/mL, 199.53 TCID50/mL, 13.18 TCID50/mL, 588.84 copy/mL, 993.25 copy/ml, 660.69 copy/mL, 389.05 copy/mL in Urine and 47.86 IFU/mL, 27.54 cfu/mL, 363.08 copy/mL, 38.90 cell/mL, 15.14 cells/mL, 245.47 copy/mL, 181.97 TCID50/mL, 8.71 TCID50/mL, 575.44 copy/mL, 741.31 copy/ml, 524.81 copy/mL, 288.40 copy/mL in vaginal swab media for CT, NG, UU, MG, TV, MH, HSV1, HSV2, UP, CA, GV, and TP, respectively. For repeatability assay, the 12 STI samples showed similar Ct value in each run, with all standard deviation (SD) were less than 2.0. In terms of cross-reactivity, there were no positive results obtained from testing bacteria and viruses using the new STI diagnostic kit.

Conclusions
The ExistationTM FA system is highly convenient platform for Real-Time PCR-based diagnostics. Particularly, our new STI diagnostic kit provides an accurate and efficient method for simultaneous detection of 12 STI pathogens.
B-267 Validation of a New Molecular Test to Quantify Plasma Cytomegalovirus Load
Authors: Dehesa-García B , García-Murillo A , Cortés-Portero I , Pérez-Grijalba V , García-Manrique B 
Published: September 2023

Abstract

Background
Cytomegalovirus (CMV) is a DNA virus that often causes severe disease in immunosuppressed or immunocompromised patients. With the expanding indications for immune-modulating agents and transplant patients, the number of patients at risk for developing CMV disease is increasing. Test results are used to determine active CMV disease, risk of developing active disease, response to therapy, risk of relapsed infection, and/or appropriate time to discontinue therapy. For this purpose, molecular assays are attractive because of their broad linear range and low limits of detection. CerTest Biotec has developed a quantitative real time PCR assay for the detection and quantification of cytomegalovirus in plasma samples.

Methods
Plasma-EDTA samples characterized as positive or negative and quantified by IU/ml were selected. The selected samples were collected from both male and female adults (except 1 sample of an infant) between July 2016 and December 2020. Two CE-marked molecular reference assays were used for the comparative analysis: RealStar® CMV PCR Kit 1.0 (Altona), using the CFX96™ Real-Time PCR Detection System (Bio-Rad) and extracted with the QIAamp® MinElute® Virus Spin Kit (QIAGEN); and cobas® CMV (Roche Diagnostics), using the cobas® 8800 system. VIASURE analysis was performed using the following workflow: the automated extraction method MagDEA® Dx SV with magLEAD® 12gC and CFX96™ Real-Time PCR Detection System (Bio-Rad).

Results
A total of 273 EDTA-plasma clinical samples were analysed, 120 samples were reported as CMV positive samples and quantified and 153 samples were reported as CMV negative samples. Comparative analysis determined &gt;99% sensitivity and specificity. Regarding quantification, all methods obtained a correlation value of R2 ≥ 0.77.

Conclusion
With this study it was possible to observe that the VIASURE CMV q Real Time PCR Detection Kit present good clinical sensitivity and specificity compared to reference molecular assays results. No false positive results were observed. However, all variables employed on the workflow contribute to final quantification results and therefore should be taken into account as accumulative error in the calculation.
B-272 Interference Assays: How Different Substances can Affect the Detection of Respiratory Viruses Using a Quick Molecular Solution
Authors: Martínez-Santolaria M , García-Manrique B , Machetti-Mareca E , Gil-Rodríguez C 
Published: September 2023

Abstract

Background
Some substances present in clinical samples could affect the performance of qPCR and significantly impact its results. If nucleic acids are obtained using quick lysis reagents, the inhibition effect could be even worse since no washing steps are included. Thence, the aim of this study was to evaluate some potentially interfering substances that may be present in respiratory samples and their effect on the diagnosis using a quick molecular solution.

Methods
Ten negative nasopharyngeal swab samples in Viral Transport Medium (VTM, Vircell) were pooled and enriched with a known concentration of viral cultures from ATCC (Influenza A: VR-95PQ™ A/Puerto Rico/8/34; Human Respiratory Syncytial virus (RSV): VR26PQ™; and SARS-CoV-2: VR-1986HK™ SARS-Related Coronavirus 2, Isolate USA-WA1/2020) to obtain positive samples around the limit of detection. The pool was divided into seven aliquots. Six of them were spiked (each) with a known concentration of potentially respiratory interfering substances, according to FDA recommendations (Table 1). Another aliquot did not contain any substance and was considered as the positive control. Each aliquot was processed in triplicate using VIASURE Resp. viruses Quick Lysis Reagent (Certest Biotec S. L), following the manufacturer’s instructions. Nucleic acids were analysed with VIASURE SARS-CoV-2, Flu & RSV Real Time PCR Detection Kit (Certest Biotec S.L) in triplicate on CFX96™ Real-Time PCR Detection System (Bio-Rad).

Results
Almost all substances did not interfere throughout the workflow at the concentrations tested. Only benzocaine had a negative effect on the three pathogens analysed (SARS-CoV-2, Influenza A and RSV), detecting less than 8/9 replicates. This problem was resolved when the substance was tested at 0.15 mg/ml.

Conclusion
Evaluation of potentially interfering substances in quick molecular solutions is important, especially if nucleic acids processing methods do not have many purification steps. Thus, the study confirmed that the diagnosis using this solution is not easily compromised.
B-281 Analytical Validation of the Mindray High Sensitivity Cardiac Troponin I Assay
Authors: Fabre-Estremera B , Schulz K M , Ladd A , Buño Soto A , Apple F S 
Published: September 2023

Abstract

Background
According to recent global guidelines for patients presenting with ischemia, high sensitivity cardiac troponin I and T (hs-cTn) are the preferred biomarkers for the diagnosis and risk assessment of acute myocardial infarction (MI) and myocardial injury. The purpose of our study was to perform an analytical validation study of the Mindray hs-cTnI assay.

Methods
Analytical studies were designed according to Clinical and Laboratory Standards Institute protocols (CLSI). We used one reagent lot and one CL1200i chemiluminescence Immunoassay Analyzer (Mindray Bio Medical Electronics Co., Shenzhen, China). Following the CLSI EP17 A2 document, the limit of blank (LoB) and limit of detection (LoD) were assessed following analysis of a) Mindray controls without cTnI concentrations added for the LoB and b) 4 lithium heparin (LiHep) fresh samples with low cTnI concentrations, for 3 days for LoB and LoD, with 5 replicates per day (60 replicates per study). For the precision study, according to the CLSI EP15-A3 protocol, we used 12 LiHep fresh samples, measured for 10 days, with 2 runs per day (separated by a minimal time of 2 h) and in replicates of 3 (n = 720). For the 12 samples for the precision study, 7 were chosen with hs-cTnI concentrations close to the manufacturer sex-specific 99th percentile upper reference limits (URL; 31 ng/L for males and 15 ng/L for females). The CLSI EP06 protocol was followed to study linearity, using 9 fresh LiHep samples analyzed the same day in replicates of 7 (n = 63). A blank sample between every sample was used to avoid carryover. Our analytical specification for linearity was an allowable deviation &lt;10%. Analyses were performed with Analyze it for Excel (Method Validation edition, version 6.15).

Results
Using both parametrical and non-parametrical analyses, LoB was &lt;0.1 ng/L, with a relative light unit (RLU) of 5394. LoD was 0.1 ng/L according to the parametrical analysis. Regarding imprecision, for samples ranging from cTnI concentrations of 1 to 106 ng/L, repeatability had a coefficient of variation (%CV) from 1.15% to 3.83%, and within-laboratory imprecision ranging from 1.69% to 5.03%. Using samples with hs-cTnI concentrations close to the manufacture’s sex-specific upper reference limits (9–36 ng/L), the repeatability and within-laboratory precision was 1.27%–1.61% and 1.92%–2.52%, respectively. The measuring interval for the linearity study ranged from 1.1 to 30 772 ng/L. All analytical parameters met CLSI limits of acceptable performance. No carry over was detected.

Conclusion
Our analytical observations of the Mindray hs-cTnI assay demonstrate excellent LoB, LoD, imprecision and linearity, that were in alignment of with the manufacturer’s claims and published guidelines for hs-cTnI.